KR102254956B1 - A molecular marker for selecting onion white bulb color and the use thereof - Google Patents
A molecular marker for selecting onion white bulb color and the use thereof Download PDFInfo
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Abstract
Description
본 발명은 백색 구피색 양파를 선별할 수 있는 신규한 서열의 분자 마커 및 그 용도에 관한 것이다.The present invention relates to a novel sequence of molecular markers capable of selecting white guppy-colored onions and their use.
국내 채소종자시장 규모는 2018년 2,872억 원 규모로 지속적인 증가세를 보이고 있다. 이중 양파는 고추와 무에 이어 지난 10여 년간 3위를 차지하는 주요 채소작물로 약 361억 원 규모의 매출 규모를 보이고 있다. 그러나 양파 종자 수입은 약 250만 불 규모로 파프리카에 이어 2위를 차지하고 있어 수입을 대체할 수 있는 우수한 품종 개발이 절실하다.The size of the domestic vegetable seed market is showing a continuous increase of 287.2 billion won in 2018. Among them, onion is a major vegetable crop that ranks third over the past 10 years after pepper and radish, showing sales of about 36.1 billion won. However, the import of onion seeds is about 2.5 million dollars, ranking second after paprika, so it is urgent to develop excellent varieties that can replace imports.
양파는 수선화과의 부추아과 부추속에 식한 식물이다. 자극적인 냄새와 매운맛이 느껴지는 식재료로써, 다양한 음식에서 사용되는 식물이다. 양파는 알리신(Allicin)을 함유하고 있어 맵고 단 맛이 난다. 양파를 섭취하면 항산화 작용 및 혈중 콜레스테롤 수치를 낮춰주는 효능이 있다.Onion is a plant in the genus Allium of the Daffodil family. It is a food ingredient that has a pungent smell and a spicy taste, and is a plant used in various foods. Onions contain Allicin, so they taste spicy and sweet. Consuming onions has antioxidant properties and lowers blood cholesterol levels.
양파의 구피(Bulb)란 양파의 비늘줄기 부분을 말하며, 구피색(Bulb color)이란 양파의 비늘줄기의 껍질 색깔을 말한다. 양파는 다양한 구피색을 가지는데 국내에서 소비되는 대부분의 양파는 황색 구피색을 가지는 품종이다. 그러나 최근 소득수준이 높아지고 식생활의 변화로 인해서 적색 구피색 품종도 아울러 늘어가고 있는 추세이다.The bulb of onion refers to the part of the scale stem of the onion, and the bulb color refers to the color of the skin of the scale stem of the onion. Onions have various guppy colors, and most onions consumed in Korea are varieties with yellow guppy colors. However, due to the recent increase in income and dietary changes, the number of red guppy varieties is also increasing.
하지만 백색 구피색의 경우 우리나라에서는 거의 유통이 되지 않고 있다. 백색 구피색 양파의 경우 색소가 전혀 없기 때문에 요리를 할 때 첨가하면 요리 본연의 색깔을 변화시키지 않는 장점이 있다. 따라서 백색 구피색 양파의 경우 미국이나 유럽에서는 주요 품종 군에 속한다. 그리고 우리나라도 이러한 백색 품종의 장점 덕분에 백색 품종이 늘어날 것으로 전망된다.However, the white guppy color is rarely distributed in Korea. In the case of white guppy-colored onions, there is no pigment at all, so adding them when cooking has the advantage of not changing the original color of the dishes. Therefore, white guppy-colored onions belong to the main cultivar group in the United States and Europe. And it is expected that the number of white varieties will increase in Korea thanks to the advantages of these white varieties.
특히 백색 구피색 양파의 경우 미국 등지에서는 양파 가루를 만드는 원료로 활용되고 있고 시장규모도 매우 큰 것으로 알려져 있다. 따라서 백색 품종 개발연구는 우리나라 보다는 미국과 유럽에서 활발하게 이루어지고 있다. 이러한 활발한 육종연구에도 불과하고 아직 양파에서 백색을 결정하는 유전에 대한 연구는 다른 작물에 비해서 매우 미진한 실정이다.In particular, white guppy-colored onions are used as raw materials to make onion powder in the United States and elsewhere, and the market is known to be very large. Therefore, research on the development of white varieties is being conducted more actively in the United States and Europe than in Korea. Even in such active breeding studies, studies on the genetics that determine whiteness in onions are still very inadequate compared to other crops.
본 발명의 제 1 측면에 따르면, 서열번호 1의 염기서열(AcWHITE)을 포함하는 백색 구피색 양파 선별용 분자 마커를 제공하는 것을 목적으로 한다.According to the first aspect of the present invention, an object of the present invention is to provide a molecular marker for selecting white guppy-colored onions comprising the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
본 발명의 제 2 측면에 있어서, 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는 백색 구피색 양파 선별용 조성물을 제공하는 것을 목적으로 한다.In a second aspect of the present invention, an object of the present invention is to provide a composition for selecting white guppy-colored onions comprising a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
본 발명의 제 3 측면에 있어서, 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는 백색 구피색 양파 선별용 키트를 제공하는 것을 목적으로 한다.In a third aspect of the present invention, an object of the present invention is to provide a kit for selecting white guppy-colored onions comprising a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
본 발명의 제 4 측면에 있어서, 서열번호 1의 염기서열(AcWHITE)이 존재하는지 여부를 분석하는 단계를 포함하는 백색 구피색 양파 선별 방법을 제공하는 것을 목적으로 한다.In a fourth aspect of the present invention, an object of the present invention is to provide a method for selecting white guppy-colored onions comprising the step of analyzing whether or not the nucleotide sequence of SEQ ID NO: 1 (AcWHITE) is present.
본 발명의 제 5 측면에 있어서, 서열번호 1의 염기서열(AcWHITE)을 포함하는 백색 구피색 양파 제조용 재조합 벡터를 제공하는 것을 목적으로 한다.In a fifth aspect of the present invention, an object of the present invention is to provide a recombinant vector for producing white guppy-colored onions comprising the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
본 발명의 제 6 측면에 있어서, 서열번호 1의 염기서열(AcWHITE)을 이용하여 양파를 형질 전환시키는 단계를 포함하는, 백색 구피색 양파 제조 방법을 제공하는 것을 목적으로 한다.In the sixth aspect of the present invention, it is an object of the present invention to provide a method for producing white guppy-colored onions, comprising transforming onions using the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
본 발명의 제 7 측면에 있어서, 본 발명의 재조합 벡터로 형질 전환된 형질 전환 양파를 제공하는 것을 목적으로 한다.In the seventh aspect of the present invention, an object of the present invention is to provide a transformed onion transformed with the recombinant vector of the present invention.
본 발명의 제 8 측면에 있어서, 본 발명의 형질 전환 양파의 종자를 제공하는 것을 목적으로 한다.In the eighth aspect of the present invention, an object of the present invention is to provide the seeds of the transformed onion of the present invention.
1. 서열번호 1의 염기서열(AcWHITE)을 포함하는, 백색 구피색 양파 선별용 분자 마커.1. A molecular marker for selecting white guppy-colored onions, including the nucleotide sequence of SEQ ID NO: 1 (AcWHITE).
2. 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는, 백색 구피색 양파 선별용 조성물.2. A composition for selecting white guppy-colored onions, including a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
3. 위 2에 있어서, 상기 프라이머는 서열번호 7 및 서열번호 8의 염기서열을 포함하는, 백색 구피색 양파 선별용 조성물.3. In the above 2, wherein the primer comprises the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, a composition for selecting a white guppy-colored onion.
4. 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는, 백색 구피색 양파 선별용 키트.4. A kit for selecting white guppy-colored onions, including a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
5. 위 4에 있어서, 상기 프라이머는 서열번호 7 및 서열번호 8의 염기서열을 포함하는, 백색 구피색 양파 선별용 키트.5. In the above 4, wherein the primer comprises the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8, white guppy-colored onion selection kit.
6. 양파의 gDNA에 서열번호 1의 염기서열(AcWHITE)이 존재하는지 여부를 분석하는 단계를 포함하는, 백색 구피색 양파 선별 방법.6. A method for selecting white guppy-colored onions comprising the step of analyzing whether the nucleotide sequence of SEQ ID NO: 1 (AcWHITE) is present in the onion gDNA.
7. 위 6에 있어서, 상기 분석하는 단계는 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 상기 gDNA에 결합시키는 단계를 포함하는, 백색 구피색 양파 선별 방법.7. In the above 6, the step of analyzing comprises the step of binding a primer capable of detecting at least a portion of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 to the gDNA, white guppy color onion selection method.
8. 위 6에 있어서, 상기 분석하는 단계는 중합효소연쇄반응(PCR)으로 유전자를 증폭시키는 단계를 포함하는, 백색 구피색 양파 선별 방법.8. In the above 6, the step of analyzing comprises a step of amplifying the gene by polymerase chain reaction (PCR), white guppy color onion selection method.
9. 위 7 또는 8에 있어서, 상기 프라이머는 서열번호 7 또는 서열번호 8의 염기서열을 포함하는, 백색 구피색 양파 선별 방법.9. The method of 7 or 8 above, wherein the primer comprises a nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8, white guppy-colored onion selection.
10. 서열번호 1의 염기서열(AcWHITE)을 포함하는 백색 구피색 양파 제조용 재조합 벡터.10. A recombinant vector for producing white guppy-colored onions containing the nucleotide sequence of SEQ ID NO: 1 (AcWHITE).
11. 서열번호 1의 염기서열(AcWHITE)을 양파에 형질 전환시키는 단계를 포함하는, 백색 구피색 양파 제조 방법.11. A method for producing a white guppy-colored onion comprising the step of transforming the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 into onions.
12. 위 11에 있어서, 상기 형질 전환 단계는 제 10 항의 재조합 벡터를 이용하여 실시하는 백색 구피색 양파 제조 방법.12. The method of 11 above, wherein the transformation step is performed using the recombinant vector of
13. 위 11에 있어서, 상기 형질 전환 단계는 CRISPR/Cas9을 이용하여 형질 전환하는 백색 구피색 양파 제조 방법.13. The method of 11 above, wherein the transformation step is transformed using CRISPR/Cas9 to produce a white guppy-colored onion.
14. 위 10의 재조합 벡터로 형질 전환된 형질 전환 양파.14. Transformed onion transformed with the recombinant vector of the above 10.
15. 위 14에 따른 양파의 형질 전환된 종자.15. Transformed seeds of onion according to 14 above.
본 발명에 따른 분자 마커를 통해 백색 구피색을 가진 양파를 효율적으로 선별할 수 있다.The onion having a white guppy color can be efficiently selected through the molecular marker according to the present invention.
본 발명에 따른 프라이머를 포함하는 백색 구피색 양파 선별용 조성물 또는 선별용 키트를 이용함으로써 백색 구피색 양파를 효율적으로 선별할 수 있다.The white guppy-colored onions can be efficiently selected by using the white guppy-colored onion selection composition or the selection kit containing the primer according to the present invention.
본 발명에 따른 백색 구피 양파 제조용 벡터를 이용함으로써 형질 전환을 통해 백색 구피색 양파 생산 원가 및 제조 원가를 절감 할 수 있다.By using the vector for producing white guppy onions according to the present invention, it is possible to reduce the production cost and production cost of white guppy onions through transformation.
도 1은 양파 염색체상의 C 유전자좌에 연결된 4 개의 콘티그(contig)의 위치를 나타낸 것이다.
도 2는 백색 양파와 황색 양파 사이의 bHLH 코딩 유전자의 전사 수준 비교한 결과를 나타낸 것이다. 도 2 (a)는 참조 전사체에 지도화함으로써 얻어진 RPKM 값을 사용하여 측정된 전사 수준을 나타낸 것이다. 도 2 (b)는 새로 조립된 콘티그(contig)에 유전자 지도화함으로써 얻어진 RPKM 값을 사용하여 측정된 전사 수준을 나타낸 것이다. 도 2 (c)는 실시간 RT-PCR을 사용하여 측정된 F2:3 개체의 B2 유전자 전사 수준을 나타낸 것이다. (W1-W5: 백색 F2:3 개체, Y1-Y5: 황색 F2:3 개체)
도 3은 양파 B2, 피튜니아 AN1, 달리아 DvIVS 및 옥수수 IN1의 유전자를 비교한 것이다. (백색 부분: 인트론, 회색 부분: 엑손, 채워진 상자 부분: bHLH 도메인, 빗금 부분: 반복 서열)
도 4는 양파 B2 유전자의 구조를 나타낸 것이다. (백색 부분: 인트론, 회색 부분: 엑손, 채워진 상자 부분: AcWHITE, 빗금 부분: LTR 서열 부분, 수평 화살표 부분: B2 분자 마커의 프라이머 결합 부위, 점선 상자 부분: Vitis vinifera에서 분리 한 gag-pol 폴리단백질과 상동성을 나타내는 영역)
도 5는 B2 마커의 PCR 결과를 나타낸 것이다. 도 5 (a)는 두 가지 다른 프라이머 조합의 밴드 패턴을 나타낸 것이다. (A 밴드: 우성 동형 접합 황색 F2:3 개체, H 밴드: 이형 접합 황색 F2:3 개체, B 밴드: 열성 동형 접합 백색 F2:3 개체) 도 5 (b)는 백색, 황색 및 적색 육종 계통의 B2 마커 유전자형의 밴드 패턴을 나타낸 것이다. (A 밴드: 우성 동형 접합 황색 F2:3 개체, H 밴드: 이형 접합 황색 F2:3 개체, B 밴드: 열성 동형 접합 백색 F2:3 개체 (TIFF 335 kb))
도 6은 C 유전자좌와 완벽하게 연결된 분자 마커인 C9860를 나타낸 것이다. 도 6 (a)는 콘티그의 전체 길이 황색과 흰색 대립 유전자의 구조를 나타낸 것이다. (회색 부분: 엑손, 백색 부분: 인트론, Yellow allele의 검은 부분: 53-bp INDEL, 수평 화살표: C9860 분자 마커의 프라이머 결합 부위) 도 6 (b)는 C9860 분자 마커의 백색, 황색 및 적색 육종 라인의 유전자형을 나타낸 것이다. (A 밴드: 우성 동형 접합 황색 F2:3 개체, H 밴드: 이형 접합 황색 F2:3 개체, B 밴드: 열성 동형 접합 열성 백색 F2:3 개체 (TIFF 358 kb))
도 7는 양파 및 다른 식물 종으로부터 분리된 단백질의 bHLH 도메인의 아미노산 서열의 정렬을 나타낸 것이다.
도 8는 AcWHITE 및 그 상류 및 하류 영역의 염기서열을 나타낸 것이다. (회색 부분: AcWHITE 염기서열, 백색 밑줄: 말단 역반복 염기서열, 직사각형: 목표 부분 복제 염기서열(Target site duplication sequence), 세로 화살표: 전사 시작 부분, 굵은 글씨: B2 유전자의 엑손1 부분, 검정색 밑줄: B2 유전자 전사 시작 코돈)1 shows the positions of four contigs linked to the C locus on the onion chromosome.
Figure 2 shows the result of comparing the transcription level of the bHLH coding gene between white onions and yellow onions. Figure 2 (a) shows the transcription level measured using the RPKM value obtained by mapping on a reference transcript. Figure 2 (b) shows the transcription level measured using the RPKM value obtained by genetic mapping to the newly assembled contig (contig). Figure 2 (c) shows the B2 gene transcription level of F 2:3 individuals measured using real-time RT-PCR. (W1-W5: white F 2:3 individual, Y1-Y5: yellow F 2:3 individual)
3 is a comparison of the genes of onion B2, petunia AN1, dahlia DvIVS and corn IN1. (White part: intron, gray part: exon, filled box part: bHLH domain, hatched part: repeat sequence)
Figure 4 shows the structure of the onion B2 gene. (White part: intron, gray part: exon, filled box part: AcWHITE, hatched part: LTR sequence part, horizontal arrow part: primer binding site of B2 molecular marker, dotted box part: gag-pol polyprotein isolated from Vitis vinifera And domains showing homology)
5 shows the PCR results of the B2 marker. Figure 5 (a) shows the band pattern of two different primer combinations. (A band: dominant homozygous yellow F 2:3 individual, H band: heterozygous yellow F 2:3 individual, B band: recessive homozygous white F 2:3 individual) Figure 5 (b) is white, yellow and red It shows the band pattern of the B2 marker genotype of the breeding line. (A band: dominant homozygous yellow F 2:3 individuals, H band: heterozygous yellow F 2:3 individuals, B band: recessive homozygous white F 2:3 individuals (TIFF 335 kb))
6 shows C9860, a molecular marker perfectly linked to the C locus. Figure 6 (a) shows the structure of the full length yellow and white alleles of the contig. (Gray part: exon, white part: intron, black part of yellow allele: 53-bp INDEL, horizontal arrow: primer binding site of C9860 molecular marker) FIG. 6(b) is a white, yellow and red sarcoma line of C9860 molecular marker It shows the genotype of. (A band: dominant homozygous yellow F 2:3 individuals, H band: heterozygous yellow F 2:3 individuals, B band: recessive homozygous recessive white F 2:3 individuals (TIFF 358 kb))
7 shows the alignment of the amino acid sequence of the bHLH domain of proteins isolated from onion and other plant species.
8 shows the base sequence of AcWHITE and its upstream and downstream regions. (Gray part: AcWHITE sequence, white underline: terminal reverse repeat sequence, rectangle: target site duplication sequence, vertical arrow: start of transcription, bold:
이하 본 발명을 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적인 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 의미를 따른다.Hereinafter, the present invention will be described in detail. Unless otherwise defined, all terms in this specification are the same as the general meanings of the terms understood by those of ordinary skill in the art to which the present invention belongs. Follow the meaning used in the specification.
양파의 구조는 줄기 부분, 구피(bulb) 부분 및 뿌리 부분으로 나누어진다. 양파는 수분이 전체의 90%를 차지하지만 단백질, 탄수화물, 비타민 C, 칼슘, 인, 철 등의 영양소가 다량 함유되어 있다. 양파의 퀘르세틴(Quercetin)이라는 성분은 지방과 콜레스테롤이 혈관에 축적되는 것을 억제하여 고혈압 예방에 도움을 준다. 또한 퀘르세틴은 활성산소와 과산화지질로부터 세포가 공격당하는 것을 막아주는 역할을 하며 세포의 염증 및 상처를 회복하는데 효과가 있다. 양파에는 크롬이 풍부하게 들어있는데, 크롬은 포도당 대사의 항상성을 유지하는 미량 무기질이며, 인슐린 작용을 촉진해주어 혈당 조절에 도움이 된다. 양파에 들어있는 알리신(Allicin)은 신경안정제 역할을 한다. 또한 알리신은 일산화질소를 배출해 혈관의 강직성을 떨어뜨려 혈압을 낮추는 효과가 있으며, 혈소판이 엉기는 것을 방지하고 혈관 내의 섬유소 용해 작용을 도와주기 때문에 혈전이나, 뇌졸중 위험을 감소시켜준다. 그 외에도 양파에 들어있는 글루타치온(Glutathione)은 비타민 B1의 흡수를 돕고, 간의 지방분해를 돕는 물질이다. 본 실험의 일 실시예에서, 양파는 Allium cepa L. 를 이용하였다.The structure of the onion is divided into a stem part, a bulb part, and a root part. Onions account for 90% of the total moisture, but contain large amounts of nutrients such as protein, carbohydrates, vitamin C, calcium, phosphorus, and iron. Onion's quercetin (Quercetin) helps prevent hypertension by inhibiting the accumulation of fat and cholesterol in blood vessels. In addition, quercetin plays a role in preventing cells from being attacked by free radicals and lipid peroxidation, and is effective in restoring inflammation and wounds of cells. Onions are rich in chromium, and chromium is a trace mineral that maintains the homeostasis of glucose metabolism, and helps to regulate blood sugar by promoting insulin action. Allicin in onions acts as a stabilizer. In addition, allicin has the effect of lowering blood pressure by releasing nitrogen monoxide, lowering the stiffness of blood vessels, preventing platelets from clogging and helping to dissolve fibrin in blood vessels, thereby reducing the risk of blood clots or stroke. In addition, glutathione contained in onions is a substance that aids in the absorption of vitamin B1 and aids in the breakdown of fat in the liver. In one example of this experiment, the onion was used Allium cepa L.
본 명세서에서 "구피(Bulb)"란 양파의 비늘줄기 부분을 말하며, 비늘줄기란 줄기의 밑 부분이나 땅을 기는 줄기의 선단에 다육화한 다수의 비늘조각이 줄기를 둘러싸고 지하 저장기관으로 되어 있는 것을 말한다.In the present specification, "bulb" refers to the part of the scale stem of the onion, and the scale stem refers to the base of the stem or the tip of the stem where a number of scale fragments succulent surround the stem and become an underground storage organ. Say that.
"구피색(Bulb color)"이란 양파의 비늘줄기의 껍질 색깔을 말한다. 구피색은 양파 육종 프로그램에서 작물학적으로 중요한 특성이다. 적색, 황색 및 백색 구피색은 양파 품종에서 일반적이다. 드물게 보고된 색상은 황금색(Kim et al. 2004b)과 샤르트뢰즈(chartreuse, 연노랑 또는 연초록) 색상(El-Shafie and Davis 1967)이다. 이는 쿼세틴과 안토시아닌과 같은 플라보노이드 화합물에 의해 나타나며, 양파 구피색에 최소 50여 가지의 유도체가 관련되어 있다고 확인되었다 (Slimestad R, Fossen T, VIM (2007) Onions: a source of unique dietary flavonoids. J Agric Food Chem 55:10067-10080)."Bulb color" refers to the color of the peel of the scales of onions. Guppy color is an important agronomic characteristic in onion breeding programs. Red, yellow and white guppy colors are common in onion varieties. Rarely reported colors are golden (Kim et al. 2004b) and chartreuse (light yellow or light green) colors (El-Shafie and Davis 1967). This is indicated by flavonoid compounds such as quercetin and anthocyanin, and it has been confirmed that at least 50 derivatives are involved in onion guppy color (Slimestad R, Fossen T, VIM (2007) Onions: a source of unique dietary flavonoids. J Agric Food Chem 55:10067-10080).
본 명세서에서 "색소"란 어떤 물체의 색깔이 나타나도록 해 주는 성분을 말한다. "색소체"는 식물과 조류에서 발견되는 세포 소기관으로 엽록체, 유색체, 백색체가 있다. 유색체는 카로티노이드계의 색소를 가진 색소체로, 당근·호박·토마토·고추 등의 색은 이것에 의해 나타나는 것이다. 한편, 백색체는 색소를 갖지 않으며, 분열조직 부근이나 지하부의 세포 등에 함유되어 있는데, 주로 녹말 저장에 관여한다. 엽록체는 광합성에 관계하며 특히 연구가 잘 되어 있어, 그 구조와 기능이 상세하게 알려져 있다.In the present specification, "pigment" refers to a component that allows the color of an object to appear. The "pigmented body" is an organelle found in plants and algae, and includes chloroplasts, colored bodies, and white bodies. A colored body is a carotenoid-based pigment, and the colors of carrots, pumpkins, tomatoes, peppers, etc. are represented by this. On the other hand, the white body does not have a pigment and is contained in cells in the vicinity of the meristem or underground, and is mainly involved in starch storage. Chloroplasts are involved in photosynthesis and are particularly well-studied, and their structure and function are known in detail.
본 명세서에서 "플라보노이드(flavonoid)"란 화학적으로 두 개의 페닐 고리(A와 B)와 하나의 헤테로순환릭 고리(C)로 구성된 15개의 탄소 골격의 일반적인 구조를 가지고 있는 화합물을 말한다. 플라보노이드, 바이오플라보노이드, 이소플라보노이드, 또는 네오플라보노이드로 분류될 수 있다. 이는 모두 케톤 함유 화합물이며, 따라서 안토잔틴(플라본 및 플라보놀)이다. 플라보노이드 백본의 3개의 순환 또는 헤테로순환은 일반적으로 고리 A, B, C로 불린다. 고리 A는 일반적으로 플로로글루시놀(phloroglucinol) 치환 패턴을 나타낸다. (화학식 1 참조) 플라보노이드는 인 비트로(In vitro) 연구에서 광범위한 생물학적 및 약리학적 활성을 갖는 것으로 나타났다. 예를 들어, 항알레르기, 항염증, 항산화 물질, 항미생균, 암, 설사 방지 활성 등이 있다.In the present specification, "flavonoid" refers to a compound having a general structure of 15 carbon skeletons chemically composed of two phenyl rings (A and B) and one heterocyclic ring (C). They can be classified as flavonoids, bioflavonoids, isoflavonoids, or neoflavonoids. These are all ketone containing compounds, and therefore antoxanthines (flavones and flavonols). The three cycles or heterocycles of the flavonoid backbone are commonly referred to as rings A, B, C. Ring A generally represents a phloroglucinol substitution pattern. (See Formula 1) Flavonoids have been shown to have a wide range of biological and pharmacological activities in in vitro studies. For example, there are anti-allergic, anti-inflammatory, antioxidant, antimicrobial, cancer, and anti-diarrhea activity.
[화학식 1][Formula 1]
양파의 주성분은 식물의 2차 대사 산물 중 하나인 플라보노이드이며, 8,000여개 이상의 플라보노이드가 보고되었다 (Veitch NC, Grayer RJ (2011) Flavonoids and their glycosides, including anthocyanins. Nat Prod Rep 28:1626-1695). 양파에서 이러한 플라보노이드 성분은 약 54가지가 보고되었다. 그 중 양파의 대표적인 성분은 쿼세틴(Quercetin)이고, 양파의 구피색을 결정하는 성분은 안토시아닌(Anthocyanin)으로 밝혀졌다.The main component of onions is flavonoids, one of the secondary metabolites of plants, and more than 8,000 flavonoids have been reported (Veitch NC, Grayer RJ (2011) Flavonoids and their glycosides, including anthocyanins. Nat Prod Rep 28:1626-1695). About 54 of these flavonoids have been reported in onions. Among them, the representative component of onions is quercetin, and the component that determines the guppy color of onions was found to be anthocyanin.
본 명세서에서 "안토시아닌(Anthocyanin)"이란 플라보노이드계 물질로 냄새와 맛이 거의 없는 것이 특징이다. 이는 꽃이나 과실 등에 주로 포함되어 있는 색소로써, 양파의 구피 세포에서 구피의 색깔을 결정하는 역할을 한다. 수소 이온 농도에 따라 적색, 보라색, 파란색 등을 띤다. 또한 식물의 각 위치에 따라 각각 다른 역할을 할 수 있다. 열매에서는 동물을 유인하는 색으로서 역할을 하며, 그 동물을 이용해 식물의 씨앗을 퍼트리게 해준다. 꽃에서는 꽃의 색깔을 나타내는 색소로 작용하여 곤충을 유인해 꽃가루를 옮기게 만든다. 잎에서는 강한 자외선을 막아주는 역할을 한다. 또한 식물세포 속에 생기는 활성산소를 없애는 항산화제로도 작용한다.In the present specification, "Anthocyanin" is a flavonoid-based substance that has almost no smell and taste. It is a pigment mainly contained in flowers and fruits, and plays a role in determining the color of the guppies in the guppy cells of onions. It is red, purple, and blue depending on the concentration of hydrogen ions. It can also play a different role depending on the location of the plant. In the fruit, it acts as a color that attracts animals, and uses the animal to spread the seeds of plants. In flowers, it acts as a pigment representing the color of the flower, attracting insects to carry pollen. In leaves, it blocks strong ultraviolet rays. It also acts as an antioxidant that removes free radicals from plant cells.
다양한 식물에서 플라보노이드의 생합성 경로가 보고되었다 (Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2-20). 생합성 경로에 관여하는 유 전자는 크게 두 가지 부류로 나눌 수 있으며, 하나는 구조유전자(Structural genes)이며 다른 하나는 조절유전자(Regulatory genes)이다. 상기 생합성 경로에서 구조유전자는 생합성 경로에 관여하는 효소를 암호화하는 유전자 그룹이다. 상기 생합성 경로에서 조절유전자는 구조유전자의 발현을 조절하는 등의 방법으로 플라보노이드 생합성 경로를 조절하는 유전자로, 대부분 MYB 단백질, bHLH 단백질, WD40 단백질로 구성된 MBW 복합체를 형성하여 조절한다.The biosynthetic pathway of flavonoids has been reported in various plants (Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2-20). Genes involved in the biosynthetic pathway can be largely divided into two classes, one is structural genes and the other is regulatory genes. In the biosynthetic pathway, the structural gene is a group of genes encoding enzymes involved in the biosynthetic pathway. In the biosynthetic pathway, the regulatory gene is a gene that regulates the flavonoid biosynthetic pathway by regulating the expression of structural genes, and is regulated by forming an MBW complex composed of mostly MYB protein, bHLH protein, and WD40 protein.
본 명세서에서 "MBW 복합체(MYB-bHLH-WD)"는 플라보노이드 색소 생합성 경로의 조절을 위한 전사 복합체 모델이다. 이는 R2R3-MYB, bHLH 및 WD40 단백질로 구성되어 있다. 상기 3개의 단백질은 R2R3 MYB 및 WD40 단백질이 bHLH 단백질에 결합하여 3원 복합체(ternary complex)를 형성한다. 예를 들어, Arabidopsis의 경우, TTG1 단백질(WD40 단백질)이 bHLH, MYB 단백질 간의 상호작용에 영향을 준다. 이 단백질들의 조합에 따라 미치는 영향이 달라지고, 그 중 영향을 미치는 핵심 단백질은 MYB 단백질이다. 세 가지 단백질은 단백질 간 상호작용 외에 서로의 발현양상을 조절하기도 한다. 예를 들어, 페츄니아의 형질 전환 실험을 통해 AN2 MYB 단백질이 an1 bHLH 유전자의 발현 유도를 확인하였다 (Spelt C, Quattrocchio F, Mol JN, Koes RE (2000) anthocyanin1 of Petunia encodes a basic helixloop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12:1619-1631).In the present specification, "MBW complex (MYB-bHLH-WD)" is a transcription complex model for regulation of the flavonoid pigment biosynthetic pathway. It is composed of R2R3-MYB, bHLH and WD40 proteins. The three proteins, R2R3 MYB and WD40 proteins bind to the bHLH protein to form a ternary complex. For example, in Arabidopsis, the TTG1 protein (WD40 protein) affects the interaction between bHLH and MYB proteins. The combination of these proteins affects differently, and among them, the key protein that affects is the MYB protein. In addition to the interactions between proteins, the three proteins also regulate their expression patterns. For example, it was confirmed that AN2 MYB protein induces the expression of an1 bHLH gene through Petunia transformation experiments (Spelt C, Quattrocchio F, Mol JN, Koes RE (2000) anthocyanin1 of Petunia encodes a basic helixloop-helix protein that directly activates transcription of structural anthocyanin genes.Plant Cell 12:1619-1631).
"MYB 단백질"은 동물 및 식물에서 발견되는 대규모 유전자 전사 조절 인자(Transcriptional regulator)이다. 식물에서 MYB 유전자로부터 보존된 MYB DNA 결합 도메인을 포함하는 단백질 패밀리가 발현되며, 이는 R2R3 타입 MYB 도메인을 특징으로 하는 MYB 단백질 서브 패밀리를 포함한다.“MYB protein” is a large-scale gene transcriptional regulator found in animals and plants. In plants, a protein family comprising the conserved MYB DNA binding domain from the MYB gene is expressed, which includes the MYB protein subfamily characterized by the R2R3 type MYB domain.
"bHLH(basic helix-loop-helix) 단백질"은 모든 진핵 생물에서 확인되는 이합체(dimer) 전사 인자이며, 가장 큰 패밀리 중 하나로서 단백질 구조 모티프(motif)이다. 13개 내지 17개 아미노산 길이로, CANNTG DNA 서열을 인식하여 결합하는 기본 도메인 및 루프를 가진다. 상기 bHLH는 식물에서 2차 대사 조절, 세포 분화 및 패턴화, 브라시노 스테로이드 및 ABA 호르몬 신호 전달 경로 조절을 통한 식물 생장 및 발달, 뿌리 철분 흡수 및 피토크롬 매개 광신호 전달 과정 등에서 매우 다양한 역할을 한다."bHLH (basic helix-loop-helix) protein" is a dimer transcription factor found in all eukaryotes, and is a protein structural motif as one of the largest families. It is 13 to 17 amino acids long and has a basic domain and loop that recognizes and binds to the CANNTG DNA sequence. The bHLH plays a wide variety of roles in plant growth and development through secondary metabolic regulation, cell differentiation and patterning, brassinosteroid and ABA hormone signaling pathway regulation, root iron absorption and phytochrome mediated optical signal transduction in plants.
"WD40 단백질"은 대략 40개 아미노산의 짧은 구조적 모티프이며, WD40 도메인을 함유한 단백질은 4개 내지 16개의 반복 단위를 가진다. 이러한 반복 단위는 원형화된 베타 프로펠러(beta-propeller) 구조를 형성하며, 상기 반복 단위는 단백질 상호 작용을 위한 단단한 비계(scaffold)로서 작용한다. WD40 반복 단백질의 기본적인 기능은 다중 단백질 복합체 조합을 조절하는 것이다. 또한 신호 전달 및 전사 조절에서 세포주기 조절, 자가 포식 및 세포자살(Apoptosis) 등 다양한 기능에 관련되어 있다.“WD40 protein” is a short structural motif of approximately 40 amino acids, and proteins containing the WD40 domain have 4 to 16 repeat units. These repeat units form a circularized beta-propeller structure, which acts as a rigid scaffold for protein interaction. The basic function of the WD40 repeat protein is to regulate the combination of multiple protein complexes. It is also involved in various functions such as cell cycle regulation, autophagy and apoptosis in signal transduction and transcription regulation.
본 발명은 AcWHITE의 염기서열을 이용하여 백색 구피색 양파를 선별할 수 있는 분자 마커에 관하여 구체적으로 설명한다.The present invention will be described in detail with respect to a molecular marker capable of selecting a white guppy color onion using the base sequence of AcWHITE.
본 명세서에서 "핵산(nucleic acid)"이란 생명체에 필수적인 생체고분자 또는 작은 생체분자 중 하나로서 DNA 및 RNA를 포함한다. 핵산은 뉴클레오타이드 단위체로 구성되어 있다.In the present specification, the term "nucleic acid" includes DNA and RNA as one of biopolymers or small biomolecules essential for living organisms. Nucleic acids are composed of nucleotide units.
본 명세서에서 "뉴클레오타이드(nucleotide)"란 핵산을 구성하는 단위체인 분자를 말한다. 이는 대사에 중추적인 역할을 하고, 화학적 에너지 공급자(ATP) 역할, 세포 내 신호계와 효소 반응의 중요 성분으로서의 역할도 수행한다. 뉴클레오타이드의 구성 성분은 염기, 당, 인산으로 구성된다. 염기란 DNA의 경우 아데닌(Adenine), 구아닌(Guanine), 사이토신(Cytosine), 티민(Thymine)으로 구성되며, RNA의 경우 아데닌, 구아닌, 사이토신, 우라실(Uracil)로 구성된다. 당 부분은 5탄당으로서, DNA의 경우 디옥시리보오스, RNA의 경우 리보오스로 되어있다. 인산은 H3PO4이나 보통 핵산 분자 안에서는 두 개의 산소로 각각 당과 결합하며 남는 두 개의 산소에는 금속 이온(보통 마그네슘 2가 이온)이 붙어 있다. 이 인산 때문에 핵산은 강한 산성을 띤다.In the present specification, "nucleotide" refers to a molecule that is a unit constituting a nucleic acid. It plays a pivotal role in metabolism, plays a role as a chemical energy provider (ATP), and also plays a role as an important component of intracellular signaling and enzymatic reactions. The constituent components of nucleotides are composed of a base, sugar, and phosphoric acid. In the case of DNA, the base is composed of adenine, guanine, cytosine, and thymine, and in the case of RNA, it is composed of adenine, guanine, cytosine, and uracil. The sugar moiety is a pentose sugar, which is deoxyribose in the case of DNA and ribose in the case of RNA. Phosphoric acid is H 3 PO 4, but usually two oxygens in a nucleic acid molecule bind to each sugar, and the remaining two oxygens have metal ions (usually magnesium divalent ions) attached. Because of this phosphoric acid, nucleic acids are strongly acidic.
본 발명에서 사용하는 "DNA"는 필요에 따라 인공적인 염기로 치환하여 사용할 수 있다. 인공적 염기는, 예를 들어, 제노핵산 (xeno nucleic acid, XNA), 펩티드 핵산 (peptide nucleic acid, PNA), 잠금 핵산 (locked nucleic acid, LNA), 글리콜 핵산 (glycol nucleic acid, GNA), 또는 트레오스 핵산 (threose nucleic acid, TNA)일 수 있으나, 이에 제한되는 것은 아니다.The "DNA" used in the present invention may be substituted with an artificial base if necessary. Artificial bases are, for example, xeno nucleic acid (XNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), or tre It may be a three nucleic acid (TNA), but is not limited thereto.
단일 가닥 형태의 핵산 분자가 적절한 온도 및 용액 이온 강도의 조건하에서 다른 핵산 분자에 결합할 수 있을 때, 핵산 분자가 cDNA, 게놈 DNA 또는 RNA와 같은 또 다른 핵산 분자에 혼성화 될 수 있다. 혼성화 및 세척 조건은 널리 공지되어 있으며, 온도 및 이온 강도 조건이 혼성화의 엄격성(stringency)을 결정한다.When a single-stranded nucleic acid molecule can bind to another nucleic acid molecule under conditions of appropriate temperature and solution ionic strength, the nucleic acid molecule can hybridize to another nucleic acid molecule such as cDNA, genomic DNA or RNA. Hybridization and washing conditions are well known, and temperature and ionic strength conditions determine the stringency of hybridization.
엄격성 조건은 관련성이 먼 생물로부터의 상동 서열과 같이 적당히 유사한 단편들 내지 관련성이 가까운 생물로부터의 기능성 효소들을 복제하는 유전자와 같이 매우 유사한 단편들을 선별하기 위해 조정될 수 있다.Stringency conditions can be adjusted to select for moderately similar fragments, such as homologous sequences from distantly related organisms, or very similar fragments, such as genes replicating functional enzymes from closely related organisms.
혼성화 염격성에 따라 염기 사이에 불일치(mismatch)가 가능함에도 불구하고, 혼성화는 두 개의 핵산이 상보적인 서열을 함유할 것을 요구한다. 두 개의 핵산이 상보적인(complementary)인 서열을 함유하는 경우란 뉴클레오타이드들이 서로 혼성화 할 수 있는 염기서열을 갖는 경우를 말한다. 예를 들어, DNA와 관련하여, 아데노신은 티민에 상보적이고 사이토신은 구아닌에 상보적이다. 따라서, 본 발명은 명세서에 개시되거나 사용된 전체 서열들에 상보적인 분리된 핵산 단편들뿐만 아니라 실질적으로 유사한 핵산 서열들도 포함한다.Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible depending on the hybridization stringency. The case where two nucleic acids contain complementary sequences refers to cases in which nucleotides have base sequences capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present invention includes not only isolated nucleic acid fragments that are complementary to the entire sequences disclosed or used in the specification, but also nucleic acid sequences that are substantially similar.
본 발명에서는 백색 구피색 양파를 식별하기 위한 분자 마커를 연구한 결과, 집단 분리 분석(Bulked segregant analysis, BSA)과 RNA 염기서열화(RNA-sequencing) 기술을 접목하여 양파의 서열번호 1로 표시되는 백색 구피색 양파를 식별할 수 있는 전이인자인 AcWHITE의 염기서열을 확인하였다. 또한 상기 염기서열을 이용하여 백색 구피색을 결정하는 유전자형을 판별할 수 있는 분자 마커를 발견하였다.In the present invention, as a result of researching molecular markers for identifying white guppy-colored onions, the white color represented by SEQ ID NO: 1 of onions was obtained by grafting bulked segregant analysis (BSA) and RNA-sequencing technology. The nucleotide sequence of AcWHITE, a transfer factor capable of identifying guppy-colored onions, was confirmed. In addition, a molecular marker capable of discriminating the genotype determining white guppy color was discovered using the nucleotide sequence.
본 명세서에서 "염기서열"이란 DNA의 기본단위 뉴클레오타이드의 구성 성분 중 하나인 염기들을 순서대로 나열해 놓은 것을 말한다. DNA 상에서 염기가 일렬로 3개씩 모이면 하나의 트리플렛 코드(Triplet code)를 형성하여 하나의 아미노산을 지정한다. 이 트리플렛 코드들이 여러 개 모이면 궁극적으로 하나의 단백질을 지정하게 된다. 즉, 염기가 3개씩 모이면 트리플렛 코드를 형성하여, 단백질 서열로 변환되는 것이다. 인간의 단백질들은 20여 가지의 아미노산이 펩타이드 결합으로 연결되어 생성된다.In the present specification, the term "base sequence" refers to a sequence of bases, which is one of the constituents of the basic unit nucleotide of DNA. When three bases are collected in a line on DNA, one triplet code is formed to designate one amino acid. When these triplet codes are gathered together, they ultimately designate a single protein. In other words, when three bases are collected, a triplet code is formed, It is converted into sequence. Human proteins are produced by linking 20 kinds of amino acids through peptide bonds.
상기 "RNA 염기서열분석(RNA sequencing, RNA-seq)"은 차세대 유전체 분석기술(Next Generation Sequencing, NGS)을 이용하여 DNA로부터 전사되는 모든 RNA 전사체(transcriptome)의 염기서열을 분석하는 기술을 말한다. RNA 염기서열 분석 기술을 통해 RNA 대체 이어 맞추기(alternative RNA splicing), 유전자의 발현량, 염기서열의 변이, 유전자 융합(gene fusion), 단일염기 다형성(single nucleotide polymorphism, SNP) 등의 정보를 얻을 수 있다. 또한 전령 RNA 뿐만 아니라 miRNA, tRNA 등의 작은 RNA의 발현 역시 확인할 수 있고, 엑손과 인트론의 경계를 확인할 때도 사용될 수 있다.The "RNA sequencing (RNA-seq)" refers to a technology that analyzes the nucleotide sequence of all RNA transcriptomes transcribed from DNA using Next Generation Sequencing (NGS). . Through RNA sequencing technology, information such as RNA replacement splicing, gene expression level, nucleotide sequence variation, gene fusion, and single nucleotide polymorphism (SNP) can be obtained. have. In addition, it is possible to check the expression of not only messenger RNA but also small RNAs such as miRNA and tRNA, and can be used to check the boundary between exons and introns.
본 발명에서는 B2 유전자의 5' 비번역부위(Untranslated Region, UTR)에 AcWHITE라 명명된 비자율적 전이인자가 존재하는 경우, bHLH의 발현과 관련되어 있다는 것을 밝혀냈다.In the present invention, it was found that the presence of a non-irritant transfer factor named AcWHITE in the 5'untranslated region (UTR) of the B2 gene is related to the expression of bHLH.
본 발명에서 "B2 유전자"란 상기 조절 MBW 복합체와 관련하여, bHLH 전사 인자를 코딩하는 유전자를 말한다. (도 8 참조)In the present invention, "B2 gene" refers to a gene encoding the bHLH transcription factor in relation to the regulatory MBW complex. (See Fig. 8)
본 발명에서 "AcWHITE"란 비자율적 DNA 전이인자로서, B2 유전자의 백색 대립 유전자의 5' 상류 영역에 포함된 577bp의 염기서열을 말한다. 상기 전이인자의 양쪽 말단은 8bp 표적 부위 복제(GTTATAAC) 및 7bp 말단 역 반복(CAAGGTT)의 서열을 가진다. (도 8 참조)In the present invention, "AcWHITE" is a non-irritating DNA transfer factor, and refers to a nucleotide sequence of 577 bp included in the 5'upstream region of the white allele of the B2 gene. Both ends of the transfer factor have a sequence of 8bp target site replication (GTTATAAC) and 7bp terminal reverse repeat (CAAGGTT). (See Fig. 8)
본 명세서에서 "AcWHITE-B2 유전자"란 AcWHITE 상류의 전사 시작 부분(Translation start site, TSS)에서부터, B2 유전자의 엑손1까지의 염기서열을 포함하는 유전자 카세트(Gene cassette)를 말한다. (도 8 참조)In the present specification, "AcWHITE-B2 gene" refers to a gene cassette including a nucleotide sequence from the translation start site (TSS) upstream of AcWHITE to
본 명세서에서 "전이인자(transposon)"란 DNA에서 RNA으로 전사된 유전정보가 역전사 효소에 의해 상보적인 DNA으로 합성되어 유전체내에 다시 삽입되는 배열의 총칭을 말한다. "역전이인자(retrotransposon)"란 DNA로 시작하여 RNA의 중간 단계를 거쳐 DNA로 다시 역전사(reverse transcription)되는 과정을 필수적으로 사용하는, 게놈 상에서 복제되어 다른 곳으로 삽입이 가능한 트랜스포존을 말한다. 레트로트랜스포존은 긴 말단 반복(long terminal repeat, LTR)을 가지는 LTR 레트로트랜스포존(LTR retrotransposon)과 그렇지 않은 비 LTR 레트로트랜스포존(non-LTR retrotransposon)이 있다.In the present specification, "transposon" refers to a generic term for an array in which genetic information transcribed from DNA to RNA is synthesized into complementary DNA by reverse transcriptase and inserted again into the genome. The term "retrotransposon" refers to a transposon that can be replicated on the genome and inserted elsewhere, which essentially uses the process of starting with DNA, going through the intermediate stage of RNA, and then back into DNA. Retrotransposons include an LTR retrotransposon with a long terminal repeat (LTR) and a non-LTR retrotransposon without a long terminal repeat (LTR).
본 발명의 일 실시예에 따르면, 상기 비자율적 전이인자인 AcWHITE가 B2 유전자의 5' 비번역부위(Untranslated Region, UTR)에 위치하는 경우, B2 유전자가 불활성화 되어 bHLH 단백질의 전사가 억제된다.According to an embodiment of the present invention, when AcWHITE, which is the non-irritant transfer factor, is located in the 5'untranslated region (UTR) of the B2 gene, the B2 gene is inactivated, thereby inhibiting the transcription of the bHLH protein.
본 명세서에서 "분자 마커(molecular marker)란 식물종과 같은 유기체가 공통적으로 가지는 분자, 예를 들어 DNA 염기서열과 같은 분자들의 차이를 이용하여 특정 형질의 표지자로 사용할 수 있는 표지 분자를 말한다. 이는 염색체 상에서 물리적 위치를 나타내며 특정 표현 형질을 암호화하고 있으므로, 특정 형질을 다른 작물 품종에 도입할 때 유전적 표지자로 사용할 수 있다. 이러한 분자 마커는 짧은 DNA서열로 구성될 수 있으며, 예를 들어 단일 염기 다형성(SNP), 제한효소 절편길이 다형성(RFLP), 단순 염기서열 길이 다형성(SSLP), 유전자 증폭산물 길이 다형성(AFLP), DNA 다형성 무작위 증폭(RAPD), 가변수 직렬반복(VNTR), 미세부수체다형성, 단순 서열 반복(SSR), 단연쇄반복(STR), 단일 특징 다형성(SFP), DArT, RAD 마커자 등이 있을 수 있으나, 이에 제한되는 것은 아니다.In the present specification, "molecular marker" refers to a molecule that can be used as a marker for a specific trait by using a molecule common to an organism such as a plant species, for example, a difference between molecules such as a DNA sequence. This refers to a marker molecule that can be used as a marker for a specific trait. Since it represents a physical location on a chromosome and encodes a specific phenotypic trait, it can be used as a genetic marker when introducing a specific trait into other crop varieties, such molecular markers can be composed of short DNA sequences, for example a single base. Polymorphism (SNP), restriction enzyme fragment length polymorphism (RFLP), simple sequence length polymorphism (SSLP), gene amplification product length polymorphism (AFLP), DNA polymorphism random amplification (RAPD), variable serial repetition (VNTR), microfractions Polymorphism, simple sequence repetition (SSR), single chain repetition (STR), single characteristic polymorphism (SFP), DArT, RAD markers, etc. may be present, but are not limited thereto.
"다형성(polymorphism)"이란, 하나의 유전자좌(locus)에 두 가지 이상의 대립유전자(allele)가 존재하는 경우를 의미하며 다형성 부위 중에서, 개체에 따라 단일 염기만이 다른 것을 단일 염기 다형성(SNP)이라 한다. 바람직한 다형성 마커는 선택된 집단에서 1% 이상, 더욱 바람직하게는 5% 또는 10% 이상의 발생빈도를 나타내는 두 가지 이상의 대립유전자를 가진다."Polymorphism" refers to the case where two or more alleles exist in one locus, and among the polymorphic sites, only a single base is different depending on the individual, which is referred to as single base polymorphism (SNP). do. Preferred polymorphic markers have two or more alleles exhibiting an incidence of at least 1%, more preferably at least 5% or 10% in the selected population.
상기 "단일 염기 다형성(Single nucleotide polymorphism, SNP)"이란 염색체의 단일부위에서 여러 가지 DNA 염기들 중의 하나에 나타나는 일반적인 돌연변이를 말한다.The "single nucleotide polymorphism (SNP)" refers to a general mutation that appears in one of several DNA bases in a single site of a chromosome.
상기 "유전자좌(locus)"란 유전학에서 특정 유전자 또는 유전자 마커가 있는 염색체의 특정 고정 위치를 말한다. 각 염색체는 많은 유전자를 가지고 있으며, 각 유전자는 다른 위치나 유전자좌를 차지하고 있다.The "locus" refers to a specific fixed position on a chromosome having a specific gene or genetic marker in genetics. Each chromosome has many genes, and each gene occupies a different location or locus.
양파 구피색을 결정한다고 알려진 유전자좌는 총 다섯 개(I, C, G, R, L)이다(El-Shafie MW, Davis GN, (1967) Inheritance of bulb color in the onion (Allium cepa L.). Hilgardia 38:607-622). "I 유전자좌"는 색깔 억제 인자(color-inhibiting factor)로, 우성 동형접합체일 때 다른 유전자좌의 유전자형과 관련 없이 백색 구피색을 나타내며 불완전 우성유전을 한다. "C 유전자좌"는 기본 색깔 인자(basic color factor)로, 열성 동형 접합체일 때 다른 유전자좌와 상관없이 백색 구피색을 나타낸다. "G 유전자좌"는 열성 동형 접합체일 때 샤틀루즈 구피색을 나타내지만, 아직 원인 유전자는 아직 밝혀지지 않았다. "R 유전자좌"와 "L 유전자좌"는 황색과 적색 구피색에 상호보완적으로 관여한다. 두 유전자좌 모두 우성 대립유전자(allele)을 가져야 적색 구피색이, 그렇지 않으면 황색 구피색이 나타난다. R 유전자좌는 Dihydroflavonol 4-reductase (DFR) 효소를 암호화하고, L 유전자좌는 Anthocyanidin synthase (ANS) 효소를 암호화한다.There are a total of five loci known to determine the onion bulb color (I, C, G, R, L) (El-Shafie MW, Davis GN, (1967) Inheritance of bulb color in the onion (Allium cepa L.). Hilgardia 38:607-622). "I locus" is a color-inhibiting factor, and when it is a dominant homozygous, it exhibits a white guppy color regardless of the genotype of other loci, and is incomplete dominant inheritance. The "C locus" is a basic color factor, and when it is a recessive homozygous locus, it exhibits a white guppy color regardless of other loci. The "G locus" exhibits a chattelose guppy color when it is a recessive homozygous, but the causative gene has not yet been identified. The "R locus" and "L locus" are complementarily involved in the yellow and red guppy colors. Both loci must have a dominant allele to give a red guppy color, otherwise a yellow guppy color. The R locus encodes the enzyme Dihydroflavonol 4-reductase (DFR), and the L locus encodes the enzyme Anthocyanidin synthase (ANS).
본 명세서에서 "우성"이란 서로 다른 대립형질(allele)을 가진 두 순종끼리 교배하여 태어난, 잡종 제1대에서 이 잡종의 어버이(두 순종) 중에서 한 쪽 형질이 주도적으로 나타나는 형질을 말한다. 반대로 나타나지 않고 숨겨져 있는 형질을 "열성"이라 한다. "완전 우성"이란 유전자가 한 개여도 같은 유전자가 두 개 있는 경우와 동등한 형질 발현 능력이 있으면 표면에 드러나는 형질이 부모 중 한쪽과 잡종 제1대가 같은 경우를 말한다. "불완전 우성"이란 한 쪽이 절반의 작용하여 잡종 제1대의 표현형이 우성 호모인 부모와 열성 호모인 부모의 중간 형으로 나타나는 것을 말한다. 그 외에 유전자간 우열 관계가 분명하지 않고, 여타 유전자에 의해 영향을 받는 경우도 불완전 우성 유전이라 할 수 있다.In the present specification, the term "dominant" refers to a trait that is born by crossing two obediences having different alleles, and one of the parents (two obediences) of this hybrid in the first generation of hybrids. Conversely, a hidden trait that does not appear is called "recessive". "Completely dominant" refers to a case in which one of the parents and the first generation of hybrids have the same trait that is revealed on the surface if there are two identical genes even if there is one gene and has the same ability to express a trait. "Imperfect dominance" refers to the fact that one side acts in half, and the phenotype of the first hybrid generation appears as an intermediate form between a parent who is a dominant homo and a parent who is a recessive homo. In addition, the dominant relationship between genes is not clear, and it can be said that it is incomplete dominant inheritance when it is affected by other genes.
상기 "대립유전자(allele)"란, 상동염색체의 동일한 유전자좌에 존재하는 한 유전자의 여러 타입을 의미한다. 대립유전자는 다형성을 나타내는데 사용되기도 하며, 예컨대, SNP는 두 종류의 대립인자(biallele)를 갖는다.The "allele" refers to several types of one gene present at the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNP has two types of alleles.
본 명세서에서 "집단 분리 분석(Bulked segregant analysis, BSA)"이란 돌연변이 표현형과 관련된 유전자 마커를 식별하는 데 사용되는 기술을 말한다. 이를 통해 질병 저항성 또는 감수성을 부여하는 유전자를 발견할 수 있다. 상기 분석은 알아보고자 하는 특성에 대한 반대되는 표현형을 표시하는 두 그룹을 형성하고, 각 그룹에 있는 모든 개체의 DNA를 모아서 두 개의 집단 DNA 샘플을 만든다. 이 2개의 집단 샘플은 제한 조각 길이 다형성(Restriction fragment length polymorphism, RFLP) 또는 RAPD와 같은 기술을 사용하여 분석되어 게놈의 다양한 유전자좌에서의 유사점 및 차이를 검출 할 수 있다. 두 그룹은 돌연변이와 관련된 유전자좌를 제외하고 게놈의 모든 유전자좌에서 대립 유전자의 무작위 분포를 갖는다. 두 집단 샘플 사이의 유전자좌에서의 일관된 차이는 유전자좌가 관심 돌연변이와 관련되어 있음을 의미한다.As used herein, "Bulked segregant analysis (BSA)" refers to a technique used to identify genetic markers associated with a mutant phenotype. Through this, genes that confer disease resistance or susceptibility can be found. The analysis forms two groups that display opposite phenotypes for the trait to be investigated, and the DNA of all individuals in each group is collected to create two population DNA samples. These two population samples can be analyzed using techniques such as Restriction fragment length polymorphism (RFLP) or RAPD to detect similarities and differences at various loci in the genome. Both groups have a random distribution of alleles at all loci in the genome, except for the loci associated with the mutation. Consistent differences in loci between the two population samples mean that the locus is associated with the mutation of interest.
상기 "제한 조각 길이 다형성(Restriction fragment length polymorphism, RFLP)"이란 DNA를 특정 제한효소(restriction enzyme)로 절단하였을 때 생기는 DNA 조각 크기의 개인 간 차이를 말한다. 염기서열의 다형성은 개인마다 평균적으로 500~1,000개의 염기서열마다 발생하는 서열 차이이다. 염기쌍의 변이는 특정 제한효소가 인지하는 부위에 변화를 가져와 제한효소로 절단된 유전자의 길이에 변화를 줄 수 있다. 이러한 차이를 이용하여 제한 조각길이 다형성은 유전자 감식이나 친자 감별 등에 사용된다.The "restriction fragment length polymorphism (RFLP)" refers to a difference between individuals in the size of a DNA fragment that occurs when DNA is cut with a specific restriction enzyme. The nucleotide sequence polymorphism is a sequence difference that occurs every 500 to 1,000 nucleotide sequences on average for each individual. Variations in base pairs can change the length of a gene cut with a restriction enzyme by causing a change in the site recognized by a specific restriction enzyme. Using this difference, restriction fragment length polymorphism is used for genetic identification or parental identification.
상기 "RAPD(Randomly amplified polymorphic DNA)"란 같은 종의 생물 집단에 공존하는 극미량의 DNA를 무작위로 증폭시켰을 때 나타나는 다양한 길이의 DNA를 말한다. 임의로 증폭된 단편의 길이가 개체마다 서로 다르므로 개체를 구분하는 핵산 지문으로 이용할 수 있다.The term "RAPD (Randomly amplified polymorphic DNA)" refers to DNA of various lengths that appear when a very small amount of DNA coexisting in a population of the same species is randomly amplified. Since the length of the randomly amplified fragment is different for each individual, it can be used as a nucleic acid fingerprint to distinguish the individual.
본 명세서에서 "RPKM(Reads Per Kilobase Million)"란 전사에서 유전자에 지도화된 READ의 수, 유전자 길이, 지도화된 READ의 총 수 만을 가지고 계산한 값을 말한다. RPKM 값은 하기와 같이 계산하였다:In the present specification, "RPKM (Reads Per Kilobase Million)" refers to a value calculated from only the number of READs mapped to a gene in transcription, the length of the gene, and the total number of mapped READs. RPKM values were calculated as follows:
RPKM = (유전자에 지도화된 READ의 수)X103X106/(주어진 라이브러리에 지도화된 READ의 총 수)X(bp 단위의 유전자 길이)RPKM = (number of reads mapped to the gene) X10 3 X10 6 / (total number of reads mapped to a given library) X (gene length in bp)
식물에서 특정 형질의 유전을 연구 및 활용하기 위해서는 분자 마커의 활용이 필요하다. 먼저 유전적으로 다양성을 나타내는 인자가 식물 종 간 또는 품종 간 무엇인지를 명확히 평가해야 한다. 이 후 특정 염기서열, 후보 원인 유전자의 위치 또는 상기 원인 유전자의 상류 또는 하류에 존재하는 DNA 변이를 분석하고 확인하여, 최종적으로 분자 마커를 개발할 수 있다. 또한 이를 활용하여 특정 형질을 나타내는 유전자의 양적 형질 지도(QTL map)를 만들 수 있다. 또한 상기 분자 마커를 이용한 분석으로 유전자좌에 있는 돌연변이나 변형에 의해 일어난 다양성을 확인할 수 있다.In order to study and utilize the inheritance of specific traits in plants, the use of molecular markers is required. First, it is necessary to clearly assess what factors that indicate genetic diversity are between plant species or between cultivars. Thereafter, a specific nucleotide sequence, the position of a candidate causative gene, or a DNA mutation present upstream or downstream of the causative gene can be analyzed and confirmed, and finally a molecular marker can be developed. In addition, it can be used to create a quantitative trait map (QTL map) of a gene representing a specific trait. In addition, the diversity caused by mutations or modifications in the locus can be confirmed by analysis using the molecular marker.
상기 분자 마커 중, 유전적 마커를 사용하여 식물에 유용한 형질을 도입할 수 있다. 식물의 색깔 같은 특정 형질은 단지 몇 개의 유전자에 의해 조절될 수 있으며, 색깔과 같은 질적 특성은 표지 활용 선발(marker assisted selection, MAS)을 사용하여 확인할 수 있다. 일단 유전적으로 명확히 구분할 수 있는 분자 마커가 개발되면, 이를 다른 세대에서 유용한 형질과 연관시켜 상기 유용한 형질이 도입된 신품종을 개발할 수 있다.Among the molecular markers, a genetic marker can be used to introduce useful traits into plants. Certain traits, such as the color of a plant, can be regulated by only a few genes, and qualitative characteristics, such as color, can be identified using marker assisted selection (MAS). Once genetically distinct molecular markers are developed, they can be associated with useful traits in other generations to develop new varieties into which the useful traits have been introduced.
본 발명의 일 실시예에서는 서열번호 1의 AcWHITE을 기초로 하는 분자 마커를 사용하였다. 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 핵산분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인(align)하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80%의 상동성, 보다 바람직하게는 90%의 상동성, 가장 바람직하게는 98%의 상동성을 나타내는 서열을 의미한다.In an embodiment of the present invention, a molecular marker based on AcWHITE of SEQ ID NO: 1 was used. Considering a mutation having biologically equivalent activity, the nucleic acid molecule of the present invention is interpreted as including a sequence exhibiting substantial identity with a sequence listed in the sequence listing. The substantial identity is at least 80 when the sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. % Homology, more preferably 90% homology, and most preferably 98% homology.
이하에서는 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는 백색 구피색 양파 선별용 조성물에 관하여 구체적으로 설명한다.Hereinafter, a composition for selecting a white guppy-colored onion including a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 will be described in detail.
상기 조성물은 백색 구피색 양파를 선별하기 위해 서열번호 1의 염기서열(AcWHITE)을 포함하는 핵산 분자를 증폭할 수 있는 제제를 포함한다.The composition includes an agent capable of amplifying a nucleic acid molecule containing the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 in order to select white guppy-colored onions.
본 발명에서 "핵산 분자를 증폭할 수 있는 제제"란, 상기 염기서열(AcWHITE)을 포함하는 핵산 분자를 증폭을 통해 확인하여 백색 구피색 양파를 식별할 수 있는 조성물을 의미한다. 상기 조성물은 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하며, 바람직하게는 서열번호 7 및 서열번호 8의 프라이머를 포함한다.In the present invention, "an agent capable of amplifying a nucleic acid molecule" refers to a composition capable of identifying a white guppy-colored onion by identifying a nucleic acid molecule containing the nucleotide sequence (AcWHITE) through amplification. The composition includes a primer capable of detecting at least a part of the nucleotide sequence of SEQ ID NO: 1 (AcWHITE), and preferably includes the primers of SEQ ID NO: 7 and SEQ ID NO: 8.
본 발명에 있어서 "프라이머(Primer)"란 복제하려는 핵산 가닥에 상보적인 단일 가닥 뉴클레오타이드 서열을 말하며, 프라이머 연장 산물의 합성을 위한 개시점으로서 작용할 수 있다. 상기 프라이머의 길이 및 서열은 연장 산물의 합성을 시작하도록 허용해야 한다. 프라이머의 구체적인 길이 및 서열은 요구되는 DNA 또는 RNA 표적의 복합도(complexity)뿐만 아니라 온도 및 이온 강도와 같은 프라이머 이용 조건에 따라 다르다.In the present invention, "Primer" refers to a single-stranded nucleotide sequence that is complementary to a nucleic acid strand to be replicated, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow the synthesis of the extension product to begin. The specific length and sequence of the primers depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the required DNA or RNA target.
이하에서는 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 포함하는 백색 구피색 양파 선별용 키트에 관하여 구체적으로 설명한다.Hereinafter, a kit for selecting a white guppy-colored onion including a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 will be described in detail.
상기 백색 구피색 양파 선별용 키트는 프라이머, 제한효소, 및 증폭 반응 수행 시약을 포함한다.The kit for selecting a white guppy color onion includes a primer, a restriction enzyme, and a reagent for performing an amplification reaction.
본 발명의 일 실시예에서, 프라이머는 서열번호 1의 염기서열(AcWHITE) 중 적어도 일부를 검출할 수 있는 프라이머를 말하며, 바람직하게는 서열번호 7 및 서열번호 8의 염기서열을 포함하는 프라이머를 포함한다.In one embodiment of the present invention, the primer refers to a primer capable of detecting at least a part of the nucleotide sequence of SEQ ID NO: 1 (AcWHITE), and preferably includes a primer comprising the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 do.
본 명세서에서 "제한효소(restriction enzyme)"는 예를 들어, EcoRⅠ, PstⅠ, AluⅠ, BamHⅠ, HindⅢ, HaeⅢ, NotⅢ, DraⅢ, BglⅠ, SfiⅠ, AhdⅠ, XcmⅠ, 또는 BstⅠ 가 사용될 수 있다. 다만, 이에 제한되는 것은 아니다.In the present specification, the "restriction enzyme" may be, for example, EcoRI, PstI, AluI, BamHI, HindIII, HaeIII, NotIII, DraIII, BglI, SfiI, AhdI, XcmI, or BstI. However, it is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 증폭 반응 수행 시약은 DNA 폴리머라제, dNTPs, 및 버퍼를 포함하는 것을 특징으로 하는, 백색 구피색 양파 선별용 키트를 제공한다.In one embodiment of the present invention, the reagent for performing the amplification reaction provides a kit for selecting white guppy-colored onions, characterized in that it comprises a DNA polymerase, dNTPs, and a buffer.
또한, 본 발명의 키트는 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다.In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, and so on. The guide includes a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.
이하에서는 본 발명에 따른 양파의 gDNA에 서열번호 1의 염기서열(AcWHITE)이 존재하는지 여부를 분석하는 단계를 포함하는 백색 구피색 양파 선별 방법에 대해 설명한다.Hereinafter, a method for selecting white guppy-colored onions comprising the step of analyzing whether the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 is present in the gDNA of onion according to the present invention will be described.
본 발명의 일 실시예에 따르면, 백색 구피색 양파 선별 방법은 양파로부터 gDNA(genomic DNA)를 수득하는 단계; 상기 수득한 양파의 gDNA를 증폭하는 단계; 상기 증폭된 gDNA와 분자 마커를 이용하여 서열번호 1의 염기서열을 포함하는 핵산 분자가 존재하는지 여부를 분석하는 단계; 및 상기 분석 단계에서 상기 서열번호 1의 염기서열을 포함하는 핵산 분자가 존재하는 경우에는 백색 구피색 양파로 판정하는 단계를 포함한다.According to an embodiment of the present invention, a method for selecting white guppy-colored onions comprises the steps of obtaining gDNA (genomic DNA) from onions; Amplifying the gDNA of the obtained onion; Analyzing whether a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 is present using the amplified gDNA and a molecular marker; And determining a white guppy-colored onion when a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 is present in the analysis step.
본 발명의 방법에 따르면, 우선 양파로부터 gDNA를 수득한다.According to the method of the present invention, gDNA is first obtained from onions.
상기 gDNA는 양파의 다양한 기관으로부터 얻을 수 있으며, 예컨대, 잎, 뿌리, 줄기, 종자, 구근, 구피, 꽃 또는 화경으로부터 얻을 수 있고, 가장 바람직하게는 잎 또는 발아된 구피로부터 얻는다. 핵산 분자의 공급원(source)로서의 양파는 어떠한 발달 단계에 있는 것이든 모두 공급원으로서 이용될 수 있다.The gDNA can be obtained from various organs of onions, for example, leaves, roots, stems, seeds, bulbs, guppies, flowers or flowers, and most preferably leaves or germinated guppies. Onions as a source of nucleic acid molecules can be used as a source of any stage of development.
양파로부터 gDNA를 분리 및/또는 수득 하는 방법은 당업계에 공지된 다양한 방법을 통하여 실시할 수 있다 (Murray MG, Thompson WF, Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8:4321-4326(1980)).A method for separating and/or obtaining gDNA from onions can be carried out through various methods known in the art (Murray MG, Thompson WF, Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8:4321- 4326 (1980)).
본 발명에 따른 증폭 단계는 공지된 다양한 유전자 증폭 방법에 의해 실시될 수 있다. 예를 들어, 염기 서열을 증폭하는 방법은 중합효소연쇄반응(PCR), 리가아제 연쇄반응(ligase chain reaction), 핵산 서열 기재 증폭(nucleic acid sequence-based amplification), 전사 기재 증폭 시스템(transcription-based amplification system), 가닥 치환 증폭(strand displacement amplification) 또는 Qβ 복제효소(replicase)를 통한 증폭 또는 당업계에 알려진 핵산 분자를 증폭하기 위한 임의의 기타 적당한 방법이 있다.The amplification step according to the present invention can be carried out by various known gene amplification methods. For example, methods of amplifying the base sequence include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification system. amplification system), strand displacement amplification or amplification via Qβ replicase or any other suitable method for amplifying nucleic acid molecules known in the art.
본 발명의 일 실시예에 따르면, 프라이머를 이용하여 중합효소연쇄반응(PCR)을 수행하여 염기 서열을 증폭하였다.According to an embodiment of the present invention, a nucleotide sequence was amplified by performing a polymerase chain reaction (PCR) using a primer.
상기 "중합효소연쇄반응(Polymerase chain reaction, PCR)"이란 중합효소 연쇄반응으로 2개의 프라이머(Primer) 사이에 낀 DNA 부분을 시험관 내에서 대량으로 증폭시킬 수 있는 방법을 말한다.The term "polymerase chain reaction (PCR)" refers to a method capable of amplifying a large amount of DNA sandwiched between two primers in a test tube through a polymerase chain reaction.
본 발명에 이용되는 프라이머는 주형의 한 부위에 혼성화 또는 어닐링되어, 이중쇄 구조를 형성한다. 이러한 이중쇄 구조를 형성하는 데 적합한 핵산 혼성화의 조건은 핵산의 길이, 뉴클레오타이드의 구성 등에 따라 달라질 수 있다.The primer used in the present invention is hybridized or annealed at one site of the template to form a double-chain structure. Conditions for hybridization of a nucleic acid suitable for forming such a double-stranded structure may vary depending on the length of the nucleic acid, the composition of the nucleotide, and the like.
본 발명의 일 실시예에서는 서열번호 7 및/또는 서열번호 8의 염기서열을 포함하는 프라이머를 이용하여 중합효소연쇄반응을 실시하였다.In an embodiment of the present invention, a polymerase chain reaction was performed using a primer including the nucleotide sequence of SEQ ID NO: 7 and/or SEQ ID NO: 8.
다양한 DNA 중합효소가 본 발명의 증폭에 이용될 수 있으며, E.coli DNA 중합효소 I의 “클레나우” 단편, 열안정성 DNA 중합효소 및 박테리오파아지 T7 DNA 중합효소를 포함한다. 바람직하게는, 중합효소는 다양한 박테리아 종으로부터 얻을 수 있는 열안정성 DNA 중합효소이고, 이는 Thermus aquaticus(Taq), Thermusthermophilus(Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, 및 Pyrococcusfuriosus(Pfu)를 포함한다.Various DNA polymerases can be used for the amplification of the present invention, and include the “Klenow” fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, including Thermus aquaticus (Taq), Thermusthermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcusfuriosus (Pfu). .
중합 반응을 실시할 때, 반응 용기에 반응에 필요한 성분들을 과량으로 제공하는 것이 바람직하다. 증폭 반응에 필요한 성분들의 과량은, 증폭반응이 성분의 농도에 실질적으로 제한되지 않는 정도의 양을 의미한다. Mg2+와 같은 조인자, dATP, dCTP, dGTP 및 dTTP를 원하는 증폭 정도가 달성될 수 있을 정도로 반응 혼합물에 제공하는 것이 요구된다. 증폭 반응에 이용되는 모든 효소들은 동일한 반응 조건에서 활성 상태일 수 있다. 사실, 완충액은 모든 효소들이 최적의 반응 조건에 근접하도록 한다. 따라서 본 발명의 증폭 과정은 반응물의 첨가와 같은 조건의 변화 없이 단일 반응물에서 실시될 수 있다.When carrying out the polymerization reaction, it is preferable to provide the reaction vessel with the components necessary for the reaction in excess. The excessive amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component. Cofactors such as Mg 2+ , dATP, dCTP, dGTP and dTTP are required to be provided to the reaction mixture to the extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.
본 발명에 따른 분석 단계는 공지된 다양한 유전자 분석 방법에 의해 실시될 수 있다. 예를 들어, southern blot, DNA 염기서열분석(DNA-sequencing), 형광 핵산 혼성화(fluorescence in situ hybridization) 등의 방법이 있다. 본 발명의 일 실시예에 따르면, 증폭된 gDNA와 본 발명에 따른 분자 마커를 southern blot 분석 방법을 이용하여 표적 염기서열이 존재하는지 여부를 분석하였다.The analysis step according to the present invention can be carried out by a variety of known genetic analysis methods. For example, there are methods such as southern blot, DNA-sequencing, and fluorescence in situ hybridization. According to an embodiment of the present invention, the amplified gDNA and the molecular marker according to the present invention were analyzed for the presence of a target nucleotide sequence using a southern blot analysis method.
본 발명에서 "DNA 염기서열분석(DNA Sequencing)"이란 DNA를 이루는 선형의 뉴클레오타이드 염기들(nucleotide bases)의 순서를 결정하기 위한 생화학적 방법이다. 맥삼-길버트법(Maxam-Gilbert sequencing)이란 DNA의 화학적 변화와 특정 염기에서의 절단을 이용한 DNA 염기서열 결정법이다. DNA 화학적 결정법이라고 불리며 정제된 이중가닥 DNA(double strand DNA)를 사용한다. DNA 5' 말단에 방사성 표식을 한 뒤 서열화 하고자 하는 부분을 정제하고 염기 특이적 화학분해 방법으로 DNA를 더 절단한다. 이후 겔 전기영동(gel electrophoresis) 및 자동방사선사진법(autoradiography)을 이용하여 염기서열을 결정한다. 그러나 이 방법은 방사성 마커를 이용하고 기술적으로 복잡하다는 이유로 사슬종료법이 발표된 이후에는 광범위하게 사용되지 못했다. 사슬종료법(Chain-termination methods) 염기서열을 결정하는 DNA 절편을 주형으로 하고, DNA 중합효소(DNA polymerase)를 이용하여 주형 DNA 절편의 상보적인 DNA 가닥을 형성시킨 후 겔 전기영동을 이용하여 분석하는 방법이다. 맥삼-길버트법에 비해 적은 종류의 유해 화학약품을 쓰고 낮은 수준의 방사성 물질을 사용한다.In the present invention, "DNA Sequencing" is a biochemical method for determining the order of linear nucleotide bases constituting DNA. The Maxam-Gilbert sequencing is a DNA sequencing method using chemical changes in DNA and cleavage at a specific base. It is called DNA chemical crystallization and uses purified double-stranded DNA. After radiolabeling the 5'end of the DNA, the part to be sequenced is purified, and the DNA is further cleaved using a base-specific chemical decomposition method. Thereafter, the base sequence is determined using gel electrophoresis and autoradiography. However, this method has not been widely used since the publication of the chain termination method due to the fact that it uses radioactive markers and is technically complex. Chain-termination methods: DNA fragments that determine base sequence are used as a template, DNA polymerase is used to form complementary DNA strands of template DNA fragments, and then analyzed by gel electrophoresis. That's how to do it. Compared to the Maxam-Gilbert method, it uses fewer types of hazardous chemicals and uses low levels of radioactive substances.
본 발명의 신규한 분자 마커를 이용하여 유전자 분석을 하는 경우에는, 종래 기술과 비교하여 보다 간편하면서도 대량 규모로 신뢰성 있게 양파의 F2 분리집단에서 백색 구피색을 가진 개체를 명확히 선별해 낼 수 있다.In the case of genetic analysis using the novel molecular markers of the present invention, it is possible to clearly select individuals with white guppy color from the F2 isolated group of onions more conveniently and reliably on a large scale compared to the prior art.
이하에서는 본 발명에 따른 백색 구피색 양파 제조용 재조합 벡터에 대해 설명한다.Hereinafter, a recombinant vector for producing white guppy-colored onions according to the present invention will be described.
본 명세서에서 "재조합 벡터(Vector)"란 적당한 숙주세포에서 목적 단백질을 발현할 수 있도록 제조된 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다.In the present specification, the term "recombinant vector" refers to a vector prepared to express a protein of interest in a suitable host cell, and refers to a genetic construct comprising essential regulatory elements operably linked to express a gene insert.
본 발명에서 사용되는 용어, "작동 가능하게 연결된(operably linked)"은 일반적 기능을 수행하도록 핵산 발현 조절 서열과 목적하는 단백질을 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어, 프로모터와 단백질을 코딩하는 핵산 서열이 작동 가능하게 연결되어 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술 분야에서 공지된 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein may be operably linked to affect the expression of the encoding nucleic acid sequence. The operative linkage with the recombinant vector can be prepared using gene recombination techniques known in the art, and site-specific DNA cleavage and linkage may be performed using enzymes generally known in the art.
본 발명에서 사용될 수 있는 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오파아지 벡터 및 아데노바이러스 벡터, 레트로바이러스 벡터, 아데노-연관 바이러스 벡터 같은 바이러스 벡터 등을 포함한다. 적합한 발현벡터는 프로모터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 등과 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호서열 또는 리더 서열을 포함할 수 있으며 목적에 따라 다양하게 제조될 수 있다.Vectors that can be used in the present invention include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as adenovirus vectors, retroviral vectors, adeno-associated virus vectors, and the like. Suitable expression vectors may include, in addition to expression control elements such as promoters, initiation codons, stop codons, polyadenylation signals and enhancers, etc., signal sequences or leader sequences for membrane targeting or secretion, and may be variously prepared according to the purpose.
본 발명의 일 실시예에서는 재조합의 대상이 되는 백색 구피색 양파 유래의 염기서열(AcWHITE)을 포함하는 재조합 플라스미드를 제조하였다. 발현 벡터의 종류는 AcWHITE의 재조합 효율에 영향을 미칠 수 있기 때문에, 상기 발현 벡터는 발현 양파의 품종뿐만 아니라 활용 분야, 특성 등 다양한 인자들을 고려하여 신중히 선택되어야 한다.In one embodiment of the present invention, a recombinant plasmid containing a nucleotide sequence (AcWHITE) derived from a white guppy-colored onion to be recombined was prepared. Since the type of expression vector may affect the recombination efficiency of AcWHITE, the expression vector should be carefully selected in consideration of various factors such as the field of application and characteristics, as well as the variety of the expression onion.
이하에서는 본 발명에 따른 백색 구피색 양파의 제조 방법에 관하여 구체적으로 설명한다. 상기 제조 방법은 서열번호 1로 표시되는 염기서열(AcWHITE)을 이용하여 양파를 형질 전환시키는 단계를 포함한다.Hereinafter, a method for producing a white guppy-colored onion according to the present invention will be described in detail. The manufacturing method includes transforming onions using the nucleotide sequence (AcWHITE) represented by SEQ ID NO: 1.
본 발명의 일 실시예에 따르면, 백색 구피색 양파의 제조 방법은 선별된 백색 구피색 양파의 구피세포의 염기서열(AcWHITE)을 포함하는 재조합 벡터를 제조하는 단계; 상기 재조합 벡터로 양파를 형질 전환하는 단계를 포함한다.According to an embodiment of the present invention, a method for producing a white guppy-colored onion comprises the steps of preparing a recombinant vector comprising the nucleotide sequence (AcWHITE) of the guppy cells of the selected white guppy-colored onion; And transforming the onion with the recombinant vector.
양파 세포의 형질 전환은 본 발명에 따른 재조합 벡터를 이용하여 수행될 수 있다. 염기서열(AcWHITE)를 추출하여 재조합 벡터를 제조하기 위한 방법으로는 유전자 가위를 이용한 기술이 이용될 수 있다.Transformation of onion cells can be performed using the recombinant vector according to the present invention. As a method for producing a recombinant vector by extracting the nucleotide sequence (AcWHITE), a technique using genetic scissors may be used.
"유전자 가위"란 동식물 유전자의 DNA를 자르는데 사용되는 것으로서, 예를 들어 제한 효소, 징크핑커 뉴클레이즈(ZFNs), 탈렌(TALENs), CRISPR/Cas9 등이 있다. 유전자 가위는 유전자의 잘못된 부분을 제거해 문제를 해결하는 유전자 편집 (Genome Editing) 기술에 사용될 수 있다. 또한 손상된 DNA를 잘라내고 정상 DNA로 갈아 끼우는 기술에도 사용될 수 있다. 다만, 상기 용도에 제한되는 것은 아니다. 본 발명의 일 실시예에서는 유전자 가위로 CRISPR/Cas9를 이용하여 재조합 벡터를 제조하였다."Gene scissors" are used to cut the DNA of animal and plant genes, and include, for example, restriction enzymes, zinc pinker nucleases (ZFNs), TALENs, CRISPR/Cas9, and the like. Genetic shears can be used in gene editing techniques to solve problems by removing faulty parts of a gene. It can also be used in techniques to cut damaged DNA and replace it with normal DNA. However, it is not limited to the above use. In an embodiment of the present invention, a recombinant vector was prepared using CRISPR/Cas9 with genetic scissors.
상기 "CRISPR/Cas9"이란 3세대 유전자 가위의 한 종류로써, RNA로 만들어진 "가이드 RNA"와 DNA를 절단하는 효소인 "Cas9"으로 이루어져 있다. 크리스퍼(CRISPR)는 박테리아와 고세균과 같은 원핵생물 유기체의 게놈(genome)에서 발견되는 DNA 서열이다. Cas9은 크리스퍼 서열에 상보적인 DNA의 특정 줄기를 인식하고 절단하기 위하여 가이드로서 크리스퍼 서열을 사용하는 효소이다. 상기 "가이드 RNA"는 DNA 중에 어떤 부분을 절단할 것인지 안내하는 가이드 역할을 한다. RNA는 DNA의 서열에 상보적 결합이 가능하다. 크리스퍼는 이러한 RNA의 특성을 활용해, 자신의 RNA의 염기서열과 상보적인 DNA 서열을 찾아 결합한다.The "CRISPR/Cas9" is a type of 3rd generation gene scissors, and consists of "guide RNA" made of RNA and "Cas9", an enzyme that cleaves DNA. CRISPR is a DNA sequence found in the genome of prokaryotic organisms such as bacteria and archaea. Cas9 is an enzyme that uses the CRISPR sequence as a guide to recognize and cut a specific stem of DNA that is complementary to the CRISPR sequence. The "guide RNA" serves as a guide for guiding which part of DNA to be cut. RNA is capable of complementary binding to the sequence of DNA. CRISPR uses these characteristics of RNA to find and combine DNA sequences that are complementary to the base sequence of his RNA.
가이드 RNA는 DNA의 이중 나선을 절단하는 제한효소인 Cas9과 복합체를 형성한다. 유전자를 조작하고 싶은 부분에 이 효소를 넣으면 목표한 DNA 서열을 찾아내 Cas9가 DNA를 절단한다. 세포는 DNA가 절단되었을 때, 복구하는 기능을 가지고 있다. 따라서, DNA가 원래 서열로 복구가 되면 또 다시 Crispr/Cas9가 작동하여 이를 절단하게 된다. 이 과정이 계속 반복되면 "복구 오류"가 나타나고 원래의 서열과 몇 개의 염기에서 차이를 보이게 된다. 이러한 차이가 생기면 크리스퍼는 작동을 멈추고, 복구 오류로 변화된 서열은 원래의 기능은 발휘하지 못하게 된다. 이런 식으로 절단하고자 하는 유전자를 정확하게 찾아내, 파괴(knockout)하는 것이 가능해진다.The guide RNA forms a complex with Cas9, a restriction enzyme that cleaves the double helix of DNA. When you put this enzyme in the part where you want to manipulate the gene, it finds the target DNA sequence and Cas9 cuts the DNA. Cells have the ability to repair when DNA is cut. Therefore, when DNA is restored to its original sequence, Crispr/Cas9 works again and cuts it. If this process is repeated over and over, a "recovery error" will appear and there will be a difference between the original sequence and a few bases. When this difference occurs, CRISPR stops working, and the sequence that has been changed due to a repair error will not be able to exert its original function. In this way, it becomes possible to accurately find and knockout the gene to be cut.
또한 이 기술을 활용해 절단뿐만 아니라 원하는 곳에 유전자를 추가할 수도 있다. Crispr/Cas9과 함께 새롭게 추가하고 싶은 DNA 서열을 넣으면, 세포가 절단된 부분을 복구하는 과정에서 추가된 DNA 서열을 흡수하게 된다.You can also use this technique to not only cut, but also add genes wherever you want. When you insert the DNA sequence you want to add with Crispr/Cas9, the added DNA sequence is absorbed in the process of repairing the cut part of the cell.
본 발명의 다른 실시예에서 형질 전환은 아그로박테리움 튜머파시엔스(Agrobacterium tumefiaciens)에 의해 매개될 수 있으나, 이에 한정되지 않는다In another embodiment of the present invention, transformation may be mediated by Agrobacterium tumefiaciens, but is not limited thereto.
또한, 본 발명의 일 실시예에서, 제조 방법은 상기 형질 전환된 양파 세포로부터 형질 전환 양파 세포를 재분화하는 단계를 포함한다. 형질 전환 양파 세포로부터 형질 전환 양파 세포를 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다 (Perez-Garcia, P., Moreno-Risueno, M. A. (2018) Stem cells and plant regeneration. Developmental Biology, 442: 3-12, Ikeuchi, M., Ogawa, Y., Iwase, A. 등 (2016) Plant regeneration: cellular origins and molecular mechanisms. Development (Cambridge, England), 143: 1442-51).In addition, in an embodiment of the present invention, the manufacturing method includes the step of regenerating transformed onion cells from the transformed onion cells. Any method known in the art may be used as a method for regenerating transformed onion cells from transformed onion cells (Perez-Garcia, P., Moreno-Risueno, MA (2018) Stem cells and plant regeneration. Developmental Biology , 442: 3-12, Ikeuchi, M., Ogawa, Y., Iwase, A. et al. (2016) Plant regeneration: cellular origins and molecular mechanisms. Development (Cambridge, England), 143: 1442-51).
이하에서는 본 발명에 따른 형질 전환된 형질 전환 양파와 그 종자에 대해 설명한다.Hereinafter, the transformed transgenic onion according to the present invention and its seeds will be described.
본 발명에서 "형질 전환"이란 원래의 세포가 가지고 있던 것과 다른 종류의 유전자가 포함된 DNA 사슬 조각, 플라스미드 같은 벡터가 침투되어 원래 세포에 존재하던 DNA와 결합하여 유전형질을 변화시키는 것을 말한다.In the present invention, "transformation" refers to a DNA chain fragment containing a gene different from that of the original cell, or a vector such as a plasmid that penetrates and binds to the DNA existing in the original cell to change the genotype.
식물의 형질 전환은 DNA를 식물에 전이시키는 임의의 방법을 의미한다. 원칙적으로, 임의의 형질 전환 방법은 본 발명에 따른 재조합 DNA를 적당한 선조 세포로 도입시키는데 이용될 수 있다. 예를 들어, 원형질체에 대한 칼슘/폴리에틸렌 글리콜 방법(Krenset al., 1982, Nature 296: 72-74; Negrutiu et al., 1987, Plant Mol. Biol. 8: 363-373), 원형질체의 전기천공법(Shillito et al., 1985, Bio/Technol. 3: 1099-1102), 식물 요소로의 현미주사법(Crossway et al.,1986, Mol. Gen. Genet. 202: 179-185), 각종 식물 요소의(DNA 또는 RNA-코팅된) 입자 충격법(Klein et al.,1987, Nature 327: 70), 식물의 침윤 또는 성숙 화분 또는 소포자의 형질 전환에 의한 아그로박테리움 투머파시엔스 매개된 유전자 전이에서(비완전성) 바이러스에 의한 감염(EP 0 301 316호) 등으로부터 적당하게 선택될 수 있다.Plant transformation refers to any method of transferring DNA into a plant. In principle, any transformation method can be used to introduce the recombinant DNA according to the invention into suitable progenitor cells. For example, the calcium/polyethylene glycol method for protoplasts (Krenset al., 1982, Nature 296: 72-74; Negrutiu et al., 1987, Plant Mol. Biol. 8: 363-373), electroporation of protoplasts. (Shillito et al., 1985, Bio/Technol. 3: 1099-1102), microinjection with plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202: 179-185), of various plant elements (DNA or RNA-coated) particle bombardment method (Klein et al., 1987, Nature 327: 70), in Agrobacterium tumerfaciens-mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles ( Incomplete) can be appropriately selected from infection by a virus (EP 0 301 316) and the like.
식물의 형질 전환에 이용되는 "식물 세포"는 어떤 식물 세포도 된다. 식물 세포는 배양 세포, 배양 조직, 배양기관 또는 전체 식물이다. "식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들어 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직배양 또는 세포 배양 상태일 수 있다. 본 발명의 일 실시예에서는 양파 세포를 형질 전환 대상이 되는 식물 세포로 이용하였다. 상기 양파 세포는, 바람직하게는, 양파 구피 세포일 수 있다.The "plant cell" used for plant transformation may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants. “Plant tissue” refers to tissues of differentiated or undifferentiated plants, such as roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used in culture, ie single cells, protoplasts, shoots. And callus tissue. The plant tissue may be in planta, organ culture, tissue culture, or cell culture. In one embodiment of the present invention, onion cells were used as plant cells to be transformed. The onion cells may be, preferably, onion guppy cells.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다. 이는 본 발명을 보다 상세하게 설명하기 위한 것일 뿐 본 발명의 권리범위가 이에 한정 되는 것은 아니다.Hereinafter, examples will be described in detail in order to describe the present invention in detail. This is only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.
실시예Example
실시예 1. 재료 및 방법Example 1. Materials and methods
1.1. 식물 재료1.1. Plant material
동일한 모계에서 유래한 2개의 F2:3 집단을 사용하여 밀접하게 연결된 분자 마커와 색상 표현형 사이의 재조합체를 검사하였다. 모든 F2:3 집단은 이형 접합성 황색 F2 양파의 자가 수분을 통해 생성되었다. 분자 마커와 표현형의 연관 관계를 분석하기 위해 총 2491 개의 F2:3 양파를 사용하였다. F2:3 양파 구피색 표현형은 구피를 수확한 후 육안으로 확인하였다. 양파 연구 연구소와 국립 원예 및 약초 과학 연구소에 의해 유지되는 육종 계통으로부터 총 30개의 백색, 황색 또는 적색 양파의 잎 조직 샘플을 수득하였다. 또한, 이전 연구에서 분리된 총 116개의 적색 또는 황색 품종의 총 게놈 DNA를 사용하여 C 유전자좌에 대한 인과적 특성을 나타내는 분자 마커를 분석하였다.Recombinants between closely linked molecular markers and color phenotypes were examined using two F 2:3 populations derived from the same maternal line. All F 2:3 populations were generated through self-pollination of heterozygous yellow F 2 onions. A total of 2491 F 2:3 onions were used to analyze the relationship between the molecular marker and the phenotype. The F 2:3 onion guppy color phenotype was visually confirmed after harvesting guppies. A total of 30 white, yellow or red onion leaf tissue samples were obtained from breeding lines maintained by the Onion Research Institute and the National Institute of Horticultural and Herbal Science. In addition, a total genomic DNA of 116 red or yellow varieties isolated in the previous study was used to analyze molecular markers representing causal characteristics for the C locus.
1.2. 실험 방법1.2. Experimental method
1.2.1. 백색과 황색 집단 사이의 SNP 및 DEG의 RNA-Seq 분석 및 식별1.2.1. RNA-Seq analysis and identification of SNPs and DEGs between white and yellow populations
RNeasy Plant Mini Kit를 사용하여 10개의 백색 및 10개의 황색 양파 구피에서 발아된 풀잎 피복 조직으로부터 총 집단 RNA를 추출하였다. GST1 및 C46747 마커의 유전자형이 동형 접합 우성인 황색 F 식물만이 황색 집단에 포함되었다. 전사체는 HiSeq 2000을 사용하여 DNA 염기서열화 하였다.Total population RNA was extracted from grass leaf-covered tissues germinated from 10 white and 10 yellow onion guppies using the RNeasy Plant Mini Kit. Only yellow F plants whose genotypes for the GST1 and C46747 markers are homozygous dominant were included in the yellow population. The transcript was DNA sequenced using HiSeq 2000.
백색 및 황색 RNA로부터 해석된 서열을 Trinity 소프트웨어를 사용하여 콘티그로 새로 조립하였다. 두 집단 RNA의 해석된 서열은 참조 전사체와 두 집단 사이의 SNP 및 차등 발현 유전자(DEG)를 확인하기 위해 본 연구에서 새로 조립된 콘티그에 개별적으로 지도화하였다. SNP 발견 및 콘티그 발현의 정량화는 SAMTools 소프트웨어 및 RSEM 소프트웨어를 사용하여 수행되었다.Sequences interpreted from white and yellow RNA were newly assembled into contigs using Trinity software. The interpreted sequences of the two populations of RNA were individually mapped onto the newly assembled contigs in this study to identify the reference transcript and the SNPs and differentially expressed genes (DEGs) between the two populations. SNP discovery and quantification of contig expression was performed using SAMTools software and RSEM software.
본 명세서에서 "콘티그(contig)"란 서로 겹치면서 연속되어 있는 DNA 절편들의 집합을 말한다. 이는 염기서열이 밝혀진 작은 DNA 조각으로, 각 콘티그는 다른 콘티그들과 동일한 염기서열 부분을 함유한다.In the present specification, "contig" refers to a set of DNA fragments that overlap each other and are continuous. This is a small piece of DNA whose nucleotide sequence has been identified, and each contig contains the same nucleotide sequence as the other contigs.
상기 "차등 발현 유전자(Differential gene expression, DEG)"란 동일한 생물체에서도 서로 다른 조직, 발달 단계, 시간, 자극에 따라 서로 다른 유전자가 발현되는 현상을 말한다.The "differential gene expression (DEG)" refers to a phenomenon in which different genes are expressed according to different tissues, developmental stages, times, and stimuli even in the same organism.
1.2.2. 실시간 RT-PCR 및 cDNA 말단의 빠른 증폭 (RACE)1.2.2. Real-time RT-PCR and fast amplification of cDNA ends (RACE)
RNeasy Plant Mini Kit (QIAGEN)를 사용하여 5개의 백색 및 5개의 황색 F2:3 구피에서 발아된 잎 덮개로부터 RNA를 추출하여 실시간 RT-PCR을 수행하였다. DFCI Allium cepa 유전자 색인에서 검색한 Tubulincoding 유전자 (TC125)를 내부 대조군으로 사용하였다. 표적 및 튜불린 유전자의 프라이머 서열은 하기와 같다. (표 1 참조)Real-time RT-PCR was performed by extracting RNA from leaf sheaths germinated from 5 white and 5 yellow F 2:3 guppies using RNeasy Plant Mini Kit (QIAGEN). The Tubulincoding gene (TC125) retrieved from the DFCI Allium cepa gene index was used as an internal control. The primer sequences of the target and tubulin genes are as follows. (See Table 1)
RACE cDNA는 SMART RACE cDNA 증폭 키트를 사용하여 제조하였다. 제조업체의 지침에 따라 RACE PCR을 수행하였다. 이후 Ethidium bromide 염색 후 1.5% 아가로스겔 상에서 RACE PCR 생성물을 시각화하였다. QIAquick PCR 정제 키트(QIAGEN)를 사용하여 PCR 생성물을 정제하였다. RACE에 사용된 프라이머는 하기와 같다. (표 1 참조)RACE cDNA was prepared using the SMART RACE cDNA amplification kit. RACE PCR was performed according to the manufacturer's instructions. After staining with Ethidium bromide, the RACE PCR product was visualized on a 1.5% agarose gel. The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN). The primers used in RACE are as follows. (See Table 1)
본 발명에서 "RACE(Rapid amplication of cDNA ends)"란 cDNA의 염기배열이 일부만이 이미 알려져 있는 경우, 기본적으로 cDNA 라이브러리 구축을 하지 않고, PCR에 따라 mRNA의 말단까지 cDNA을 증폭하여 클로닝하는 방법을 말한다.1.2.3. PCR 증폭 및 Genome walking In the present invention, "RACE (Rapid amplication of cDNA ends)" refers to a method of amplifying and cloning cDNA to the end of mRNA according to PCR, without basically constructing a cDNA library when only a part of the cDNA nucleotide sequence is known. Say. 1.2.3. PCR amplification and genome walking
Cetyl trimethylammonium bromide(CTAB) 방법을 사용하여 발아된 구피의 잎 또는 구피 비늘의 끝 부분에서 전체 게놈 DNA를 추출하였다. RNA-seq에 의해 검출된 SNP의 확인과 단단히 연결된 콘티그 및 B2 유전자의 전체 길이의 게놈 DNA 서열의 분리를 위해, 0.05μg 주형, 2.5μL 10×PCR 완충액, 0.2μL 정방향 프라이머(10μM), 0.2μL 역방향 프라이머(10μM), 0.2μL dNTP(각각 10mM) 및 0.25μL 혼합된 중합효소를 함유하는 25μL의 반응 혼합물에서 PCR을 수행하였다. PCR 증폭은 4분 동안 95℃에서 초기 변성 단계, 30초 동안 95℃에서 10 순환, 30초 동안 65℃ (각 순환에서 0.8℃ 감소), 및 1분 동안 72℃, 95℃에서 30초 동안 35 순환, 30초 동안 57℃, 및 1분 동안 72℃, 그리고 72℃에서 최종 10분 연장으로 이루어졌다. 이를 Ethidium bromide 염색 후 1.5 % 아가로스겔 상에서 PCR 생성물을 가시화하고 상기 한 바와 같이 서열 분석하였다. (표 2 참조)Whole genomic DNA was extracted from the leaves of germinated guppies or the tips of guppy scales using the Cetyl trimethylammonium bromide (CTAB) method. For identification of SNPs detected by RNA-seq and separation of full-length genomic DNA sequences of tightly linked contigs and B2 genes, 0.05 μg template, 2.5 μL 10×PCR buffer, 0.2 μL forward primer (10 μM), 0.2 PCR was performed in 25 μL of the reaction mixture containing μL reverse primer (10 μM), 0.2 μL dNTP (10 mM each) and 0.25 μL mixed polymerase. PCR amplification was performed at 95° C. for 4 minutes in the initial denaturation step, 10 cycles at 95° C. for 30 seconds, 65° C. for 30 seconds (0.8° C. decrease in each cycle), and 72° C. for 1 minute, 35° C. for 30 seconds at 95° C. Cycling, at 57° C. for 30 seconds, and 72° C. for 1 minute, and a final 10 minute extension at 72° C. consisted of. After staining with Ethidium bromide, the PCR product was visualized on a 1.5% agarose gel and sequenced as described above. (See Table 2)
분자 마커 분석의 경우, 0.05μg 주형, 1.0μL 10×PCR 완충액, 0.2μL 정방향 프라이머(10μM), 0.2μL 역방향 프라이머(10μM), 0.2μL dNTP (각각 10mM) 및 0.25 U Taq 중합효소를 포함하는 10μL 반응 혼합물에서 PCR 증폭을 수행하였다. PCR 증폭은 4분 동안 95℃에서 초기 변성 단계, 30초 동안 95℃에서 10 순환, 30초 동안 65℃ (각 순환에서 0.8℃ 감소), 및 1분 동안 72℃, 95℃에서 30초 동안 35순환, 30초 동안 57℃, 및 1분 동안 72℃, 그리고 72℃에서 최종 10분 연장으로 이루어졌다. 이를 Ethidium bromide 염색 후 1.5% 아가로스겔 상에서 B2 마커의 유전자형 분석을 수행하였다. 분자 마커의 프라이머 서열은 하기와 같다. (표 2 참조)For molecular marker analysis, 10 μL containing 0.05 μg template, 1.0 μL 10×PCR buffer, 0.2 μL forward primer (10 μM), 0.2 μL reverse primer (10 μM), 0.2 μL dNTP (10 mM each) and 0.25 U Taq polymerase. PCR amplification was performed in the reaction mixture. PCR amplification was performed at 95° C. for 4 minutes in the initial denaturation step, 10 cycles at 95° C. for 30 seconds, 65° C. for 30 seconds (0.8° C. decrease in each cycle), and 72° C. for 1 minute, 35° C. for 30 seconds at 95° C. Cycling, at 57° C. for 30 seconds, and 72° C. for 1 minute, and a final 10 minute extension at 72° C. consisted of. After staining with Ethidium bromide, genotyping of the B2 marker was performed on a 1.5% agarose gel. The primer sequence of the molecular marker is as follows. (See Table 2)
긴 PCR 및 genome walking을 수행하여 B2 유전자의 인트론 5를 수득 하였다. 긴 PCR의 경우, 0.25μg 주형, 5μL 10×PCR 완충액, 0.2μM 정방향 프라이머, 0.2μM 역방향 프라이머, 0.2mM dNTP 및 0.5μL Taq 중합효소를 함유하는 50μL 혼합물에서 반응을 수행하였다. 긴 PCR 증폭은 10초 동안 98℃ 및 15분 동안 68℃의 40 순환을 수행하였다. Genome walking은 제조사의 지시에 따라 Universal GenomeWalker 키트(Clontech)를 사용하여 수행하였다.본 발명에서 "Genome walking"이란 알려진 DNA 서열의 영역 상류(upstream)에 존재하는 알려지지 않은 게놈 영역의 DNA 서열을 결정하는 방법을 말한다. 한 번의 수행으로 6-7 kb의 서열을 포착 할 수 있으며, 프로모터 영역을 식별하는 데 유용하다. 또한 특정 종에 대해 강한 서열 보존을 갖는 표적 유전자에 영역이 있을 때 새로운 종에서 상동성 유전자를 포획하는데 상당히 유용하다.Long PCR and genome walking were performed to obtain
1.2.4. 계통수의 구성1.2.4. Composition
bHLH 코딩 유전자의 감소된 아미노산 서열을 BioEdit 소프트웨어를 사용하여 정렬하였다. 정렬에서의 차이는 Gblocks 소프트웨어를 사용하여 제거하였다. 계통수는 neighbor-joining 방법으로 MEGA 버전 7을 사용하여 구성하였다. 계통수의 마디 지지는 1000 부트스트랩 복제에 의해 평가하였다.The reduced amino acid sequence of the bHLH coding gene was aligned using BioEdit software. Differences in alignment were eliminated using Gblocks software. The phylogenetic tree was constructed using MEGA version 7 as a neighbor-joining method. Node support of the phylogenetic tree was evaluated by 1000 bootstrap replicates.
1.2.5. 비자율적 DNA 전이인자 AcWHITE의 복제수 측정1.2.5. Measurement of copy number of non-irritating DNA transfer factor AcWHITE
AcWHITE의 복제수를 측정하기 위해 3개의 양파 육종 계통(JNU1306, JNU1305 및 JNU1101)의 총 게놈 DNA를 사용하여, Illumina NextSeq 500 플랫폼을 이용해 150bp paired-end 서열의 원시 해석값을 얻었다. 원시 해석값은 Trimmomatic 소프트웨어 버전 0.38을 사용하여 다듬었다. 다듬어진 해석값을 B2 유전자의 백색 대립 유전자에 지도화하였다.In order to measure the copy number of AcWHITE, the total genomic DNA of three onion breeding lines (JNU1306, JNU1305 and JNU1101) was used to obtain a raw analysis value of a 150bp paired-end sequence using the Illumina NextSeq 500 platform. Raw analysis values were refined using Trimmomatic software version 0.38. The refined analysis values were mapped onto the white allele of the B2 gene.
실시예 2. 실험 결과Example 2. Experimental results
2.1. BSA 및 RNA-Seq 분석을 통한 분자 마커 개발2.1. Molecular marker development through BSA and RNA-Seq analysis
2.1.1. BSA 및 RNA-Seq 분석2.1.1. BSA and RNA-Seq analysis
황색 F2:3 개체의 집단 RNA는 동형 접합 우성 및 이형 접합 유전자형을 모두 함유하여, 황색 및 백색 집단 사이에 제한된 수의 신뢰할 수 있는 SNP를 생성하였다. C 유전자좌를 담당하는 후보 유전자를 확인하기 위해, C 유전자좌의 상류 및/또는 하류에 밀접하게 위치한 GST1 및 C46747 마커를 사용하여 이형 접합 황색 F2:3 개체를 제거한 후, 또 다른 라운드의 BSA 및 RNA-Seq 분석을 수행하였다.Population RNA of yellow F 2:3 individuals contained both homozygous dominant and heterozygous genotypes, resulting in a limited number of reliable SNPs between the yellow and white populations. To identify the candidate gene responsible for the C locus, the heterozygous yellow F 2:3 individuals were removed using the GST1 and C46747 markers located closely upstream and/or downstream of the C locus, followed by another round of BSA and RNA. -Seq analysis was performed.
측정값은 참조 전사체에 개별적으로 지도화하였고, C 유전자좌의 원인 유전자는 참조 전사체에 포함되지 않을 수 있기 때문에 214,961개의 콘티그가 조립되었다. 백색과 황색 집단의 측정값이 새로운 콘티그의 조합에 사용되었다. 참조 전사체와 비교하여 대략 10% 더 많은 측정이 이들 새로 조립된 콘티그에 지도화하였다. 백색 및 황색 집단 사이의 동형 접합 SNP를 함유하는 총 114개의 콘티그를 단계적 공정을 사용하여 분석하였다. 그 중 33개의 콘티그가 참조 및 새로 조립된 콘티그 모두에서 검출되었다. 동형 접합성 SNP를 함유하는 이들 114개의 콘티그가 C 유전자좌에 연결된 것으로 측정되었다. 그 결과 4개의 콘티그만이 염색체 5에서 81.3 내지 120.0cM 범위의 4개의 유전자좌를 갖는 95% 초과의 뉴클레오타이드 서열 동일성을 나타내었고, 이는 C 유전자좌가 염색체 5의 중간에 위치함을 나타낸다.The measurements were individually mapped to the reference transcript, and 214,961 contigs were assembled because the causative gene of the C locus may not be included in the reference transcript. Measurements of the white and yellow populations were used for the new contig combination. Approximately 10% more measurements compared to the reference transcript were mapped onto these newly assembled contigs. A total of 114 contigs containing homozygous SNPs between the white and yellow populations were analyzed using a step-by-step process. Of those, 33 contigs were detected in both the reference and newly assembled contigs. These 114 contigs containing homozygous SNPs were determined to be linked to the C locus. As a result, only 4 contigs showed greater than 95% nucleotide sequence identity with 4 loci ranging from 81.3 to 120.0 cM on
2.1.2 분자 마커 개발 및 관련성 분석2.1.2 Molecular Marker Development and Relevance Analysis
후보 원인 유전자를 확인하고 C 유전자좌에 밀접하게 연결된 분자 마커를 개발하기 위해 1905개의 개체로 구성된 3개의 F2:3 집단을 구성하였다. 이들 집단에서 구피색 표현형의 분리 비율을 단일 유전자 유전 패턴에 맞추었다. 2 개의 분자 마커(C46747 및 GST1)를 사용하여 재조합체를 분석하였다. C46747과 C 유전자좌 사이에 총 21개의 재조합체가 확인되었고, GST1과 C 유전자좌 사이에는 단일 재조합체만이 확인되었다.To identify candidate causative genes and develop molecular markers closely linked to the C locus, three F 2:3 populations of 1905 individuals were constructed. The percentage of segregation of the guppy phenotype in these populations was fitted to a single gene genetic pattern. Recombinants were analyzed using two molecular markers (C46747 and GST1). A total of 21 recombinants were identified between the C46747 and C loci, and only a single recombinant was identified between the GST1 and C loci.
114개의 분석된 콘티그 중에서, 백색 집단의 판독에서 다수의 SNP를 함유하는 40개의 콘티그가 PCR 및 서열 분석을 위해 선택되었다. 이들 중 34개의 콘티그는 PCR 산물의 염기서열 분석을 통해 집단 DNA간에 동형 접합 SNP를 갖는 것으로 확인되었다. 이들 34개 콘티그의 SNP의 C 유전자좌에서의 위치를 확인하기 위해 2개의 선택된 재조합체를 추가로 분석하였다. 그 결과, 단 하나의 콘티그만이 C 유전자좌와 완벽한 연계를 보여주었다. 이 밀접하게 연결된 콘티그의 전체 길이의 게놈 DNA 서열을 각각 백색 및 황색 집단 DNA로부터 얻었고, 인트론 10에서 53bp InDel을 사용하여 간단한 PCR 마커를 개발하였다. 그러나, 마커 유전자형과 구피색 표현형 사이의 유의한 관계는 다양한 적색, 황색 또는 백색 육종 계통에서 관찰되지 않았으며, 이는 이러한 콘티그가 C 유전자좌의 원인 유전자가 아닐 수 있음을 의미한다.Of the 114 analyzed contigs, 40 contigs containing multiple SNPs in the read of the white population were selected for PCR and sequencing. Of these, 34 contigs were confirmed to have homozygous SNPs between population DNAs through nucleotide sequence analysis of PCR products. Two selected recombinants were further analyzed to confirm the position at the C locus of the SNPs of these 34 contigs. As a result, only one Contig showed perfect association with the C locus. The full-length genomic DNA sequence of this closely linked contig was obtained from the white and yellow population DNA, respectively, and a simple PCR marker was developed using 53 bp InDel at
상기 "Indel"이란 유기체의 게놈에서 염기의 삽입(Insertion) 또는 결실(Deletion)을 말한다.The "Indel" refers to the insertion or deletion of a base in the genome of an organism.
2.2. 콘티크 분석 및 백색 구피색 유전자 식별2.2. Concrete analysis and white guppy gene identification
2.2.1. 백색 및 황색 집단의 콘티그 분석2.2.1. Contig analysis of white and yellow populations
백색 및 황색 집단 RNA에서 참조 및 새로 조립된 콘티그의 발현 수준을 RPKM(Reads per kilobase of transcript per milion mapped reads) 값을 사용하여 평가하였다. 백색 및 황색 집단 RNA 사이의 모든 콘티그의 발현 수준의 상관 계수는 참조에 비해 0.957, 및 새로 조립된 콘티그에 비해 0.964만큼 높았다. 황색 집단 RNA에서 5배 이상의 증가 및 RPKM 값의 5 이상을 모두 나타내는 DEG를 분석하였다. 총 84 및 90 DEG가 각각 참조 및 새로 조립된 콘티그로부터 확인되었고, 28개의 콘티그는 두 전사체 모두로부터 확인되었다. 이들 DEG 중 하나의 콘티그도 SNP 함유 콘티그로부터 확인되었다. 그러나 이 콘티그의 SNP는 C 유전자좌와는 완벽하게 연관되지 않았다.Expression levels of reference and newly assembled contigs in white and yellow population RNAs were evaluated using RPKM (Reads per kilobase of transcript per milion mapped reads) values. The correlation coefficient of the expression level of all contigs between the white and yellow population RNAs was as high as 0.957 compared to the reference and 0.964 compared to the freshly assembled contigs. DEGs showing both a 5-fold or more increase in the yellow population RNA and a 5 or more of the RPKM value were analyzed. A total of 84 and 90 DEGs were identified from the reference and newly assembled contigs, respectively, and 28 contigs were identified from both transcripts. Contigs of one of these DEGs were also identified from contigs containing SNPs. However, this contig's SNP was not completely associated with the C locus.
2.2.2. 비자율적 전이인자 AcWHITE의 발견2.2.2. Discovery of AcWHITE, a non-irritating transfer factor
안토시아닌 생합성 경로에서 효소를 코딩하는 모든 구조적 유전자의 전사 수준은 백색 집단에서 상당히 감소했다. 조절 MBW 복합체와 관련하여, bHLH 전사 인자를 코딩하는 B2 유전자는 백색 집단에서 전사의 가장 현저히 감소하였지만, 일부 다른 유전자의 상대적으로 감소된 발현 수준이 MYB 및 WD40 코딩 유전자에서도 관찰되었다. 백색 F 개체에서 B2 유전자의 감소된 전사는 실시간 RT-PCR에 의해 확인되었다. 그리고 WRKY 전사 인자를 코딩하는 또 다른 유전자(AcWRKY1) 또한 백색 집단에서 전사의 상당한 감소를 보였다.The levels of transcription of all structural genes encoding enzymes in the anthocyanin biosynthetic pathway were significantly reduced in the white population. Regarding the regulatory MBW complex, the B2 gene encoding the bHLH transcription factor had the most marked reduction in transcription in the white population, but relatively reduced expression levels of some other genes were also observed in the MYB and WD40 coding genes. Reduced transcription of the B2 gene in white F individuals was confirmed by real-time RT-PCR. And another gene encoding the WRKY transcription factor (AcWRKY1) also showed a significant decrease in transcription in the white population.
이들 2개의 유전자(B2 및 AcWRKY1)의 코딩 영역에서 SNP가 검출되지 않았기 때문에, B2 및 AcWRKY1의 1638bp 및 1787bp 추정 프로모터 서열은 각각 genome walking을 통해 얻었다. 황색 및 백색 집단 DNA 사이의 AcWRKY1 유전자의 5' 상류 영역에서 SNP가 검출되지 않았지만, B2 유전자의 백색 대립 유전자의 5' 상류 영역에서 577bp 삽입이 발견되었다. 5' RACE PCR 생성물의 서열은 이 삽입이 5' UTR 영역의 5' 말단에 위치함을 보여주었다. (도 8 참조) 이들 삽입된 서열은 임의의 공지된 서열과 유의한 상동성을 나타내지 않았지만, 이들 서열은 비자율적 DNA 전이인자에 속하는 것으로 가정하였다. 8bp 표적 부위 복제(GTTATAAC) 및 7bp 말단 역반복(CAAGGTT)이 이 삽입의 양쪽 말단에서 확인되었다. 또한, 이러한 삽입의 복제 수는 B2 유전자의 코딩 영역의 복제 수보다 500배 이상 더 높았다. 이러한 비자율적 DNA 전이인자를 AcWHITE이라 명명하였다.Since SNPs were not detected in the coding regions of these two genes (B2 and AcWRKY1), 1638bp and 1787bp putative promoter sequences of B2 and AcWRKY1 were obtained through genome walking, respectively. No SNP was detected in the 5'upstream region of the AcWRKY1 gene between the yellow and white population DNA, but a 577 bp insertion was found in the 5'upstream region of the white allele of the B2 gene. The sequence of the 5'RACE PCR product showed that this insertion was located at the 5'end of the 5'UTR region. (See Fig. 8) These inserted sequences did not show significant homology with any known sequence, but these sequences were assumed to belong to the non-irritating DNA transfer factor. 8bp target site replication (GTTATAAC) and 7bp terminal reverse repeat (CAAGGTT) were identified at both ends of this insertion. In addition, the number of copies of this insert was more than 500 times higher than the number of copies of the coding region of the B2 gene. This non-irritating DNA transfer factor was named AcWHITE.
2.2.3. B2 유전자의 분석2.2.3. Analysis of the B2 gene
양파 B2 유전자의 감소된 아미노산 서열은 옥수수 IN1 및 안토시아닌 생합성 경로의 주요 조절 인자로 잘 알려진 페튜니아 AN1의 염기서열과 높은 상동성을 나타냈다. 또한, B2 유전자는 안토시아닌 생합성 경로를 조절하는 것으로 알려진 다른 bHLH 단백질 중에서 보존된 bHLH 도메인을 함유했다. 긴 PCR 및 genome walking에 의해 B2 유전자의 백색 및 황색 대립 유전자의 전체 길이의 게놈 DNA 서열을 얻었다. 백색 및 황색 대립 유전자의 뉴클레오타이드 서열을 각각 MK636837 및 MK636838의 수탁 번호로 GenBank에 수탁하였다. B2 유전자의 엑손-인트론 구성은 다른 이종 상동 유전자의 구성과 유사하였다. B2 유전자의 엑손-인트론 경계는 엑손 6과 인트론 6 사이의 접합부를 제외하고 옥수수 IN1의 것과 동일했다. 다른 한편으로, 양파 B2 유전자는 매우 긴 (13,852bp) 인트론 5를 독특하게 보유하였다. 인트론 5에서 96% 뉴클레오타이드 서열 동일성을 나타내는 2개의 2516bp 반복 서열이 확인되어, 이 긴 인트론 5의 서열 분석이 어려워졌다. (도 3 참조) 이들 반복은 5'-TG-3 '으로 시작하며 다른 알려진 긴 말단 반복(LTR) 역전이인자와 같이 5'-CA-3'으로 끝나고, 이 추정 LTR 역전이인자는 5bp 표적 부위 복제 (GTAAA)에 의해 측면에 위치한다. 또한, 수천 번의 판독이 이들 반복에 지도화되었고, 반복 사이에서 gag/pol 폴리단백질과의 상동성을 나타내는 부분 서열이 확인되었다. 따라서 이 요소는 비활성 LTR 역전이인자일 가능성이 높아 보였다.The reduced amino acid sequence of the onion B2 gene showed high homology to the nucleotide sequence of corn IN1 and petunia AN1, which is well known as a major regulator of the anthocyanin biosynthetic pathway. In addition, the B2 gene contained a conserved bHLH domain among other bHLH proteins known to regulate the anthocyanin biosynthetic pathway. Full-length genomic DNA sequences of the white and yellow alleles of the B2 gene were obtained by long PCR and genome walking. The nucleotide sequences of the white and yellow alleles were deposited with GenBank under the accession numbers of MK636837 and MK636838, respectively. The exon-intron configuration of the B2 gene was similar to that of other orthologous genes. The exon-intron boundary of the B2 gene was the same as that of maize IN1 except for the junction between
2.2.4. AcWHITE 마커 제작 및 이를 이용한 분석2.2.4. Preparation of AcWHITE marker and analysis using the same
프로모터 영역에서 18개의 SNP가 확인되었지만, 황색 및 백색 대립 유전자 사이의 인트론에서는 2개의 SNP만이 확인되었다. 따라서 AcWHITE 삽입을 기반으로 간단한 PCR 마커를 개발하였다. (도 4 참조) AcWHITE를 추적하는 프라이머는 알 수 없는 이유 때문에 PCR 증폭의 효율이 낮아서, 이를 보완하기 위해 단일 불일치 뉴클레오타이드를 프라이머 B2-F1-2의 8번째 위치에 도입하여 이종 접합성 개체에 대해 두 PCR 산물의 강도를 비슷하게 만들 수 있었다. B2-F1-1 프라이머의 8 번째 뉴클레오티드 '사이토신'을 B2-F1-2 프라이머의 '구아닌'으로 변경하여 사용하였다. (도 5 참조)18 SNPs were identified in the promoter region, but only 2 SNPs were identified in the intron between the yellow and white alleles. Therefore, a simple PCR marker was developed based on the AcWHITE insertion. (See Fig. 4) Primers tracking AcWHITE have low PCR amplification efficiency for an unknown reason, so to compensate for this, a single mismatch nucleotide was introduced at the 8th position of primer B2-F1-2, and two for heterozygous individuals. The strength of the PCR products could be made similar. The 8th nucleotide'cytosine' of the B2-F1-1 primer was changed to'guanine' of the B2-F1-2 primer and used. (See Fig. 5)
대규모 F2:3 집단으로부터 확인된 모든 28개의 재조합체를 이 마커로 분석 하였다. 결과는 이 마커가 재조합체 없이 C 유전자좌에 완벽하게 연결되어 있음을 보여 주었고, 이는 이 유전자가 C 유전자좌와 관련이 있음을 말한다. 더욱이 AcWHITE 삽입은 20개의 적색 또는 황색 자손에서 나타나지 않았지만, 일부 백색 자손에서는 나타났다. (도 5 참조) 백색 대립 유전자의 마커 유전자형은 추가적인 116개의 적색 또는 황색 양파 품종에서도 검출되지 않았다. AcWHITE를 포함하지 않은 백색 구피색 자손은 우성 백색을 제어하는 I 유전자좌 또는 다른 유형의 돌연변이를 포함하는 다른 B2 대립 유전자에 의해 발생한다는 것을 나타낸다.All 28 recombinants identified from the large F 2:3 population were analyzed with this marker. The results showed that this marker is completely linked to the C locus without recombinants, suggesting that this gene is related to the C locus. Moreover, AcWHITE insertion was not seen in the 20 red or yellow offspring, but in some white offspring. (See Fig. 5) The marker genotype of the white allele was not detected even in the additional 116 red or yellow onion varieties. White guppy progeny that do not contain AcWHITE indicate that they are caused by the I locus that controls dominant white or other B2 alleles containing other types of mutations.
종합하면, 본 연구의 결과는 양파에서 C 유전자좌에 의해 부여된 백색 구피색이 비자율적 DNA 전이인자인 AcWHITE의 삽입으로 인한 bHLH 전사 인자를 코딩하는 B2 유전자의 불활성화에 의해 발현되는 것을 의미한다.Taken together, the results of this study imply that the white guppy color imparted by the C locus in onions is expressed by inactivation of the B2 gene encoding the bHLH transcription factor due to the insertion of the non-irritant DNA transfer factor, AcWHITE.
논의Argument
1. 양파에서 열성 백색 구피색 유전자의 식별1. Identification of Recessive White Guppy Color Genes in Onions
bHLH 전사 인자를 코딩하는 양파 B2 유전자는 본 연구에서 열성 백색 구피색을 제어하는 C 유전자좌에 대한 원인 유전자로 제안되었다. 안토시아닌 생합성 경로의 다른 공지된 조절 인자와 상동성을 나타내는 4개의 추정 bHLH 코딩 유전자(B1, B2, B3 및 B4)가 이전 연구에서 양파 전사체로부터 확인되었다. 이 중 B1 및 B2 유전자는 bHLH 유전자와의 상동성이 상대적으로 높았다. bHLH 전사 인자 패밀리는 효모, 동물 및 식물로부터 확인된 두 번째로 큰 전사 인자 군이다. 식물 bHLH 패밀리는 500개 이상의 bHLH 유전자의 계통발생학적 분석에 기초하여 26개의 하위 군으로 분류된다. 플라보노이드 생합성 경로의 직접적인 조절에 관여하는 모든 알려진 bHLH 유전자는 하위군 IIIf에 속한다. 하위군 IIIf의 bHLH 유전자는 계통수에서 2개의 단계통군으로 세분된다. 일반적으로, 각각의 단계통군에 속하는 2개의 bHLH 유전자는 단일 식물종으로부터 확인된다.The onion B2 gene, which encodes the bHLH transcription factor, was proposed in this study as the causative gene for the C locus that controls recessive white guppy color. Four putative bHLH coding genes (B1, B2, B3 and B4) that exhibit homology with other known regulatory factors of the anthocyanin biosynthetic pathway have been identified from onion transcripts in previous studies. Among them, the B1 and B2 genes had relatively high homology with the bHLH gene. The bHLH transcription factor family is the second largest family of transcription factors identified from yeast, animals and plants. The plant bHLH family is divided into 26 subgroups based on phylogenetic analysis of more than 500 bHLH genes. All known bHLH genes involved in the direct regulation of flavonoid biosynthetic pathways belong to subgroup IIIf. The bHLH gene of subgroup IIIf is subdivided into two subgroups in the phylogenetic tree. In general, the two bHLH genes belonging to each stage population are identified from a single plant species.
양파에서, B1 유전자는 피튜니아 JAF13 및 아라비돕시스 GL3과 군집되는 반면, B2 유전자는 피튜니아 AN1 및 아라비돕시스 TT8과 직교할 가능성이 있다. 각각의 단계통군에 속하는 두 유전자가 플라보노이드 생합성 경로의 조절에 관여하지만, AN1/TT8 단계통군의 유전자는 일부 종에서 안토시아닌 생산에 필수적인 것으로 알려져 있다. 백색 구피색을 담당하는 양파 B2 유전자도 AN1/TT8 단계통군에 속한다. 따라서, B2 유전자는 양파에서 안토시아닌 색소의 생산에 필수적인 역할을 하는 것으로 보인다. 아그로박테리움 매개 형질 전환에 대한 저항성 작물은 양파가 대부분이기 때문에 보완 테스트를 수행하기는 어렵지만, 본 연구는 안토시아닌 생산에서 B2 유전자의 필수 역할을 지지하는 몇몇 증거를 제시한다.In onions, the B1 gene clusters with petunia JAF13 and Arabidopsis GL3, while the B2 gene is likely orthogonal to petunia AN1 and Arabidopsis TT8. Although two genes belonging to each stage control group are involved in the regulation of the flavonoid biosynthetic pathway, the genes of the AN1/TT8 stage control group are known to be essential for anthocyanin production in some species. The onion B2 gene responsible for the white guppy color also belongs to the AN1/TT8 stage group. Thus, the B2 gene appears to play an essential role in the production of anthocyanin pigments in onions. Complementary testing is difficult because onions are the majority of crops resistant to Agrobacterium-mediated transformation, but this study provides some evidence supporting the essential role of the B2 gene in anthocyanin production.
첫째, 계통발생학적 분석은 양파 B2 유전자가 피튜니아 AN1, 아라비돕시스 TT8 및 달리아 DvIVS와 같은 다른 필수 bHLH 전사 인자에 대한 동원체(ortholog)임을 나타냈다. 또한, B2 유전자의 엑손-인트론 구성은 옥수수 IN1 유전자의 구성과 거의 동일하였다. bHLH 도메인을 포함하는 133 Arabidopsis 유전자의 분석은 동일한 하위 그룹 내의 유전자가 유사한 엑손-인트론 조직을 공유하며, 상대적으로 보존된 위치의 bHLH 도메인을 나타냈다.First, phylogenetic analysis revealed that the onion B2 gene is an ortholog to other essential bHLH transcription factors such as petunia AN1, Arabidopsis TT8 and Dahlia DvIVS. In addition, the exon-intron configuration of the B2 gene was almost the same as that of the maize IN1 gene. Analysis of 133 Arabidopsis genes containing the bHLH domain revealed that genes within the same subgroup share similar exon-intron tissues, and a relatively conserved location of the bHLH domain.
둘째, B2 유전자 서열 내에 AcWHITE 삽입을 기반으로 설계된 분자 마커는 2491 개체로 구성된 대규모 분리 집단에서 C 유전자좌와의 완벽한 관련성을 보여 주었다. C9860과는 달리, 또 다른 완벽하게 관련된 마커인 B2 유전자의 백색 대립 유전자는 136개의 적색 또는 황색 육종 계통 또는 품종에서 검출되지 않았으며, 이는 B2 마커가 열성 백색 구피색을 제어하는 C 유전자좌와 관련 불균형에 있음을 암시한다. (도 6 참조)Second, molecular markers designed based on the insertion of AcWHITE within the B2 gene sequence showed a perfect association with the C locus in a large isolated population of 2491 individuals. Unlike C9860, another perfectly related marker, the white allele of the B2 gene, was not detected in 136 red or yellow sarcoma lineages or cultivars, which is an imbalance associated with the C locus where the B2 marker controls recessive white guppy color. Implies that it is in. (See Fig. 6)
마지막으로, AcWHITE의 삽입에 의한 B2 유전자의 손상은 안토시아닌 생합성 경로에서 B2 및 모든 구조 유전자의 전사 수준에 대한 돌연변이의 영향을 끼친다. 비자율적 DNA 전이인자인, AcWHITE는 전사 시작 부위의 4bp 하류(downstream)에 삽입된다. AcWHITE가 프로모터 영역 대신 5' UTR에 삽입되었지만, B2 유전자의 전사 수준은 백색 RNA 규모 및 백색 F2:3 개체에서 상당히 감소하였다. 유전자 발현에서 5' UTR의 중요한 역할이 일부 식물 종에서 보고되었다. B2 유전자에 더하여, 안토시아닌 생합성 경로에서 CHS로부터 글루코스 플라보노이드 글루코실 전이효소(UDP-glucose flavonoid glucosyltransferase, UFGT)로의 효소를 코딩하는 모든 구조적 유전자의 전사 수준은 백색 집단 RNA에서 상당히 감소하였다. 이 결과는 모든 구조 유전자의 발현이 양파에서 MBW 복합체의 제어 하에 있을 수 있고, B2 유전자가 MBW 복합체의 구성원일 가능성이 있음을 의미한다.Finally, damage to the B2 gene by the insertion of AcWHITE affects the mutations on the transcription levels of B2 and all structural genes in the anthocyanin biosynthetic pathway. AcWHITE, a non-irritating DNA transfer factor, is inserted 4bp downstream of the transcription start site. Although AcWHITE was inserted into the 5'UTR instead of the promoter region, the level of transcription of the B2 gene was significantly reduced in white RNA scale and white F 2:3 individuals. An important role of 5'UTR in gene expression has been reported in some plant species. In addition to the B2 gene, transcription levels of all structural genes encoding enzymes from CHS to glucose flavonoid glucosyltransferase (UFGT) in the anthocyanin biosynthetic pathway were significantly reduced in white population RNA. This result implies that the expression of all structural genes can be under the control of the MBW complex in onions, and that the B2 gene is likely a member of the MBW complex.
2. AcWHITE DNA 전이인자의 확인 및 분석을 위한 분자 마커의 개발2. Development of molecular markers for identification and analysis of AcWHITE DNA transfer factors
2.1. AcWHITE DNA 전이인자의 확인2.1. Identification of AcWHITE DNA transfer factors
B2 유전자의 코딩 영역에서 다형성이 없었기 때문에, B2에 상응하는 콘티그는 SNP 분석 과정에 의해 검출되지 않았다. 인트론에서 오직 2개의 SNP만이 확인되었는데, 이는 백색 돌연변이 대립 유전자가 AcWHITE DNA 전이인자의 최근의 전위에서 유래한 것임을 나타낸다. 이배체 양파는 대략 16Gb의 게놈을 함유하기 때문에, 양파 게놈의 대부분은 이식 가능한 요소로 구성 될 수 있다. 비록 비자율적 DNA 전사인자인 AcWHITE는 전이효소를 인코딩하는 개방형 해독틀(Open reading frame, ORF)을 포함하지 않지만, DNA 전이인자의 전형적인 특징으로서 7bp 말단 역반복 및 8bp 표적 부위 복제를 포함한다. 비자율적 DNA 전이인자의 복제수는 일반적으로 온전한 DNA 전이인자보다 더 많다. 이 연구에서 AcWHITE의 복제 수는 500 이상으로 측정되었다. 유사하게, ANS 유전자의 프로모터 영역에서 확인된, 또 다른 양파의 비자율적 DNA 전이인자인 AcPINK의 복제수는, 3개의 다른 완전한 전이 가능 요소와 비교하여 매우 높았다.Since there was no polymorphism in the coding region of the B2 gene, contigs corresponding to B2 were not detected by the SNP analysis process. Only two SNPs were identified in the intron, indicating that the white mutant allele was derived from the recent translocation of the AcWHITE DNA transfer factor. Since diploid onions contain approximately 16 Gb of genome, most of the onion genome can be made up of implantable elements. Although the non-irritating DNA transcription factor AcWHITE does not contain an open reading frame (ORF) encoding a transferase, it includes 7bp terminal reverse repeat and 8bp target site replication as typical features of the DNA transfer factor. The number of copies of non-irritating DNA transfer factors is generally higher than that of intact DNA transfer factors. In this study, the number of copies of AcWHITE was measured to be over 500. Similarly, the copy number of AcPINK, another onion's non-irritant DNA transfer factor, identified in the promoter region of the ANS gene was very high compared to the three other fully transferable elements.
2.2. C 유전자좌의 유전자형 분석을 위한 분자 마커의 개발2.2. Development of molecular markers for genotyping of the C locus
B2 대립 유전자의 엑손 및 인트론에는 다형성이 거의 없으므로, 본 연구에서 AcWHITE 삽입을 기초로 간단한 PCR 마커가 개발되었다. 예상한 바와 같이, AcWHITE를 추적하는 PCR 산물은 본 실험의 적색 또는 황색 육종 계통 또는 품종에서 검출되지 않았다.Since there are few polymorphisms in the exons and introns of the B2 allele, a simple PCR marker was developed based on the AcWHITE insertion in this study. As expected, no PCR product tracking AcWHITE was detected in the red or yellow breeding lines or varieties of this experiment.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A molecular marker for selecting onion white bulb color and the use thereof <130> p10095 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 577 <212> DNA <213> Allium cepa <400> 1 caaggttcaa aattccggcg gaattccggt tattccggtg attccgttcc ggttccggtc 60 ataaccggaa cggaatccgg tcaaaattcc ggttccggtc taaattccgg atttaatttt 120 caaatattga aaaaaaatta taaaaaaata aaaaaattca acaaacatat agcaattaaa 180 aaagaaaagc aaattaaaca ggacaataaa taaattgaca aaaaatcatg tgtatgaata 240 aaaaaagttg ttatattttt gttaatttac gtcgaatcat aataaaaatt tcaaaaatcg 300 accaaaattt taaaatctta aatgagcaat acagactttt taaacaatgg ttttttaatc 360 aaagtgtatt ttacacgtaa tttaaataat gtactataaa tttatgattg tattgaatat 420 ttattagcga aatagcaaat taaagcaaaa aagtaccgga attagaaaac cggaattaaa 480 acggaacttc cgaaatttcc ggaacggaac cgaaatttta aaattaccaa tgaccgaaac 540 ggaaacggaa aacggaattt caaaccttgg ttataac 577 <210> 2 <211> 19374 <212> DNA <213> Allium cepa <400> 2 cggaccggat cgaaactgaa ccgtattaca ttcaaaccgg accgaaaccg aaccgtttaa 60 aggattggta taatggtttg tatttttgat taaccgttta atgcgatgcg gaagcggttt 120 ggactccaaa ccgcgccaaa ccgatccgcg ccacccataa gcaaaattag tttgagaatt 180 ttatcgtgtg tactacatat tataatcaaa acaaagttta gaatatgaca acctatcttt 240 attatataag gagtaaaaaa tgacgctata gtagtagatg cttttaagta gaaaggaata 300 ttagttacca ttagtgcagc atacggtctg aaagtcatat gcaattattt cccagcgatt 360 gaagatgtca aatactagat tggcagtttc tggttaaaca ttttcatgga aataattcct 420 aaatttggcc tgctatttat tacagattac cccagcattt atcacaattt ttaggcttac 480 gcaatgcatc tatcagtatc tgcagtaggt agcaagcacc aaatgacaca cccatatata 540 ctaacttttc aattgaagaa ttttgcaacg aatctggaaa tttttgtata aatttatttg 600 agaaatggga tacaaaaggt tcatggaaag atcaaatgtt tttattatga aactttgtta 660 tttgtattat cagttatatt aagtttaatg gtatttatga agagttctta ctggtgacac 720 actacaccaa ttaccaaatc ctcaattcca ttaacaaaca aataactgat tcgtgtggtt 780 ctataataaa ctataacaat ttacctaata ctctttacgt acgtattctc aatacacata 840 aagtactgac ctttattgac ataatagtaa aagagtacat gcgtttaaaa caggcatcag 900 ttcatacttc attttgttaa tacacgtgat aaatagattt gtcagtttat taccgacctg 960 tagaaccaac cattcaactc ataaacagtc catggcatgt agaatatcac tttctcattc 1020 ctattatctg tttccctcca catctattac cgaaccatat caaactgttt catcttatgt 1080 ggacaatatc acaaatgata taaaatgtag gaattcaatt aaaacgttac atgttataac 1140 ataaagcttt ggatatggca aaggactcga gtagtaatag caagcttcag gagatcctgc 1200 agctggctgt gcaaagtgtt caatggactt acggcatttt ttggcagctc tgcccaaatc 1260 aaaggtgtgt tggtttttct tgaaaagttt ccactaagcg ctcaactgac ctaattaata 1320 tggatttgaa atgggtaagg atgttaatat ggggagatgg atactacaat ggagcaataa 1380 agacaagaaa gacagtgcaa ggagttgaaa ttagcgcaga agatgtacct atacatagaa 1440 gccaacagtt aagagagctc tacgagtcac tttcaaatgc aggcgacgct gaacaaccca 1500 gtcataggtc gtgcacttct ttgtcccccg aagatttgtc tgagtatgaa tggttctacc 1560 tcatgtgttt ctcctttgct tttcctccag gtcatgggta tgtttatagt atctttgtta 1620 tttgtaattt tgaagtttta tttattttca ggtgttcaat ttatacctct aatgatttgt 1680 tttgttgccg cactacttaa ttgcaatcat gatcctcgac atcaaaacat taaaccaaag 1740 acacacatga aattttgttt tccaaagtag aaagatttga catgaaggat aggtattgat 1800 taactgttag caaccaaaat atttgaaaat attgaattgt ataaagatta tttataaaag 1860 aatcttaatt gtctgtatta aactttaatt aactacattt tctgcgaaaa gacattcttt 1920 gatgcctagg agtacgatgc attaactttt cgagatgttt aaatagtgtg atttgtttcc 1980 aggcatcttt tttccattcc tagaagtatg agatcctaat attttaactc aaaactgaag 2040 tatggattag aaagttgcca ttcaataata tgtaaatgaa tattttgcac tacgacaagg 2100 gatttggagc agtttactaa gtacttaatt aacgactgaa atcaattaga gcaagatccg 2160 tagagaagta atgtttgatg aagagagata cactcaatac taagaacgta agtgttacag 2220 tacctggtct ccaattcaag tcccagacac atatccgatt gaaaatttat caagagatga 2280 ttaatatagt accgagatct cttacagaag atacatgtta agcttttaag atatataaca 2340 tagtagtgtg tatagagaat agtcttacta cgtgatacca taataagttt cctatgttat 2400 agtgcaagag taatgtggtc gttttactgc atactgcata tgtgatacca taaatccaaa 2460 tagctccggt gtaaatgcta tataaggaag tagttggaga tgtctattac ataggaatat 2520 gtacgatgat aataatacac gttatattta caagtacagg ctaccaggaa aagcacttga 2580 actgcagcag cctgtttggc ttacaaaagc aaatggggca gacagtggaa tattcagcag 2640 ggccattctt gcaaaggtac ctcaattaaa tagaagtata gttttcttgt gggcccacaa 2700 accctacaaa ttaacagata attaagactg attttcggtg caaactattg aaacaaacat 2760 ttgaataatt ggtaatatca agtgatcaaa ttgtggaaga aactatcgca ataaagtaat 2820 gttggaaatt tcaagaggat ttttacgttc tgataccacc attgagaaat ttctatagaa 2880 agaagaaaga gaaagaagta acttctaatt tactaatgaa attgaaagaa tattttgtaa 2940 agaaggaaat atgacttatt ttctctttgt actttttttg cttgtatata attacatttt 3000 ccacatcaag tgagatagca catctcaagt tataataaag tatattatct gggcttttct 3060 atttctagac ttttttttca attttcaatt ttctaaattt ttcttgacaa accattttaa 3120 ttctccaaat tacatattat tcttaatttt aacattttct aaatataagg attaaatttg 3180 taacagaacc aatgttttct aaaaacaaag tttatttcaa acaagtaatg gtatagatgc 3240 attgcaacta tccaagtgtg ctctataaat atcacatcat aatagcgact aagcaataag 3300 tttcggtttc aaaaagtcct ttgtgccatc aaggttgatc attcagaact gagatagtaa 3360 acaaaattgt acataacaac aagaacaatc aacaaaagta tctaactatc atagaaccaa 3420 gaactccgtg atatttgtac tttttatcct tattctaact taatttctca ttcttttgtt 3480 tgtgatacat caatgtgacg aattaaaccg aattcggtat gcataatcag agcgcgcaca 3540 ttcaggttat attttttctc gaaaccaatc tccataattt tatttttttg ttcaaaagaa 3600 gaaaacgtct aaccataatg cttgatgttg cagacagtaa tctgcattcc tgtaatgcat 3660 ggggtatttg aacttggaac gctagaaaat gtgagttcaa atttgttatt tccgccaact 3720 atttttgtca ttacctattt ctgttaaagg tttaacttta gtgaaaaaga tctatcagtt 3780 atacataaag caagtaaact taagttacct tttattttca tccacggtct ctttcactta 3840 tccgcaaata taattccatt aagttattaa ctcaccaatt attctaaact aatccgcatg 3900 tgggttagct gacagaataa ttaaggtatt ctaattttca tttatttgct tactctttta 3960 ctttgctgtt tataattcta cttctattca tgaaatacaa aatctgtaca ttagagtcaa 4020 aattatgaga aaagctactc aaatagtcag ctaaaaaaca attgatacaa accaaacata 4080 ctaggtgtca taatataaga ttttagcaga aagattacac atttcttaca tatataaaat 4140 ctccaatcca tacaccattt aatgcgacta caataaaatc agaagatggt catttatctt 4200 tgtaagccta aatcattttc agatttgaac tgcagtggaa aagaaagagt gacttatatt 4260 acaagaagaa agaagtatac ttggtggata aactaatcat tttttcaaaa ttatataaaa 4320 gtttgaagac gctatttttg tgggcggatt agaagactaa aaatgttgat tcatgtaaaa 4380 cttaccgacc ttaaaaatac aacgagtatc aaatgaaagt atagagccgc agttatcgca 4440 gttatcaaat ataagcatca caatataatt gaaacgggtt gtgataatgg accatcccga 4500 agcccaattt tctttttata aatatcaagt tgtaaacagt tcactgtatt atcaaaagaa 4560 caaatacatt ctgtaactac agaatatttt tcttgttcac tgtttttcaa atattaatca 4620 ataaaatcaa gattcaaact ctctcatatt ctttcttggt atcaaagcgt gtacattgct 4680 taacagcaaa agaaaagttc atcagaaaat caacagagaa gaagagaaca gcaaaagaaa 4740 agctcatcag aaaatcaaca caaaaaggag gaaaacacaa atcgtgccat gtcattaacc 4800 tctcaaagaa gatctcatca cagagattag gctgcactta aaatcatcag aaaattaaaa 4860 ttttcatcat ctgattgtga atcatcagag aattaacatt ctcttcatct gattgtgaat 4920 atatttgtct aacctgggtt tttgtcaaat ctcctcctct ctgttcaaca tttctgatca 4980 cttcactcta ctgcctcgtc aagaaatcgg ttgtcagaga caatttcatc actcaattga 5040 tttctggctg cacttttctc taagtgtatt cattcccaca aatttttctg taaaaaattc 5100 tttaatccca taagttcttg aatatggttg aaggggatat taacaactca gcttcttctg 5160 aagcaagaac aaccaattca gcatcttaaa ctgccaatac ccaagatctc atcaaagcca 5220 tcataaagtc acaaagtcaa atagtcagtg ttcaactaga tgaaaacaat tacttgctct 5280 ggaaattcca gatagaaaca gcagtgagag gctatggcct tgaggatcat gttcttggaa 5340 cattaattac accatcaaaa tacactgcag ataaagatga caaattagtg agtaatcaag 5400 attatgttac acaccaatgg aaagacagtc taattagtgc ttggttactg tcttttataa 5460 gtgcaaacat tctaccatag ctaattgagt gcaagagtgc acatgaaata tagtcaacag 5520 tggagcagat ctttaacaca cagtctgctg caaaaatcat gtattacaaa agacaactac 5580 aaagcataag aaaagaaaac atgtcaatga gagagtacat gaccaagatc aagacactgt 5640 gtgatcaact tgcatcaatg ggtcataaaa tctcagacac tgggcaagtc ttatgcattc 5700 taggtggttt aaatgaagaa tatgaggcag tggtagcagt tatttcctcg aaggaaacaa 5760 caccaaatat acaatatgtt cactcaactc tactagccca tgaaggaaga atagaacaaa 5820 gaagatcaac aatttcatat cttattgtaa atgtgacgcc ccacctctat actcacgcta 5880 gtcaaggaca ctacaggata ataccgtcaa ctaacatgga gaataaggct gattaggagc 5940 gaccatctat atccttgctc taactatcta ttgaatacat caataataga cataccaagc 6000 aggtcgacat acccaattaa tacttaaaac aaaaccatag gtggttgaag gaactatcag 6060 aacataatat aacaactcaa atgtaatata catcacagaa cagtagatca tcatatacac 6120 atatattgat acataacaga atttacaaac tgatacacgt caagtggtat agcaaaccac 6180 aataaacagg caaatccaaa agaacgcata tatcagagtt acaacaaaaa ggactacaca 6240 taaagcttta acaaatagga gcaagctgaa tagtccatgc agaagcaaaa taggctaagt 6300 ccaaaagcac ctaacctgaa acatctcggt ctgacccgag tataatacac gagtcagtct 6360 agctaaacaa atcagttaga taaactgaac aacaataacc tgcaaagtta caaagtataa 6420 caaatatctc taaaagagat aacacaatca tctaacaaaa agatgaatca tatcataaca 6480 agcctcgtat catcggcaac tcagagaaac aaaggatagg aatatcccaa aacaagccat 6540 aaagacaaat aacaaatacc aatgaaggta acagaagttc ctcaaggtaa tcgctatata 6600 ttgtgcggct caaaaagacc aacaccgaag agtgatacct agggaatata catatcaatg 6660 aaacatcaga aaattttaat tgcaacaaac ccaaatatat catgagcggt ggtgaagaaa 6720 ccactccaat atctctcgag cggtggtgaa gaaaaaccac tccgaaagca atactaaagg 6780 taaccgacaa ccccaaatat ctctcgagcc ggggcaacgg accaactcca aaagcgatat 6840 taaaggcaac aagccagaag gagatagtct gaagacacta tcccaaaggc aacatgtgtc 6900 acaaactccc agaaaggaat ccgaaaagtg gtaacgtgag tctcataacc acccgttcgc 6960 attaaacggt ttactatccg ttagttaatt tcacactcaa agcagtacaa agtttacctc 7020 atctcatcgg taaaccagac acaaaggtca aaacgttcaa acgaatagat tgagaattaa 7080 ctgagaagat gaacgcacgt gttcattgca cgtgttttat ctaatcattt gattctcctc 7140 ccacttcgct cggagtgcga aaccgtgtgt gttttaaata gaatttataa attggagaaa 7200 cgagctttta atgcaattta aagggattat tttataaaaa acccgtggat gaagtcaaaa 7260 aggtatcatc aaggtcaaat atgcaaaaac aactaattgg aatcaatgtg gatcgataaa 7320 agttggaatc acagacaaat aggcaaagta ggaaaaacag gggaacatga gcaggtcggt 7380 tgaccggact ctgagctcgg tcgaccggcc caaaaacaga gaagtgctct ggtagcaggt 7440 cggtcaccgg cctactgagt tcgatcggcc gaaccctgtt tccagcactt cccagcctat 7500 caaaacgagg gccaatctcg accaaattgc aatcgttcga gcaacgacgc caagggttta 7560 tgtcaatcga ctacccgatg ctatatgctc ctcgatgcat caccaaaaac atgtttaaac 7620 atcaaagtta tgatctttaa agatttgaac ccaaaacact aaaaattggg atattcacca 7680 attcaatcca aatcaagagc aaatgcacat gaaaaagtgt taggaattca tatctgacaa 7740 tcaaagatag atataagcat agatgtaaga taaaaatccc acctttaacc ctaaaaaaaa 7800 gaagaagaag aagctcaatt gcaaggaaaa gatcaaccac tctctacttc aactcgacta 7860 gcttcaatct aagatcaaaa caagtatttc ctaagttagg tttaggaaac ttctgaaatt 7920 gaaggagaaa gagttggaga agatgaagag gaggcttacc aatagcttct tttgattttg 7980 gagcaagaat ttgagctagc aagagaaact agagcttaga gtttctaatt ccctttcctt 8040 tccaccattt tcagttagag cttgagtttg aggagtggga atgagtgaat ggagaggaga 8100 gagctgagtt agggtattaa gttagctagt gggttgaacc caaacccaaa acccaactag 8160 aaaaagtagc aaaattcaac ttactccaaa catatcaaag cttatactca aaaagatcac 8220 aaagccattt tggactcaat ggatgtggtc ttgagtccca attcgaccgt ttgcagaaag 8280 tagccttttg gactataatc cgacaaacga cgttatattt aagtaaaata tttaaatata 8340 caaccgataa atatgcgggt tgttacagac tcccctcctt aaaaaaattg cgtcctcgca 8400 attcttaaga agaaaataaa gagtaacaac ccaaaaacat atcaacacat aagaataaca 8460 gaataagaag ttcaccataa agtgcacgaa aggataagcc ctaacttgtt tgctcataga 8520 attttgtgga ttcctaagct caaaataatt cggaacaccg acatggcgca acaatacgtc 8580 aaatgaacca aagctcaaca aaatgaagta caacaaaacg tccgatgaac ttggtaaaaa 8640 gatcattcca ttcggacatc aacaaagaaa caatcacttg ttgcttacaa ctaaatgcat 8700 tggcaacacg atcccccttt caggaataaa aagaatgaga gtattggaaa agagttcttt 8760 taaataatca actttcccga aaagctaaaa ctttcaaaat cacaattatc tttatgtaat 8820 tatttttcta gaaaattttt cttatttcaa atcaattcac tgtgtctgat tgtctcatca 8880 aagctttagg tgattcctat cgataacgtt gattaagcgc atcgaccaga attcttaaag 8940 cacttaatgg gcaatacaat cacaaaacaa tcttctatat gataaacata tttctaaaaa 9000 cttttaaaaa ttatctttgt tttttttaag aataaaaatc ccatttatac ttataaataa 9060 atcttatgaa aaagatttgc aaaaattttg aattgctaat ttccatccaa aaccaataaa 9120 tgctaaaatg gaatcatcga gatattcaca aaatactcga ttcatacaat ggatgaaatg 9180 agctagacaa actgaatgac ataataaatc attcataaga atcacaacga gtgcggaatg 9240 cggtaagtgg agtgctaact tccgcaactc gtagccaaat caaaaccggt ggcaaggtcg 9300 agctgaaaag accttaatac cactcaaagg attgcttagg agataatcct aagactagac 9360 ccaaaatacc agaaaattct cgaactataa gaatgtgaga aacaacactt tcctcatcat 9420 gagggattaa agaaataaaa tgatgccatc aaattggaaa tcgaatcaca aagaaaagta 9480 gaaacctacc aaaatagatg gagacataac ttatccctga gaagaacctc cagaagcatt 9540 tgttacattt aacttaccag atggagaatt attcggagca atgggacaag tggaactacg 9600 atggtcttcg ttcttgcaat agcggcagaa gagaggctta ggacaagctc gaatcatatg 9660 atcaggagaa ccacaattga aacacttccg aatggaaggc gttccagcag gctgagactg 9720 agaatcagat tcaacggact ttttcttctt ggaagggaaa aattcccgct catccaatgg 9780 agactgcttt ttgttggggt tcttacgaac ctcaacatca tcaccactat cacaatcctt 9840 ctcgatggtt tcaagatgaa gtgcagaatc taccaactcc tcaaaagaaa gcttaaactg 9900 gagcaataaa gacttcttaa gatatttctt aagaccagaa acaaacttca aattcttctt 9960 ataagaagtg caaatgaatt catgaccata cttagataaa gcctcaaatg atgaaacata 10020 ctcagaaaca gaagacttcc cttacttcaa cacgaggaag cgacgggtca ataaaacttt 10080 ttcagattca ggaacaaact tgagttcgaa tcgtccccga aactgagtcc aaaaaagagt 10140 agactcatca atttcggcta caagaagctc ataccaatca agagcacgct attgtaataa 10200 gctagaagtt acaagaaccc tatcatgatc agaaagatcc atgcgagaga aaatacgctc 10260 gcacgtgcga agccaagctt ggacttcaag aacagactcg gagcctttaa acggctttcc 10320 accaagatga gaaaaatcac ttcacaggcg tgcaaaagta gacttgcgag gtggagatac 10380 acgagtagga gctggaggct gaatgctcct aaacaactcc ataaactgat cggcagacat 10440 ataaacgcca tccggatgtg agccactgga ttgcccagca tccgaacgtc gtcccctgcc 10500 tcttccagcc atacgttgaa agttaagata agaacaagat tattcacaag gcagatgatt 10560 caatatggaa tactacagga tttaaatgca ttcctagacg tattcccaca ttctatcaac 10620 caccaagaca caaaagaaat atagaggcat gatgattcaa agatttgtat cacttataca 10680 ctttgacgta ccggaatatg tgcgtctatt ctgtgtttat catattttgt tctaaacctc 10740 gctctgatac caagctgtga cgcctcacct ctatacccac gctagccaag gacactacag 10800 ggtaatgccg tcaactaaca cggagaataa ggctggttag gagcgaccat ctatagcctt 10860 gctctaacta tctattgaat acatcgacaa tagacatacc aagcaggtcg acgtacctaa 10920 tgaatactta aaacaaaacc ataggtggtt ggaggatcta tcagaacata atataacaac 10980 tcaaatgtaa tatacatcac agaacagtag atcatcatat acacatatat tgatacataa 11040 cagagtttac aaactgatac acgtcaagtg atatagcaaa ccacaataaa caggcaaatc 11100 caaaagaata catatatcag agttacaaca aaaaggacta cacataaagc tttaacaaaa 11160 aggagcaagc tgaatagtcc atgcggaagc aaaatagcta agtccaaaag cacctaacct 11220 gaaacatctc ggtctgaccc gagtataata cacgggtcag tctagctaaa caaatcagtt 11280 agataaactg aacaacaata acctgcaaag tcacaaagta taacaaatat ctctaaaaga 11340 gataacacaa tcatctaaca aaaagatgaa tcatatcata acaagcctcg tatcatcggc 11400 aactcataga aacaaaggat aggaatatcc caaaacaagc cataaaggca aataacaaat 11460 actagtgaag gtaacacaag ttccacaagg taatcgctat atatcgtgcg gctcaaaaag 11520 accaacaccg aagagcgata cctagggaat atacatatca atgaaacatc ataaaatttt 11580 aattgcaaca aacccaaata tctcatgagc ggtggtgaag aaaccagtcc aatatctctc 11640 gagcggtggt gaagaaaaac cactccgaaa gcaatactaa aggtaaccga caaccccaaa 11700 tatctctcga gccggggcaa aggaccaact ccaaaagcga tatttaaggc aacaagccag 11760 aaggagacag tccaaagaca ctatcccaaa ggcaacatgt gtcacaaact ctcagaaagg 11820 aatccgaaaa gtggtaacgt gagtctcata accacccgtt cgcactaaac ggtttcttat 11880 ccgttagtta atttcacact caaagcagta caaagtttac ctcatctcat cggtaaacca 11940 gacacaaagg tcaaaacgtt caaacgaata tattgggaat taactgagaa tatgaacgca 12000 cgtgttcatt gcacgcgttt tatctaatca tttgattctc ctcccacttc gctcggagtg 12060 cgaaaccgtg tgtgttttaa atagaattta taaattggag aaatgtgctt ttaatgcaac 12120 ttaaagggat tattttataa aaatcccgtg gatgaagtca aaaaggaatc atcaaggtca 12180 aatatgcata aacaactaat tggaatcaac gtgcatcgat aaaagttgga atcacagaca 12240 aataggcaaa gtagaaaaaa caggggaaca ggagcaggtc ggttgaccgg actttgagct 12300 cggtcgaccg gcccaaaaac agagaagtgc tctggtagca ggtcggtcga ccggcctact 12360 gagttcgatc ggccaaaccc tgtttccagc acttcccagc ctatcaaaac gagggccaat 12420 ctcgaccaaa ttgcaatcgt tcgagcaaag ccgccaaggg tttatgccaa tcgactaccc 12480 gacgctatat gctcctcgat gcatcaccaa aaacatgttt aaacatcaaa gttatgatct 12540 ttaaagattt gaacccaaaa cactaaaaat tgggatattc accaattcaa tccaaatcaa 12600 gagcaaatgc acatgaaaaa gtgttaggaa ttcatacctg acaatcaaag atagatataa 12660 gcatagatgt gtgataaaaa tctcaccttt aaccctaaaa aaagaagaag aagacgctca 12720 attgtaagga aaagatcaac cactctccac ttcaactcta ctagcttcaa tctaagatca 12780 aaacaagtat ttcttaagtt aggtttagga aacttctgaa attgaaggag aaagagttgg 12840 agaagatgaa gaggaggctt accaatagct tcttttgatt ttggagcaag aatttgagct 12900 agcaagagaa actagggctt ggagtttcta attccctttc ctttccacca ttttcagtta 12960 gagcttgagt ttgagaagtg ggaatgagtg aatggagagg agagagctga gttagggtat 13020 taagttagct agtgggctga acccaaaccc aaaacccaac tagaaaaagt agcaaaattt 13080 aacttactcc aaacatatca aagcttatat tcaaaaagat tgcaaacgcc attttggact 13140 caatggaggt ggtcttgagt cccaattcga ccgtttgcag aaagtagctt tttggactat 13200 aatccgacaa atgacgttat atttaaataa aatatttaaa tatgcaaccg ataaatatgc 13260 gggttgttac agtaaacttc gccagcaata acagattcaa gaatcaagaa agaagcaaca 13320 acaataaaaa tcagcctaat tttcaaagaa atggtcaatc ctctagatcc agaggaagaa 13380 atggaagata taacaacagc aataacagac tatggtgtca aatttgtgaa aagtttggtc 13440 atgcagccaa cgaatgttac ttcagatttg atgccaacta cattccatct cagcaaaata 13500 acaacagttc aagaaatcac aatctcaatg caaacatggt aaatacttat ccagattcta 13560 aagaacaagc agataccaaa agtacagaga ctgcaaagat tgatgatatc taggatgaaa 13620 gctagtatcc agattatgag gctaccaatc atgtaactaa taatctcaac aatctaaact 13680 tgggaagcag ggagtacaga ggtaggcaat taattcatat gggtaatggt gaatcagtaa 13740 aaattacaca tattggaaat gcaagtttca gtggcagaaa ataactcttt ctaaaaaatc 13800 tgctaagagt tccatctatt agaaaaacct tattgagtgt ttcgcatttt gcccaaggca 13860 atgatgtgta ttttatccca aatactgttt aattagagat attctaacta aggaaataat 13920 tttgcagggt aagaaattta aaggattgta caaattcgat gttgcaaaat caggaaattc 13980 cgaatttcat ggaaaatggt gtaatgttac tgaaattgaa catgaagata actataaaga 14040 ttttgatatt tgacattcca aacttggaca tccaagtgta agcattgtaa aaaaggtgtt 14100 aagtaatgat aatatttctg ttaaagatgt tgcaatgcct tatgtatgta catattgtca 14160 aatgggcaaa agccataaac ttgcatttcc aatctcaaaa ataaactata caaaaccctt 14220 agattaattg cttgagatct atggggtcca tcttctgtta acactgatta tggctataaa 14280 tactttatat catttgtcga tgcatactct agatatgtat ggatttattt cttgaaatca 14340 aaatctgaaa cttatgatgc agtcttacag tttataactc aagctgagag gcaaacaaat 14400 tgccaattaa aagtcttgca gactgatggt ggaaccgagt ttcaaccact taaggaatac 14460 cttcaaaaga aaggtataat tcacagaaca aactgtccat acacatcaga acaaaatgga 14520 ctggttgaaa gaaaacacag acatattgtt gagactgggc ttactttact agcccaagca 14580 tctcttcctc taaagttttg accagatgcc tttgccacta ctgtgttttt aataaataga 14640 ttatccataa aagttttaca gttcaaaagt ccagtggaaa tcttatttaa agttaagcca 14700 gaatatcata gtttgaaagt ttttggatgt ttatgctttc cctatttaag gccatataat 14760 acacacaagt taagtttcag gtcatcacca tgtaccttct taggctatgc agccaataag 14820 aagggataca aatgcttgga tagcaatgaa ttttgttttt gtttctatac atgtggtgtt 14880 taatgaaaga atatttccct ttaatcaatc aaacataaaa acatcacaga aagtgtataa 14940 ttcaaaactt tctataccac cagtacctga gcaacttgat gtcataactc aagttgaaga 15000 tgaaagaatg cagcacaata taactttaga gtctcctgtc aaatccactt cacaatacac 15060 tgtgcccaga cctgagccag gtcatgaaga agatgaacaa gctggaagca caagtgaggc 15120 atctcatatg tgccccatct atcagtcaga ctgcagtcag tcattagatg attcacaatc 15180 atctaatgat caacaggaag tcacagctga agagccaagt caatcacctg cacaagctag 15240 acatcacatg ataacaaggg ccaagagtgg tatattcaca ccaaaaatat acagtgttgc 15300 tatgaacaca gaagagccag atacctatca tcaagctatg cagaatgaaa actggatgtc 15360 tgctatggaa gaagaataca atgcattgat aaaaaataaa acatgggatc taacttcatt 15420 gccaagcaat aaaaatgtca taggctgtaa acggacatat agaataaaga gaagtggtga 15480 tggttcggtt tctaaatata aggcaaggct agtagccgaa gggtattcat agcaaagtgg 15540 ctttgacttt actgaaactt tttcaccagt tgtgaaaccc tcaactatta gaattgttct 15600 tactattgcc ttacatacaa actgaaacat caagcaatta gatgtaaata atgccctttt 15660 gtggcatttt acaggaggaa gtctacatgt aacagcctct aggttttgaa caaaaaaggg 15720 gtggaaaact tgtatgcaaa ttgaacaaag cgatctatag actcaaacat gcaccaaggg 15780 tctggtttga aaagctgaag tcaatactaa tcagacttgg ctacagctca acaaaatcat 15840 acaattcttt atttacaaaa tttaaaaaca atgcaaccat gtatgtacta atatatgttg 15900 atgattttat tattacaagc agcagtgaac cagagatgaa taaactcaca gacctcttaa 15960 ataaagaatt ttcaatcaaa gacttgggga acttaaatta ctttcttggc attgaggtta 16020 aaagactttc acacatagaa atgcttctaa gtcaaaggaa atacatctca gaaatcttac 16080 agaaagcaaa aacggataaa gcaaatgcaa tatctactcc tatggcaagc aattcacatc 16140 tgtcaacgta taaaggagaa tccatatcac atggcaagca gtacagaagt attgtaggag 16200 cattacagta tgtaacaatc actaagccag aaatttcatt tagtatcaat aaagtgagtc 16260 aatatatgca aatttcactg gatgaacatt ggaaagcagt taagagaatt ctaagatatc 16320 tcaaaggaac tataaatcat ggattaaaga tgcaatcatg caaatctatc catataaaag 16380 ggtatgcaga tgcagattgg gccacagaca tggatgacag aagatctacc actggttatt 16440 gcatatttct aggcaacaat ccagtttcat ggtgctcaaa aaaacagcct acagtatctc 16500 ggtcaagtac tgaagccgag tacagaaacg ttgctaatgc aacagctgaa ataatatgga 16560 taatgtcctt gctcagtgaa cttcatatca agtttacaga aaaaccgaca atttggtgcg 16620 acaatctgag caccgttctg ccctcagcaa atctagttct acatgctaga accaagcaca 16680 tagaactgga tctgtatttt gtcagagaac aagtaacaga ggaaaaaatt aacataagtc 16740 atctaccatc atcttaccaa atcgctgatg ttatgataaa acctctatca aagaagaact 16800 ttgtttgatt taagcaagaa ttgaaggtta ctgagttcga cagcaacgaa aaaggcaaga 16860 tctatgataa ctcaacaaat tgaagcttga cagcatcgtc tactccaatt gaagagaaat 16920 tccacttgac aatcaagtga aatttcccct gatgaagaaa gcaagtctcg atatacggca 16980 atttaaccaa aatcgaaaca cgacagctta atctctccaa cttcagcaaa aagagggctt 17040 ccgtatcact gcaaggtcct gagtggtaaa tctacacact cgagaaaccg aagccatcat 17100 attttattcg tgcggctgaa gaaattgcaa accctaaccc taagttaggt gtgaggggag 17160 gtatcaaatg aaagtataga gccgcactta tcaaatataa gcatcacaat ataattggaa 17220 cgggttgtga taatggacca tcccgaagcc caattttctt tttataaata tcaagttgta 17280 aacagttcag tgtattatca aaagaacaaa aacattctgt aactacggaa tatttttctt 17340 gttttctgtt tttcaaatat taatcaataa aatcaagatt caaactctct caatattctt 17400 tttacgagct aaaaatatat catattttct gtgtccgaac gctgatattg ttttttactt 17460 tggactctga tacttagcta acattataca cgtgagaaat aggaaactta tatctagtga 17520 aaattaccca tgaatctgac aggttgctga agatataaat ttaatcagac atataaagag 17580 ccttttcgtg gatcattatg attgccatcc aaagcctgcc ctctcagaac agtcttcgtc 17640 taattcaaaa gcacatgcac agcatggccc ctttgatatt caagaaaaca taacaataat 17700 caagccaact cgtaacaacg caaataaaga aaacgaagat gaatgcatag gtgatgaaga 17760 aaatgggaat gatgaaacag aaggagaaac tgatactgaa acatatagca cacccataac 17820 gccagcagat attcaaccaa gcgagcatat gcaaatcgat acatgcgaaa atattccatt 17880 tggttcatct catgagtact attcaaacaa tttggataat gaaccacaga tgttagatag 17940 agtggatgat tcttccagag aatggcacca tttgaatgat gatacatgtg gtggtctacc 18000 agagctatcc ggtacgtacc aactgtgtta taacacataa taattgaagt gtctttcgga 18060 ccctaaccgt gcagggataa atcaataatt tcatatgttt gtacgtagct cctcggccag 18120 ttatatgtag cttgcaataa attcatcttt tacattcacc agttgcataa ttttctcctt 18180 aggttcaagt ttacttcaac agttatctgc agaggattcc cagtactcag aaacagtttc 18240 tacaattttg cataagacct caagtcaatg gactggacct attactcgaa atcatctggt 18300 ttgttctcag caatcagcat tctcaaaatg gaatgatact gatcatatac ttcacatttc 18360 atctgaggat acatcacaga aaactctgaa atatatcttg ctcagtgttc ccaagctata 18420 cagcaaggac aaaacaaaaa agttttcagc atctaaagat agaattactt ctcaagagga 18480 actatgtgct aaccatgtac ttgctgaacg taaacgtagg gagaagctta atgaaaagtt 18540 cattattctt cgatcgttgg ttccttttgt taccaaggta ttgatgactt gtagacatga 18600 cacaagttta aagtaagaaa atacatagag ctcagagtca aaacaacgca tgtttccata 18660 ataatttaat tattatttaa tctgtaatgt ggatcacgat ataagtatat aactgctgaa 18720 taatatatgg tcattgacgc aggtggataa agcttctata ttgggtgaca caattgagta 18780 tataaagcaa ttaacgtgga aaatacaaga actcggatca caaaacaagt ctactgaaag 18840 tgaaaacagt ttcagaccag gcaaagttca aagacaaact actactgcaa aaagcaaggt 18900 taaatcaaat gtgactgcga aatcaataga aaatcatcgc gttcatttgc aagtttcaat 18960 tattgaggca gacgcacttc tcgaaatgca gtgcccatac aaagaagggt tgatactgca 19020 aattatacag acactagata aacttgggct agaaattact tcgattcaat caacatcaaa 19080 caatggatta atgaaggcag aatttagggc taaggcaagt gtattccttc ttatatgcat 19140 ttttggttca atttatgcaa ttaatatgac tacatttgca acttatatgc aggttaaaga 19200 catcggagga agaaaggcca caattgtcaa agtgaagaaa gcaatttacc atattctctc 19260 acagtaattt ataatacata ctgatataac agtgacatta taatatatat atatatatat 19320 atatatatat atatatatat atatatatat atatatatat atatatatat atat 19374 <210> 3 <211> 19915 <212> DNA <213> Allium cepa <400> 3 cggaccggat cgaaactgaa ccgtattaca ttcaaaccga accgaaaccg aaccgtttaa 60 agaattggta taatagtttg tatttttgat taaccgttta atgcggtgcg gaagcggttt 120 ggactccaaa ccgcaccaaa ccgatccgcg ccacccataa gcaaaattag tttgagaatt 180 ttatcgtgtg tactacatat tataatcaaa acaaagttta gaatatgaca acctatcttt 240 attatataag gagtaaaaaa tgacgctata gtagtagatg cttttaagta gaaaggaata 300 ttagttacca ttagtgcagc atacggtctg aaagtcatat gcaattattt cccagcgatt 360 gaagatgtca aatactagat tggcagtttc tggttaaaca ttttcatgga aataattcct 420 aaatttggcc tgctatttat tacagattac cccagcattt atcacaattt ttaggcttac 480 gcaatgcatc tatcagtatc tgcagtaggt agcaagcacc aaatgacaca cccatatata 540 ctaacttttc aattgaagaa ttttgcaacg aatctggaaa tttttgtata aatttatttg 600 agaaatggga tacaaaaggt tcatggaaag atcaaatgtt tttattatga aactttgtta 660 tttgtattat cagttatgtt aagtttaatg gtatttatga agagttctta ctggtgacac 720 actacaccaa ttaccaaatc ctcaatttca ttaacaaaca aataactgat tcgtgtggtt 780 ttataataaa ctataacaat ttacctaata ctctttacgc acgtattctc aatacacata 840 aagtactgag ctttagtgac aaaatagtaa aagagtacat gcgtttaaaa caggcgtcag 900 ttcatacttc atattgttaa tacatgtgat aaatagattt gtcagtttat taccgacctg 960 tagaaccaac cattcaactc ataaacaatc catggcatgt agaatatcac tttctcattc 1020 ctattatttg tttccctcca cgtctattac cgaaccatat caaactgttt catcttatgt 1080 ggacaatatc acaaatgata taaaatgtag gaattcaatt aaaacgttac atgttataac 1140 caaggttcaa aattccggcg gaattccggt tattccggtg attccgttcc ggttccggtc 1200 ataaccggaa cggaatccgg tcaaaattcc ggttccggtc taaattccgg atttaatttt 1260 caaatattga aaaaaaatta taaaaaaata aaaaaattca acaaacatat agcaattaaa 1320 aaagaaaagc aaattaaaca ggacaataaa taaattgaca aaaaatcatg tgtatgaata 1380 aaaaaagttg ttatattttt gttaatttac gtcgaatcat aataaaaatt tcaaaaatcg 1440 accaaaattt taaaatctta aatgagcaat acagactttt taaacaatgg ttttttaatc 1500 aaagtgtatt ttacacgtaa tttaaataat gtactataaa tttatgattg tattgaatat 1560 ttattagcga aatagcaaat taaagcaaaa aagtaccgga attagaaaac cggaattaaa 1620 acggaacttc cgaaatttcc ggaacggaac cgaaatttta aaattaccaa tgaccgaaac 1680 ggaaacggaa aacggaattt caaaccttgg ttataacata aagctttgga tatggcaaag 1740 gactcgagta gtaatagcaa gcttcaggag atcctgcagc tggctgtgca aagtgttcaa 1800 tggacttacg gcattttttg gcagctctgc ccaaatcaaa ggtgtgttgg tttttcttga 1860 aaagtttcca ctaagcgctc aactgaacta attaatatgg atttgaaatg ggtaaggatg 1920 ttaatatggg gagatggata ctacaatgga gcaataaaga caagaaagac agtgcaagga 1980 gttgaaatta gcgcagaaga tgtacctata catagaagcc aacagttaag agagctctac 2040 gagtcacttt caaatgcagg cgacgctgaa caacccagtc ataggtcgtg cacttctttg 2100 tcccccgaag atttgtctga gtatgaatgg ttctacctca tgtgtttctc ctttgctttt 2160 cctccaggtc atgggtatgt ttatagtatc tttgttattt gtaattttga agttttattt 2220 attttcaggt gttcaattta tacctctaat gatttgtttt gttgccgcac tacttaattg 2280 caatcatgat cctcgacatc aaaacattaa accaaagaca cacatgaaat tttgttttcc 2340 aaagtagaaa gatttgacat gaaggatagg tattgattaa ctgttagcaa ccaaaatatt 2400 tgaaaatatt gaattgtata aagattattt ataaaagaat cttaattgtc tgtattaaac 2460 tttaattaac tacattttct gcgaaaagac attctttgat gcctaggagt acgatgcatt 2520 aacttttcga gatgtttaaa tagtgtgatt tgtttccagg catctttttt ccattcctag 2580 aagtatgaga tcctaatatt ttaactcaaa actgaagtat ggattagaaa gttgccattc 2640 aataatatgt aaatgaatat tttgcactac gacaagggat ttggagcagt ttactaagta 2700 cttaattaac gactgaaatc aattagagca agatccgtag agaagtaatg tttgatgaag 2760 agagatacac tcaatactaa gaacgtaagt gttacagtac ctggtctcca attcaagtcc 2820 cagacacata tccgattgaa aatttatcaa gagatgatta atatagtacc gagatctctt 2880 acagaagata catgttaagc ttttaagata tataacatag tagtgtgtat agagaatagt 2940 cttactacgt gataccataa taagtttcct atgttatagt gcaagagtaa tgtggtcgtt 3000 ttactgcata ctgcatatgt gataccataa atccaaatag ctccggtgta aatgctatat 3060 aaggaagtag ttggagatgt ctattacata ggaatatgta cgatgataat aatacacgtt 3120 atatttacaa gtacaggcta ccaggaaaag cacttgaact gcagcagcct gtttggctta 3180 caaaagcaaa tggggcagac agtggaatat tcagcagggc cattcttgca aaggtacctc 3240 aattaaatag aagtatagtt ttcttgtggg cccacaaacc ctacaaatta acagataatt 3300 aagactgatt ttcggtgcaa actattgaaa caaacatttg aataattggt aatatcaagt 3360 gatcaaattg tggaagaaac tatcgcaata aagtaatgtt ggaaatttca agaggatttt 3420 tacgttctga taccaccatt gagaaatttc tatagaaaga agaaagagaa agaagtaact 3480 tctaatttac taatgaaatt gaaagaatat tttgtaaaga aggaaatatg acttattttc 3540 tctttgtact ttttttgctt gtatataatt acattttcca catcaagtga gatagcacat 3600 ctcaagttat aataaagtat attatctggg cttttctatt tctagacttt tttttcaatt 3660 ttcaattttc taaatttttc ttgacaaacc attttaattc tccaaattac atattattct 3720 taattttaac attttctaaa tataaggatt aaatttgtaa cagaaccaat gttttctaaa 3780 aacaaagttt atttcaaaca agtaatggta tagatgcatt gcaactatcc aagtgtgctc 3840 tataaatatc acatcataat agcgactaag caataagttt cggtttcaaa aagtcctttg 3900 tgccatcaag gttgatcatt cagaactgag atagtaaaca aaattgtaca taacaacaag 3960 aacaatcaac aaaagtatct aactatcata gaaccaagaa ctccgtgata tttgtacttt 4020 ttatccttat tctaacttaa tttctcattc ttttgtttgt gatacatcaa tgtgacgaat 4080 taaaccgaat tcggtatgca taatcagagc gcgcacattc aggttatatt ttttctcgaa 4140 accaatctcc ataattttat ttttttgttc aaaagaagaa aacgtctaac cataatgctt 4200 gatgttgcag acagtaatct gcattcctgt aatgcatggg gtatttgaac ttggaacgct 4260 agaaaatgtg agttcaaatt tgttatttcc gccaactatt tttgtcatta cctatttctg 4320 ttaaaggttt aactttagtg aaaaagatct atcagttata cataaagcaa gtaaacttaa 4380 gttacctttt attttcatcc acggtctctt tcacttatcc gcaaatataa ttccattaag 4440 ttattaactc accaattatt ctaaactaat ccgcatgtgg gttagctgac agaataatta 4500 aggtattcta attttcattt atttgcttac tcttttactt tgctgtttat aattctactt 4560 ctattcatga aatacaaaat ctgtacatta gagtcaaaat tatgagaaaa gctactcaaa 4620 tagtcagcta aaaaacaatt gatacaaacc aaacatacta ggtgtcataa tataagattt 4680 tagcagaaag attacacatt tcttacatat ataaaatctc caatccatac accatttaat 4740 gcgactacaa taaaatcaga agatggtcat ttatctttgt aagcctaaat cattttcaga 4800 tttgaactgc agtggaaaag aaagagtgac ttatattaca agaagaaaga agtatacttg 4860 gtggataaac taatcatttt ttcaaaatta tataaaagtt tgaagacgct atttttgtgg 4920 gcggattaga agactaaaaa tgttgattca tgtaaaactt accgacctta aaaatacaac 4980 gagtatcaaa tgaaagtata gagccgcagt tatcgcagtt atcaaatata agcatcacaa 5040 tataattgaa acgggttgtg ataatggacc atcccgaagc ccaattttct ttttataaat 5100 atcaagttgt aaacagttca ctgtattatc aaaagaacaa atacattctg taactacaga 5160 atatttttct tgttcactgt ttttcaaata ttaatcaata aaatcaagat tcaaactctc 5220 tcatattctt tcttggtatc aaagcgtgta cattgcttaa cagcaaaaga aaagttcatc 5280 agaaaatcaa cagagaagaa gagaacagca aaagaaaagc tcatcagaaa atcaacacaa 5340 aaaggaggaa aacacaaatc gtgccatgtc attaacctct caaagaagat ctcatcacag 5400 agattaggct gcacttaaaa tcatcagaaa attaaaattt tcatcatctg attgtgaatc 5460 atcagagaat taacattctc ttcatctgat tgtgaatata tttgtctaac ctgggttttt 5520 gtcaaatctc ctcctctctg ttcaacattt ctgatcactt cactctactg cctcgtcaag 5580 aaatcggttg tcagagacaa tttcatcact caattgattt ctagctgcac ttttctctaa 5640 gtgtattcat tcccacaaat ttttctgtaa aaaattcttt aatcccataa gttcttgaat 5700 atggttgaag gggatattaa caactcagct tcttctgaag caagaacaac caattcagca 5760 tcttaaactg ccaataccca agatctcatc aaagccatca taaagtcaca aagtcaaata 5820 gtcagtgttc aactagatga aaacaattac ttgctctgga aattccagat agaaacagca 5880 gtgagaggct atggccttga ggatcatgtt cttggaacat taattacacc atcaaaatac 5940 actgcagata aagatgacaa attagtgagt aatcaagatt atgttacaca ccaatggaaa 6000 gacagtctaa ttagtgcttg gttactgtct tttataagtg caaacattct accatagcta 6060 attgagtgca agagtgcaca tgaaatatag tcaacagtgg agcagatctt taacacacag 6120 tctgctgcaa aaatcatgta ttacaaaaga caactacaaa gcataagaaa agaaaacatg 6180 tcaatgagag agtacatgac caagatcaag acactgtgtg atcaacttgc atcaatgggt 6240 cataaaatct cagacactgg gcaagtctta tgcattctag gtggtttaaa tgaagaatat 6300 gaggcagtgg tagcagttat ttcctcgaag gaaacaacac caaatataca atatgttcac 6360 tcaactctac tagcccatga aggaagaata gaacaaagaa gatcaacaat ttcatatctt 6420 attgtaaatg tgacgcccca cctctatact cacgctagtc aaggacacta caggataata 6480 ccgtcaacta acatggagaa taaggctgat taggagcgac catctatatc cttgctctaa 6540 ctatctattg aatacatcaa taatagacat accaagcagg tcgacatacc caattaatac 6600 ttaaaacaaa accataggtg gttgaaggaa ctatcagaac ataatataac aactcaaatg 6660 taatatacat cacagaacag tagatcatca tatacacata tattgataca taacagaatt 6720 tacaaactga tacacgtcaa gtggtatagc aaaccacaat aaacaggcaa atccaaaaga 6780 acgcatatat cagagttaca acaaaaagga ctacacataa agctttaaca aataggagca 6840 agctgaatag tccatgcaga agcaaaatag gctaagtcca aaagcaccta acctgaaaca 6900 tctcggtctg acccgagtat aatacacgag tcagtctagc taaacaaatc agttagataa 6960 actgaacaac aataacctgc aaagttacaa agtataacaa atatctctaa aagagataac 7020 acaatcatct aacaaaaaga tgaatcatat cataacaagc ctcgtatcat cggcaactca 7080 gagaaacaaa ggataggaat atcccaaaac aagccataaa gacaaataac aaataccaat 7140 gaaggtaaca gaagttcctc aaggtaatcg ctatatattg tgcggctcaa aaagaccaac 7200 accgaagagt gatacctagg gaatatacat atcaatgaaa catcagaaaa ttttaattgc 7260 aacaaaccca aatatatcat gagcggtggt gaagaaacca ctccaatatc tctcgagcgg 7320 tggtgaagaa aaaccactcc gaaagcaata ctaaaggtaa ccgacaaccc caaatatctc 7380 tcgagccggg gcaacggacc aactccaaaa gcgatattaa aggcaacaag ccagaaggag 7440 atagtctgaa gacactatcc caaaggcaac atgtgtcaca aactcccaga aaggaatccg 7500 aaaagtggta acgtgagtct cataaccacc cgttcgcatt aaacggttta ctatccgtta 7560 gttaatttca cactcaaagc agtacaaagt ttacctcatc tcatcggtaa accagacaca 7620 aaggtcaaaa cgttcaaacg aatagattga gaattaactg agaagatgaa cgcacgtgtt 7680 cattgcacgt gttttatcta atcatttgat tctcctccca cttcgctcgg agtgcgaaac 7740 cgtgtgtgtt ttaaatagaa tttataaatt ggagaaacga gcttttaatg caatttaaag 7800 ggattatttt ataaaaaacc cgtggatgaa gtcaaaaagg tatcatcaag gtcaaatatg 7860 caaaaacaac taattggaat caatgtggat cgataaaagt tggaatcaca gacaaatagg 7920 caaagtagga aaaacagggg aacatgagca ggtcggttga ccggactctg agctcggtcg 7980 accggcccaa aaacagagaa gtgctctggt agcaggtcgg tcaccggcct actgagttcg 8040 atcggccgaa ccctgtttcc agcacttccc agcctatcaa aacgagggcc aatctcgacc 8100 aaattgcaat cgttcgagca acgacgccaa gggtttatgt caatcgacta cccgatgcta 8160 tatgctcctc gatgcatcac caaaaacatg tttaaacatc aaagttatga tctttaaaga 8220 tttgaaccca aaacactaaa aattgggata ttcaccaatt caatccaaat caagagcaaa 8280 tgcacatgaa aaagtgttag gaattcatat ctgacaatca aagatagata taagcataga 8340 tgtaagataa aaatcccacc tttaacccta aaaaaaagaa gaagaagaag ctcaattgca 8400 aggaaaagat caaccactct ctacttcaac tcgactagct tcaatctaag atcaaaacaa 8460 gtatttccta agttaggttt aggaaacttc tgaaattgaa ggagaaagag ttggagaaga 8520 tgaagaggag gcttaccaat agcttctttt gattttggag caagaatttg agctagcaag 8580 agaaactaga gcttagagtt tctaattccc tttcctttcc accattttca gttagagctt 8640 gagtttgagg agtgggaatg agtgaatgga gaggagagag ctgagttagg gtattaagtt 8700 agctagtggg ttgaacccaa acccaaaacc caactagaaa aagtagcaaa attcaactta 8760 ctccaaacat atcaaagctt atactcaaaa agatcacaaa gccattttgg actcaatgga 8820 tgtggtcttg agtcccaatt cgaccgtttg cagaaagtag ccttttggac tataatccga 8880 caaacgacgt tatatttaag taaaatattt aaatatacaa ccgataaata tgcgggttgt 8940 tacagactcc cctccttaaa aaaattgcgt cctcgcaatt cttaagaaga aaataaagag 9000 taacaaccca aaaacatatc aacacataag aataacagaa taagaagttc accataaagt 9060 gcacgaaagg ataagcccta acttgtttgc tcatagaatt ttgtggattc ctaagctcaa 9120 aataattcgg aacaccgaca tggcgcaaca atacgtcaaa tgaaccaaag ctcaacaaaa 9180 tgaagtacaa caaaacgtcc gatgaacttg gtaaaaagat cattccattc ggacatcaac 9240 aaagaaacaa tcacttgttg cttacaacta aatgcattgg caacacgatc cccctttcag 9300 gaataaaaag aatgagagta ttggaaaaga gttcttttaa ataatcaact ttcccgaaaa 9360 gctaaaactt tcaaaatcac aattatcttt atgtaattat ttttctagaa aatttttctt 9420 atttcaaatc aattcactgt gtctgattgt ctcatcaaag ctttaggtga ttcctatcga 9480 taacgttgat taagcgcatc gaccagaatt cttaaagcac ttaatgggca atacaatcac 9540 aaaacaatct tctatatgat aaacatattt ctaaaaactt ttaaaaatta tctttgtttt 9600 ttttaagaat aaaaatccca tttatactta taaataaatc ttatgaaaaa gatttgcaaa 9660 aattttgaat tgctaatttc catccaaaac caataaatgc taaaatggaa tcatcgagat 9720 attcacaaaa tactcgattc atacaatgga tgaaatgagc tagacaaact gaatgacata 9780 ataaatcatt cataagaatc acaacgagtg cggaatgcgg taagtggagt gctaacttcc 9840 gcaactcgta gccaaatcaa aaccggtggc aaggtcgagc tgaaaagacc ttaataccac 9900 tcaaaggatt gcttaggaga taatcctaag actagaccca aaataccaga aaattctcga 9960 actataagaa tgtgagaaac aacactttcc tcatcatgag ggattaaaga aataaaatga 10020 tgccatcaaa ttggaaatcg aatcacaaag aaaagtagaa acctaccaaa atagatggag 10080 acataactta tccctgagaa gaacctccag aagcatttgt tacatttaac ttaccagatg 10140 gagaattatt cggagcaatg ggacaagtgg aactacgatg gtcttcgttc ttgcaatagc 10200 ggcagaagag aggcttagga caagctcgaa tcatatgatc aggagaacca caattgaaac 10260 acttccgaat ggaaggcgtt ccagcaggct gagactgaga atcagattca acggactttt 10320 tcttcttgga agggaaaaat tcccgctcat ccaatggaga ctgctttttg ttggggttct 10380 tacgaacctc aacatcatca ccactatcac aatccttctc gatggtttca agatgaagtg 10440 cagaatctac caactcctca aaagaaagct taaactggag caataaagac ttcttaagat 10500 atttcttaag accagaaaca aacttcaaat tcttcttata agaagtgcaa atgaattcat 10560 gaccatactt agataaagcc tcaaatgatg aaacatactc agaaacagaa gacttccctt 10620 acttcaacac gaggaagcga cgggtcaata aaactttttc agattcagga acaaacttga 10680 gttcgaatcg tccccgaaac tgagtccaaa aaagagtaga ctcatcaatt tcggctacaa 10740 gaagctcata ccaatcaaga gcacgctatt gtaataagct agaagttaca agaaccctat 10800 catgatcaga aagatccatg cgagagaaaa tacgctcgca cgtgcgaagc caagcttgga 10860 cttcaagaac agactcggag cctttaaacg gctttccacc aagatgagaa aaatcacttc 10920 acaggcgtgc aaaagtagac ttgcgaggtg gagatacacg agtaggagct ggaggctgaa 10980 tgctcctaaa caactccata aactgatcgg cagacatata aacgccatcc ggatgtgagc 11040 cactggattg cccagcatcc gaacgtcgtc ccctgcctct tccagccata cgttgaaagt 11100 taagataaga acaagattat tcacaaggca gatgattcaa tatggaatac tacaggattt 11160 aaatgcattc ctagacgtat tcccacattc tatcaaccac caagacacaa aagaaatata 11220 gaggcatgat gattcaaaga tttgtatcac ttatacactt tgacgtaccg gaatatgtgc 11280 gtctattctg tgtttatcat attttgttct aaacctcgct ctgataccaa gctgtgacgc 11340 ctcacctcta tacccacgct agccaaggac actacagggt aatgccgtca actaacacgg 11400 agaataaggc tggttaggag cgaccatcta tagccttgct ctaactatct attgaataca 11460 tcgacaatag acataccaag caggtcgacg tacctaatga atacttaaaa caaaaccata 11520 ggtggttgga ggatctatca gaacataata taacaactca aatgtaatat acatcacaga 11580 acagtagatc atcatataca catatattga tacataacag agtttacaaa ctgatacacg 11640 tcaagtgata tagcaaacca caataaacag gcaaatccaa aagaatacat atatcagagt 11700 tacaacaaaa aggactacac ataaagcttt aacaaaaagg agcaagctga atagtccatg 11760 cggaagcaaa atagctaagt ccaaaagcac ctaacctgaa acatctcggt ctgacccgag 11820 tataatacac gggtcagtct agctaaacaa atcagttaga taaactgaac aacaataacc 11880 tgcaaagtca caaagtataa caaatatctc taaaagagat aacacaatca tctaacaaaa 11940 agatgaatca tatcataaca agcctcgtat catcggcaac tcatagaaac aaaggatagg 12000 aatatcccaa aacaagccat aaaggcaaat aacaaatact agtgaaggta acacaagttc 12060 cacaaggtaa tcgctatata tcgtgcggct caaaaagacc aacaccgaag agcgatacct 12120 agggaatata catatcaatg aaacatcata aaattttaat tgcaacaaac ccaaatatct 12180 catgagcggt ggtgaagaaa ccagtccaat atctctcgag cggtggtgaa gaaaaaccac 12240 tccgaaagca atactaaagg taaccgacaa ccccaaatat ctctcgagcc ggggcaaagg 12300 accaactcca aaagcgatat ttaaggcaac aagccagaag gagacagtcc aaagacacta 12360 tcccaaaggc aacatgtgtc acaaactctc agaaaggaat ccgaaaagtg gtaacgtgag 12420 tctcataacc acccgttcgc actaaacggt ttcttatccg ttagttaatt tcacactcaa 12480 agcagtacaa agtttacctc atctcatcgg taaaccagac acaaaggtca aaacgttcaa 12540 acgaatatat tgggaattaa ctgagaatat gaacgcacgt gttcattgca cgcgttttat 12600 ctaatcattt gattctcctc ccacttcgct cggagtgcga aaccgtgtgt gttttaaata 12660 gaatttataa attggagaaa tgtgctttta atgcaactta aagggattat tttataaaaa 12720 tcccgtggat gaagtcaaaa aggaatcatc aaggtcaaat atgcataaac aactaattgg 12780 aatcaacgtg catcgataaa agttggaatc acagacaaat aggcaaagta gaaaaaacag 12840 gggaacagga gcaggtcggt tgaccggact ttgagctcgg tcgaccggcc caaaaacaga 12900 gaagtgctct ggtagcaggt cggtcgaccg gcctactgag ttcgatcggc caaaccctgt 12960 ttccagcact tcccagccta tcaaaacgag ggccaatctc gaccaaattg caatcgttcg 13020 agcaaagccg ccaagggttt atgccaatcg actacccgac gctatatgct cctcgatgca 13080 tcaccaaaaa catgtttaaa catcaaagtt atgatcttta aagatttgaa cccaaaacac 13140 taaaaattgg gatattcacc aattcaatcc aaatcaagag caaatgcaca tgaaaaagtg 13200 ttaggaattc atacctgaca atcaaagata gatataagca tagatgtgtg ataaaaatct 13260 cacctttaac cctaaaaaaa gaagaagaag acgctcaatt gtaaggaaaa gatcaaccac 13320 tctccacttc aactctacta gcttcaatct aagatcaaaa caagtatttc ttaagttagg 13380 tttaggaaac ttctgaaatt gaaggagaaa gagttggaga agatgaagag gaggcttacc 13440 aatagcttct tttgattttg gagcaagaat ttgagctagc aagagaaact agggcttgga 13500 gtttctaatt ccctttcctt tccaccattt tcagttagag cttgagtttg agaagtggga 13560 atgagtgaat ggagaggaga gagctgagtt agggtattaa gttagctagt gggctgaacc 13620 caaacccaaa acccaactag aaaaagtagc aaaatttaac ttactccaaa catatcaaag 13680 cttatattca aaaagattgc aaacgccatt ttggactcaa tggaggtggt cttgagtccc 13740 aattcgaccg tttgcagaaa gtagcttttt ggactataat ccgacaaatg acgttatatt 13800 taaataaaat atttaaatat gcaaccgata aatatgcggg ttgttacagt aaacttcgcc 13860 agcaataaca gattcaagaa tcaagaaaga agcaacaaca ataaaaatca gcctaatttt 13920 caaagaaatg gtcaatcctc tagatccaga ggaagaaatg gaagatataa caacagcaat 13980 aacagactat ggtgtcaaat ttgtgaaaag tttggtcatg cagccaacga atgttacttc 14040 agatttgatg ccaactacat tccatctcag caaaataaca acagttcaag aaatcacaat 14100 ctcaatgcaa acatggtaaa tacttatcca gattctaaag aacaagcaga taccaaaagt 14160 acagagactg caaagattga tgatatctag gatgaaagct agtatccaga ttatgaggct 14220 accaatcatg taactaataa tctcaacaat ctaaacttgg gaagcaggga gtacagaggt 14280 aggcaattaa ttcatatggg taatggtgaa tcagtaaaaa ttacacatat tggaaatgca 14340 agtttcagtg gcagaaaata actctttcta aaaaatctgc taagagttcc atctattaga 14400 aaaaccttat tgagtgtttc gcattttgcc caaggcaatg atgtgtattt tatcccaaat 14460 actgtttaat tagagatatt ctaactaagg aaataatttt gcagggtaag aaatttaaag 14520 gattgtacaa attcgatgtt gcaaaatcag gaaattccga atttcatgga aaatggtgta 14580 atgttactga aattgaacat gaagataact ataaagattt tgatatttga cattccaaac 14640 ttggacatcc aagtgtaagc attgtaaaaa aggtgttaag taatgataat atttctgtta 14700 aagatgttgc aatgccttat gtatgtacat attgtcaaat gggcaaaagc cataaacttg 14760 catttccaat ctcaaaaata aactatacaa aacccttaga ttaattgctt gagatctatg 14820 gggtccatct tctgttaaca ctgattatgg ctataaatac tttatatcat ttgtcgatgc 14880 atactctaga tatgtatgga tttatttctt gaaatcaaaa tctgaaactt atgatgcagt 14940 cttacagttt ataactcaag ctgagaggca aacaaattgc caattaaaag tcttgcagac 15000 tgatggtgga accgagtttc aaccacttaa ggaatacctt caaaagaaag gtataattca 15060 cagaacaaac tgtccataca catcagaaca aaatggactg gttgaaagaa aacacagaca 15120 tattgttgag actgggctta ctttactagc ccaagcatct cttcctctaa agttttgacc 15180 agatgccttt gccactactg tgtttttaat aaatagatta tccataaaag ttttacagtt 15240 caaaagtcca gtggaaatct tatttaaagt taagccagaa tatcatagtt tgaaagtttt 15300 tggatgttta tgctttccct atttaaggcc atataataca cacaagttaa gtttcaggtc 15360 atcaccatgt accttcttag gctatgcagc caataagaag ggatacaaat gcttggatag 15420 caatgaattt tgtttttgtt tctatacatg tggtgtttaa tgaaagaata tttcccttta 15480 atcaatcaaa cataaaaaca tcacagaaag tgtataattc aaaactttct ataccaccag 15540 tacctgagca acttgatgtc ataactcaag ttgaagatga aagaatgcag cacaatataa 15600 ctttagagtc tcctgtcaaa tccacttcac aatacactgt gcccagacct gagccaggtc 15660 atgaagaaga tgaacaagct ggaagcacaa gtgaggcatc tcatatgtgc cccatctatc 15720 agtcagactg cagtcagtca ttagatgatt cacaatcatc taatgatcaa caggaagtca 15780 cagctgaaga gccaagtcaa tcacctgcac aagctagaca tcacatgata acaagggcca 15840 agagtggtat attcacacca aaaatataca gtgttgctat gaacacagaa gagccagata 15900 cctatcatca agctatgcag aatgaaaact ggatgtctgc tatggaagaa gaatacaatg 15960 cattgataaa aaataaaaca tgggatctaa cttcattgcc aagcaataaa aatgtcatag 16020 gctgtaaacg gacatataga ataaagagaa gtggtgatgg ttcggtttct aaatataagg 16080 caaggctagt agccgaaggg tattcatagc aaagtggctt tgactttact gaaacttttt 16140 caccagttgt gaaaccctca actattagaa ttgttcttac tattgcctta catacaaact 16200 gaaacatcaa gcaattagat gtaaataatg cccttttgtg gcattttaca ggaggaagtc 16260 tacatgtaac agcctctagg ttttgaacaa aaaaggggtg gaaaacttgt atgcaaattg 16320 aacaaagcga tctatagact caaacatgca ccaagggtct ggtttgaaaa gctgaagtca 16380 atactaatca gacttggcta cagctcaaca aaatcataca attctttatt tacaaaattt 16440 aaaaacaatg caaccatgta tgtactaata tatgttgatg attttattat tacaagcagc 16500 agtgaaccag agatgaataa actcacagac ctcttaaata aagaattttc aatcaaagac 16560 ttggggaact taaattactt tcttggcatt gaggttaaaa gactttcaca catagaaatg 16620 cttctaagtc aaaggaaata catctcagaa atcttacaga aagcaaaaac ggataaagca 16680 aatgcaatat ctactcctat ggcaagcaat tcacatctgt caacgtataa aggagaatcc 16740 atatcacatg gcaagcagta cagaagtatt gtaggagcat tacagtatgt aacaatcact 16800 aagccagaaa tttcatttag tatcaataaa gtgagtcaat atatgcaaat ttcactggat 16860 gaacattgga aagcagttaa gagaattcta agatatctca aaggaactat aaatcatgga 16920 ttaaagatgc aatcatgcaa atctatccat ataaaagggt atgcagatgc agattgggcc 16980 acagacatgg atgacagaag atctaccact ggttattgca tatttctagg caacaatcca 17040 gtttcatggt gctcaaaaaa acagcctaca gtatctcggt caagtactga agccgagtac 17100 agaaacgttg ctaatgcaac agctgaaata atatggataa tgtccttgct cagtgaactt 17160 catatcaagt ttacagaaaa accgacaatt tggtgcgaca atctgagcac cgttctgccc 17220 tcagcaaatc tagttctaca tgctagaacc aagcacatag aactggatct gtattttgtc 17280 agagaacaag taacagagga aaaaattaac ataagtcatc taccatcatc ttaccaaatc 17340 gctgatgtta tgataaaacc tctatcaaag aagaactttg tttgatttaa gcaagaattg 17400 aaggttactg agttcgacag caacgaaaaa ggcaagatct atgataactc aacaaattga 17460 agcttgacag catcgtctac tccaattgaa gagaaattcc acttgacaat caagtgaaat 17520 ttcccctgat gaagaaagca agtctcgata tacggcaatt taaccaaaat cgaaacacga 17580 cagcttaatc tctccaactt cagcaaaaag agggcttccg tatcactgca aggtcctgag 17640 tggtaaatct acacactcga gaaaccgaag ccatcatatt ttattcgtgc ggctgaagaa 17700 attgcaaacc ctaaccctaa gttaggtgtg aggggaggta tcaaatgaaa gtatagagcc 17760 gcacttatca aatataagca tcacaatata attggaacgg gttgtgataa tggaccatcc 17820 cgaagcccaa ttttcttttt ataaatatca agttgtaaac agttcagtgt attatcaaaa 17880 gaacaaaaac attctgtaac tacggaatat ttttcttgtt ttctgttttt caaatattaa 17940 tcaataaaat caagattcaa actctctcaa tattcttttt acgagctaaa aatatatcat 18000 attttctgtg tccgaacgct gatattgttt tttactttgg actctgatac ttagctaaca 18060 ttatacacgt gagaaatagg aaacttatat ctagtgaaaa ttacccatga atctgacagg 18120 ttgctgaaga tataaattta atcagacata taaagagcct tttcgtggat cattatgatt 18180 gccatccaaa gcctgccctc tcagaacagt cttcgtctaa ttcaaaagca catgcacagc 18240 atggcccctt tgatattcaa gaaaacataa caataatcaa gccaactcgt aacaacgcaa 18300 ataaagaaaa cgaagatgaa tgcataggtg atgaagaaaa tgggaatgat gaaacagaag 18360 gagaaactga tactgaaaca tatagcacac ccataacgcc agcagatatt caaccaagcg 18420 agcatatgca aatcgataca tgcgaaaata ttccatttgg ttcatctcat gagtactatt 18480 caaacaattt ggataatgaa ccacagatgt tagatagagt ggatgattct tccagagaat 18540 ggcaccattt gaatgatgat acatgtggtg gtctaccaga gctatccggt acgtaccaac 18600 tgtgttataa cacataataa ttgaagtgtc tttcggaccc taaccgtgca gggataaatc 18660 aataatttca tatgtttgta cgtagctcct cggccagtta tatgtagctt gcaataaatt 18720 catcttttac attcaccagt tgcataattt tctccttagg ttcaagttta cttcaacagt 18780 tatctgcaga ggattcccag tactcagaaa cagtttctac aattttgcat aagacctcaa 18840 gtcaatggac tggacctatt actcgaaatc atctggtttg ttctcagcaa tcagcattct 18900 caaaatggaa tgatactgat catatacttc acatttcatc tgaggataca tcacagaaaa 18960 ctctgaaata tatcttgctc agtgttccca agctatacag caaggacaaa acaaaaaagt 19020 tttcagcatc taaagataga attacttctc aagaggaact atgtgctaac catgtacttg 19080 ctgaacgtaa acgtagggag aagcttaatg aaaagttcat tattcttcga tcgttggttc 19140 cttttgttac caaggtattg atgacttgta gacatgacac aagtttaaag taagaaaata 19200 catagagctc agagtcaaaa caacgcatgt ttccataata atttaattat tatttaatct 19260 gtaatgtgga tcacgatata agtatataac tgctgaataa tatatggtca ttgacgcagg 19320 tggataaagc ttctatattg ggtgacacaa ttgagtatat aaagcaatta acgtggaaaa 19380 tacaagaact cggatcacaa aacaagtcta ctgaaagtga aaacagtttc agaccaggca 19440 aagttcaaag acaaactact actgcaaaaa gcaaggttaa atcaaatgtg actgcgaaat 19500 caatagaaaa tcatcgcgtt catttgcaag tttcaattat tgaggcagac gcacttctcg 19560 aaatgcagtg cccatacaaa gaagggttga tactgcaaat tatacagaca ctagataaac 19620 ttgggctaga aattacttcg attcaatcaa catcaaacaa tggattaatg aaggcagaat 19680 ttagggctaa ggcaagtgta ttccttctta tatgcatttt tggttcaatt tatgcaatta 19740 atatgactac atttgcaact tatatgcagg ttaaagacat cggaggaaga aaggccacaa 19800 ttgtcaaagt gaagaaagca atttaccata ttctctcaca gtaatttata atacatactg 19860 atataacagt gacattataa tatatatata tatatatata tatatatata tatat 19915 <210> 4 <211> 713 <212> DNA <213> Allium cepa <400> 4 acatgttata accaaggttc aaaattccgg cggaattccg gttattccgg tgattccgtt 60 ccggttccgg tcataaccgg aacggaatcc ggtcaaaatt ccggttccgg tctaaattcc 120 ggatttaatt ttcaaatatt gaaaaaaaat tataaaaaaa taaaaaaatt caacaaacat 180 atagcaatta aaaaagaaaa gcaaattaaa caggacaata aataaattga caaaaaatca 240 tgtgtatgaa taaaaaaagt tgttatattt ttgttaattt acgtcgaatc ataataaaaa 300 tttcaaaaat cgaccaaaat tttaaaatct taaatgagca atacagactt tttaaacaat 360 ggttttttaa tcaaagtgta ttttacacgt aatttaaata atgtactata aatttatgat 420 tgtattgaat atttattagc gaaatagcaa attaaagcaa aaaagtaccg gaattagaaa 480 accggaatta aaacggaact tccgaaattt ccggaacgga accgaaattt taaaattacc 540 aatgaccgaa acggaaacgg aaaacggaat ttcaaacctt ggttataaca taaagctttg 600 gatatggcaa aggactcgag tagtaatagc aagcttcagg agatcctgca gctggctgtg 660 caaagtgttc aatggactta cggcattttt tggcagctct gcccaaatca aag 713 <210> 5 <211> 21 <212> DNA <213> Allium cepa <400> 5 tgggatacaa aaggttcatg g 21 <210> 6 <211> 21 <212> DNA <213> Allium cepa <400> 6 tgggatagaa aaggttcatg g 21 <210> 7 <211> 23 <212> DNA <213> Allium cepa <400> 7 aaaatttcaa aaatcgacca aaa 23 <210> 8 <211> 20 <212> DNA <213> Allium cepa <400> 8 tgccgtaagt ccattgaaca 20 <210> 9 <211> 13852 <212> DNA <213> Allium cepa <400> 9 gtgagttcaa atttgttatt tccgccaact atttttgtca ttacctattt ctgttaaagg 60 tttaacttta gtgaaaaaga tctatcagtt atacataaag caagtaaact taagttacct 120 tttattttca tccacggtct ctttcactta tccgcaaata taattccatt aagttattaa 180 ctcaccaatt attctaaact aatccgcatg tgggttagct gacagaataa ttaaggtatt 240 ctaattttca tttatttgct tactctttta ctttgctgtt tataattcta cttctattca 300 tgaaatacaa aatctgtaca ttagagtcaa aattatgaga aaagctactc aaatagtcag 360 ctaaaaaaca attgatacaa accaaacata ctaggtgtca taatataaga ttttagcaga 420 aagattacac atttcttaca tatataaaat ctccaatcca tacaccattt aatgcgacta 480 caataaaatc agaagatggt catttatctt tgtaagccta aatcattttc agatttgaac 540 tgcagtggaa aagaaagagt gacttatatt acaagaagaa agaagtatac ttggtggata 600 aactaatcat tttttcaaaa ttatataaaa gtttgaagac gctatttttg tgggcggatt 660 agaagactaa aaatgttgat tcatgtaaaa cttaccgacc ttaaaaatac aacgagtatc 720 aaatgaaagt atagagccgc agttatcgca gttatcaaat ataagcatca caatataatt 780 gaaacgggtt gtgataatgg accatcccga agcccaattt tctttttata aatatcaagt 840 tgtaaacagt tcactgtatt atcaaaagaa caaatacatt ctgtaactac agaatatttt 900 tcttgttcac tgtttttcaa atattaatca ataaaatcaa gattcaaact ctctcatatt 960 ctttcttggt atcaaagcgt gtacattgct taacagcaaa agaaaagttc atcagaaaat 1020 caacagagaa gaagagaaca gcaaaagaaa agctcatcag aaaatcaaca caaaaaggag 1080 gaaaacacaa atcgtgccat gtcattaacc tctcaaagaa gatctcatca cagagattag 1140 gctgcactta aaatcatcag aaaattaaaa ttttcatcat ctgattgtga atcatcagag 1200 aattaacatt ctcttcatct gattgtgaat atatttgtct aacctgggtt tttgtcaaat 1260 ctcctcctct ctgttcaaca tttctgatca cttcactcta ctgcctcgtc aagaaatcgg 1320 ttgtcagaga caatttcatc actcaattga tttctggctg cacttttctc taagtgtatt 1380 cattcccaca aatttttctg taaaaaattc tttaatccca taagttcttg aatatggttg 1440 aaggggatat taacaactca gcttcttctg aagcaagaac aaccaattca gcatcttaaa 1500 ctgccaatac ccaagatctc atcaaagcca tcataaagtc acaaagtcaa atagtcagtg 1560 ttcaactaga tgaaaacaat tacttgctct ggaaattcca gatagaaaca gcagtgagag 1620 gctatggcct tgaggatcat gttcttggaa cattaattac accatcaaaa tacactgcag 1680 ataaagatga caaattagtg agtaatcaag attatgttac acaccaatgg aaagacagtc 1740 taattagtgc ttggttactg tcttttataa gtgcaaacat tctaccatag ctaattgagt 1800 gcaagagtgc acatgaaata tagtcaacag tggagcagat ctttaacaca cagtctgctg 1860 caaaaatcat gtattacaaa agacaactac aaagcataag aaaagaaaac atgtcaatga 1920 gagagtacat gaccaagatc aagacactgt gtgatcaact tgcatcaatg ggtcataaaa 1980 tctcagacac tgggcaagtc ttatgcattc taggtggttt aaatgaagaa tatgaggcag 2040 tggtagcagt tatttcctcg aaggaaacaa caccaaatat acaatatgtt cactcaactc 2100 tactagccca tgaaggaaga atagaacaaa gaagatcaac aatttcatat cttattgtaa 2160 atgtgacgcc ccacctctat actcacgcta gtcaaggaca ctacaggata ataccgtcaa 2220 ctaacatgga gaataaggct gattaggagc gaccatctat atccttgctc taactatcta 2280 ttgaatacat caataataga cataccaagc aggtcgacat acccaattaa tacttaaaac 2340 aaaaccatag gtggttgaag gaactatcag aacataatat aacaactcaa atgtaatata 2400 catcacagaa cagtagatca tcatatacac atatattgat acataacaga atttacaaac 2460 tgatacacgt caagtggtat agcaaaccac aataaacagg caaatccaaa agaacgcata 2520 tatcagagtt acaacaaaaa ggactacaca taaagcttta acaaatagga gcaagctgaa 2580 tagtccatgc agaagcaaaa taggctaagt ccaaaagcac ctaacctgaa acatctcggt 2640 ctgacccgag tataatacac gagtcagtct agctaaacaa atcagttaga taaactgaac 2700 aacaataacc tgcaaagtta caaagtataa caaatatctc taaaagagat aacacaatca 2760 tctaacaaaa agatgaatca tatcataaca agcctcgtat catcggcaac tcagagaaac 2820 aaaggatagg aatatcccaa aacaagccat aaagacaaat aacaaatacc aatgaaggta 2880 acagaagttc ctcaaggtaa tcgctatata ttgtgcggct caaaaagacc aacaccgaag 2940 agtgatacct agggaatata catatcaatg aaacatcaga aaattttaat tgcaacaaac 3000 ccaaatatat catgagcggt ggtgaagaaa ccactccaat atctctcgag cggtggtgaa 3060 gaaaaaccac tccgaaagca atactaaagg taaccgacaa ccccaaatat ctctcgagcc 3120 ggggcaacgg accaactcca aaagcgatat taaaggcaac aagccagaag gagatagtct 3180 gaagacacta tcccaaaggc aacatgtgtc acaaactccc agaaaggaat ccgaaaagtg 3240 gtaacgtgag tctcataacc acccgttcgc attaaacggt ttactatccg ttagttaatt 3300 tcacactcaa agcagtacaa agtttacctc atctcatcgg taaaccagac acaaaggtca 3360 aaacgttcaa acgaatagat tgagaattaa ctgagaagat gaacgcacgt gttcattgca 3420 cgtgttttat ctaatcattt gattctcctc ccacttcgct cggagtgcga aaccgtgtgt 3480 gttttaaata gaatttataa attggagaaa cgagctttta atgcaattta aagggattat 3540 tttataaaaa acccgtggat gaagtcaaaa aggtatcatc aaggtcaaat atgcaaaaac 3600 aactaattgg aatcaatgtg gatcgataaa agttggaatc acagacaaat aggcaaagta 3660 ggaaaaacag gggaacatga gcaggtcggt tgaccggact ctgagctcgg tcgaccggcc 3720 caaaaacaga gaagtgctct ggtagcaggt cggtcaccgg cctactgagt tcgatcggcc 3780 gaaccctgtt tccagcactt cccagcctat caaaacgagg gccaatctcg accaaattgc 3840 aatcgttcga gcaacgacgc caagggttta tgtcaatcga ctacccgatg ctatatgctc 3900 ctcgatgcat caccaaaaac atgtttaaac atcaaagtta tgatctttaa agatttgaac 3960 ccaaaacact aaaaattggg atattcacca attcaatcca aatcaagagc aaatgcacat 4020 gaaaaagtgt taggaattca tatctgacaa tcaaagatag atataagcat agatgtaaga 4080 taaaaatccc acctttaacc ctaaaaaaaa gaagaagaag aagctcaatt gcaaggaaaa 4140 gatcaaccac tctctacttc aactcgacta gcttcaatct aagatcaaaa caagtatttc 4200 ctaagttagg tttaggaaac ttctgaaatt gaaggagaaa gagttggaga agatgaagag 4260 gaggcttacc aatagcttct tttgattttg gagcaagaat ttgagctagc aagagaaact 4320 agagcttaga gtttctaatt ccctttcctt tccaccattt tcagttagag cttgagtttg 4380 aggagtggga atgagtgaat ggagaggaga gagctgagtt agggtattaa gttagctagt 4440 gggttgaacc caaacccaaa acccaactag aaaaagtagc aaaattcaac ttactccaaa 4500 catatcaaag cttatactca aaaagatcac aaagccattt tggactcaat ggatgtggtc 4560 ttgagtccca attcgaccgt ttgcagaaag tagccttttg gactataatc cgacaaacga 4620 cgttatattt aagtaaaata tttaaatata caaccgataa atatgcgggt tgttacagac 4680 tcccctcctt aaaaaaattg cgtcctcgca attcttaaga agaaaataaa gagtaacaac 4740 ccaaaaacat atcaacacat aagaataaca gaataagaag ttcaccataa agtgcacgaa 4800 aggataagcc ctaacttgtt tgctcataga attttgtgga ttcctaagct caaaataatt 4860 cggaacaccg acatggcgca acaatacgtc aaatgaacca aagctcaaca aaatgaagta 4920 caacaaaacg tccgatgaac ttggtaaaaa gatcattcca ttcggacatc aacaaagaaa 4980 caatcacttg ttgcttacaa ctaaatgcat tggcaacacg atcccccttt caggaataaa 5040 aagaatgaga gtattggaaa agagttcttt taaataatca actttcccga aaagctaaaa 5100 ctttcaaaat cacaattatc tttatgtaat tatttttcta gaaaattttt cttatttcaa 5160 atcaattcac tgtgtctgat tgtctcatca aagctttagg tgattcctat cgataacgtt 5220 gattaagcgc atcgaccaga attcttaaag cacttaatgg gcaatacaat cacaaaacaa 5280 tcttctatat gataaacata tttctaaaaa cttttaaaaa ttatctttgt tttttttaag 5340 aataaaaatc ccatttatac ttataaataa atcttatgaa aaagatttgc aaaaattttg 5400 aattgctaat ttccatccaa aaccaataaa tgctaaaatg gaatcatcga gatattcaca 5460 aaatactcga ttcatacaat ggatgaaatg agctagacaa actgaatgac ataataaatc 5520 attcataaga atcacaacga gtgcggaatg cggtaagtgg agtgctaact tccgcaactc 5580 gtagccaaat caaaaccggt ggcaaggtcg agctgaaaag accttaatac cactcaaagg 5640 attgcttagg agataatcct aagactagac ccaaaatacc agaaaattct cgaactataa 5700 gaatgtgaga aacaacactt tcctcatcat gagggattaa agaaataaaa tgatgccatc 5760 aaattggaaa tcgaatcaca aagaaaagta gaaacctacc aaaatagatg gagacataac 5820 ttatccctga gaagaacctc cagaagcatt tgttacattt aacttaccag atggagaatt 5880 attcggagca atgggacaag tggaactacg atggtcttcg ttcttgcaat agcggcagaa 5940 gagaggctta ggacaagctc gaatcatatg atcaggagaa ccacaattga aacacttccg 6000 aatggaaggc gttccagcag gctgagactg agaatcagat tcaacggact ttttcttctt 6060 ggaagggaaa aattcccgct catccaatgg agactgcttt ttgttggggt tcttacgaac 6120 ctcaacatca tcaccactat cacaatcctt ctcgatggtt tcaagatgaa gtgcagaatc 6180 taccaactcc tcaaaagaaa gcttaaactg gagcaataaa gacttcttaa gatatttctt 6240 aagaccagaa acaaacttca aattcttctt ataagaagtg caaatgaatt catgaccata 6300 cttagataaa gcctcaaatg atgaaacata ctcagaaaca gaagacttcc cttacttcaa 6360 cacgaggaag cgacgggtca ataaaacttt ttcagattca ggaacaaact tgagttcgaa 6420 tcgtccccga aactgagtcc aaaaaagagt agactcatca atttcggcta caagaagctc 6480 ataccaatca agagcacgct attgtaataa gctagaagtt acaagaaccc tatcatgatc 6540 agaaagatcc atgcgagaga aaatacgctc gcacgtgcga agccaagctt ggacttcaag 6600 aacagactcg gagcctttaa acggctttcc accaagatga gaaaaatcac ttcacaggcg 6660 tgcaaaagta gacttgcgag gtggagatac acgagtagga gctggaggct gaatgctcct 6720 aaacaactcc ataaactgat cggcagacat ataaacgcca tccggatgtg agccactgga 6780 ttgcccagca tccgaacgtc gtcccctgcc tcttccagcc atacgttgaa agttaagata 6840 agaacaagat tattcacaag gcagatgatt caatatggaa tactacagga tttaaatgca 6900 ttcctagacg tattcccaca ttctatcaac caccaagaca caaaagaaat atagaggcat 6960 gatgattcaa agatttgtat cacttataca ctttgacgta ccggaatatg tgcgtctatt 7020 ctgtgtttat catattttgt tctaaacctc gctctgatac caagctgtga cgcctcacct 7080 ctatacccac gctagccaag gacactacag ggtaatgccg tcaactaaca cggagaataa 7140 ggctggttag gagcgaccat ctatagcctt gctctaacta tctattgaat acatcgacaa 7200 tagacatacc aagcaggtcg acgtacctaa tgaatactta aaacaaaacc ataggtggtt 7260 ggaggatcta tcagaacata atataacaac tcaaatgtaa tatacatcac agaacagtag 7320 atcatcatat acacatatat tgatacataa cagagtttac aaactgatac acgtcaagtg 7380 atatagcaaa ccacaataaa caggcaaatc caaaagaata catatatcag agttacaaca 7440 aaaaggacta cacataaagc tttaacaaaa aggagcaagc tgaatagtcc atgcggaagc 7500 aaaatagcta agtccaaaag cacctaacct gaaacatctc ggtctgaccc gagtataata 7560 cacgggtcag tctagctaaa caaatcagtt agataaactg aacaacaata acctgcaaag 7620 tcacaaagta taacaaatat ctctaaaaga gataacacaa tcatctaaca aaaagatgaa 7680 tcatatcata acaagcctcg tatcatcggc aactcataga aacaaaggat aggaatatcc 7740 caaaacaagc cataaaggca aataacaaat actagtgaag gtaacacaag ttccacaagg 7800 taatcgctat atatcgtgcg gctcaaaaag accaacaccg aagagcgata cctagggaat 7860 atacatatca atgaaacatc ataaaatttt aattgcaaca aacccaaata tctcatgagc 7920 ggtggtgaag aaaccagtcc aatatctctc gagcggtggt gaagaaaaac cactccgaaa 7980 gcaatactaa aggtaaccga caaccccaaa tatctctcga gccggggcaa aggaccaact 8040 ccaaaagcga tatttaaggc aacaagccag aaggagacag tccaaagaca ctatcccaaa 8100 ggcaacatgt gtcacaaact ctcagaaagg aatccgaaaa gtggtaacgt gagtctcata 8160 accacccgtt cgcactaaac ggtttcttat ccgttagtta atttcacact caaagcagta 8220 caaagtttac ctcatctcat cggtaaacca gacacaaagg tcaaaacgtt caaacgaata 8280 tattgggaat taactgagaa tatgaacgca cgtgttcatt gcacgcgttt tatctaatca 8340 tttgattctc ctcccacttc gctcggagtg cgaaaccgtg tgtgttttaa atagaattta 8400 taaattggag aaatgtgctt ttaatgcaac ttaaagggat tattttataa aaatcccgtg 8460 gatgaagtca aaaaggaatc atcaaggtca aatatgcata aacaactaat tggaatcaac 8520 gtgcatcgat aaaagttgga atcacagaca aataggcaaa gtagaaaaaa caggggaaca 8580 ggagcaggtc ggttgaccgg actttgagct cggtcgaccg gcccaaaaac agagaagtgc 8640 tctggtagca ggtcggtcga ccggcctact gagttcgatc ggccaaaccc tgtttccagc 8700 acttcccagc ctatcaaaac gagggccaat ctcgaccaaa ttgcaatcgt tcgagcaaag 8760 ccgccaaggg tttatgccaa tcgactaccc gacgctatat gctcctcgat gcatcaccaa 8820 aaacatgttt aaacatcaaa gttatgatct ttaaagattt gaacccaaaa cactaaaaat 8880 tgggatattc accaattcaa tccaaatcaa gagcaaatgc acatgaaaaa gtgttaggaa 8940 ttcatacctg acaatcaaag atagatataa gcatagatgt gtgataaaaa tctcaccttt 9000 aaccctaaaa aaagaagaag aagacgctca attgtaagga aaagatcaac cactctccac 9060 ttcaactcta ctagcttcaa tctaagatca aaacaagtat ttcttaagtt aggtttagga 9120 aacttctgaa attgaaggag aaagagttgg agaagatgaa gaggaggctt accaatagct 9180 tcttttgatt ttggagcaag aatttgagct agcaagagaa actagggctt ggagtttcta 9240 attccctttc ctttccacca ttttcagtta gagcttgagt ttgagaagtg ggaatgagtg 9300 aatggagagg agagagctga gttagggtat taagttagct agtgggctga acccaaaccc 9360 aaaacccaac tagaaaaagt agcaaaattt aacttactcc aaacatatca aagcttatat 9420 tcaaaaagat tgcaaacgcc attttggact caatggaggt ggtcttgagt cccaattcga 9480 ccgtttgcag aaagtagctt tttggactat aatccgacaa atgacgttat atttaaataa 9540 aatatttaaa tatgcaaccg ataaatatgc gggttgttac agtaaacttc gccagcaata 9600 acagattcaa gaatcaagaa agaagcaaca acaataaaaa tcagcctaat tttcaaagaa 9660 atggtcaatc ctctagatcc agaggaagaa atggaagata taacaacagc aataacagac 9720 tatggtgtca aatttgtgaa aagtttggtc atgcagccaa cgaatgttac ttcagatttg 9780 atgccaacta cattccatct cagcaaaata acaacagttc aagaaatcac aatctcaatg 9840 caaacatggt aaatacttat ccagattcta aagaacaagc agataccaaa agtacagaga 9900 ctgcaaagat tgatgatatc taggatgaaa gctagtatcc agattatgag gctaccaatc 9960 atgtaactaa taatctcaac aatctaaact tgggaagcag ggagtacaga ggtaggcaat 10020 taattcatat gggtaatggt gaatcagtaa aaattacaca tattggaaat gcaagtttca 10080 gtggcagaaa ataactcttt ctaaaaaatc tgctaagagt tccatctatt agaaaaacct 10140 tattgagtgt ttcgcatttt gcccaaggca atgatgtgta ttttatccca aatactgttt 10200 aattagagat attctaacta aggaaataat tttgcagggt aagaaattta aaggattgta 10260 caaattcgat gttgcaaaat caggaaattc cgaatttcat ggaaaatggt gtaatgttac 10320 tgaaattgaa catgaagata actataaaga ttttgatatt tgacattcca aacttggaca 10380 tccaagtgta agcattgtaa aaaaggtgtt aagtaatgat aatatttctg ttaaagatgt 10440 tgcaatgcct tatgtatgta catattgtca aatgggcaaa agccataaac ttgcatttcc 10500 aatctcaaaa ataaactata caaaaccctt agattaattg cttgagatct atggggtcca 10560 tcttctgtta acactgatta tggctataaa tactttatat catttgtcga tgcatactct 10620 agatatgtat ggatttattt cttgaaatca aaatctgaaa cttatgatgc agtcttacag 10680 tttataactc aagctgagag gcaaacaaat tgccaattaa aagtcttgca gactgatggt 10740 ggaaccgagt ttcaaccact taaggaatac cttcaaaaga aaggtataat tcacagaaca 10800 aactgtccat acacatcaga acaaaatgga ctggttgaaa gaaaacacag acatattgtt 10860 gagactgggc ttactttact agcccaagca tctcttcctc taaagttttg accagatgcc 10920 tttgccacta ctgtgttttt aataaataga ttatccataa aagttttaca gttcaaaagt 10980 ccagtggaaa tcttatttaa agttaagcca gaatatcata gtttgaaagt ttttggatgt 11040 ttatgctttc cctatttaag gccatataat acacacaagt taagtttcag gtcatcacca 11100 tgtaccttct taggctatgc agccaataag aagggataca aatgcttgga tagcaatgaa 11160 ttttgttttt gtttctatac atgtggtgtt taatgaaaga atatttccct ttaatcaatc 11220 aaacataaaa acatcacaga aagtgtataa ttcaaaactt tctataccac cagtacctga 11280 gcaacttgat gtcataactc aagttgaaga tgaaagaatg cagcacaata taactttaga 11340 gtctcctgtc aaatccactt cacaatacac tgtgcccaga cctgagccag gtcatgaaga 11400 agatgaacaa gctggaagca caagtgaggc atctcatatg tgccccatct atcagtcaga 11460 ctgcagtcag tcattagatg attcacaatc atctaatgat caacaggaag tcacagctga 11520 agagccaagt caatcacctg cacaagctag acatcacatg ataacaaggg ccaagagtgg 11580 tatattcaca ccaaaaatat acagtgttgc tatgaacaca gaagagccag atacctatca 11640 tcaagctatg cagaatgaaa actggatgtc tgctatggaa gaagaataca atgcattgat 11700 aaaaaataaa acatgggatc taacttcatt gccaagcaat aaaaatgtca taggctgtaa 11760 acggacatat agaataaaga gaagtggtga tggttcggtt tctaaatata aggcaaggct 11820 agtagccgaa gggtattcat agcaaagtgg ctttgacttt actgaaactt tttcaccagt 11880 tgtgaaaccc tcaactatta gaattgttct tactattgcc ttacatacaa actgaaacat 11940 caagcaatta gatgtaaata atgccctttt gtggcatttt acaggaggaa gtctacatgt 12000 aacagcctct aggttttgaa caaaaaaggg gtggaaaact tgtatgcaaa ttgaacaaag 12060 cgatctatag actcaaacat gcaccaaggg tctggtttga aaagctgaag tcaatactaa 12120 tcagacttgg ctacagctca acaaaatcat acaattcttt atttacaaaa tttaaaaaca 12180 atgcaaccat gtatgtacta atatatgttg atgattttat tattacaagc agcagtgaac 12240 cagagatgaa taaactcaca gacctcttaa ataaagaatt ttcaatcaaa gacttgggga 12300 acttaaatta ctttcttggc attgaggtta aaagactttc acacatagaa atgcttctaa 12360 gtcaaaggaa atacatctca gaaatcttac agaaagcaaa aacggataaa gcaaatgcaa 12420 tatctactcc tatggcaagc aattcacatc tgtcaacgta taaaggagaa tccatatcac 12480 atggcaagca gtacagaagt attgtaggag cattacagta tgtaacaatc actaagccag 12540 aaatttcatt tagtatcaat aaagtgagtc aatatatgca aatttcactg gatgaacatt 12600 ggaaagcagt taagagaatt ctaagatatc tcaaaggaac tataaatcat ggattaaaga 12660 tgcaatcatg caaatctatc catataaaag ggtatgcaga tgcagattgg gccacagaca 12720 tggatgacag aagatctacc actggttatt gcatatttct aggcaacaat ccagtttcat 12780 ggtgctcaaa aaaacagcct acagtatctc ggtcaagtac tgaagccgag tacagaaacg 12840 ttgctaatgc aacagctgaa ataatatgga taatgtcctt gctcagtgaa cttcatatca 12900 agtttacaga aaaaccgaca atttggtgcg acaatctgag caccgttctg ccctcagcaa 12960 atctagttct acatgctaga accaagcaca tagaactgga tctgtatttt gtcagagaac 13020 aagtaacaga ggaaaaaatt aacataagtc atctaccatc atcttaccaa atcgctgatg 13080 ttatgataaa acctctatca aagaagaact ttgtttgatt taagcaagaa ttgaaggtta 13140 ctgagttcga cagcaacgaa aaaggcaaga tctatgataa ctcaacaaat tgaagcttga 13200 cagcatcgtc tactccaatt gaagagaaat tccacttgac aatcaagtga aatttcccct 13260 gatgaagaaa gcaagtctcg atatacggca atttaaccaa aatcgaaaca cgacagctta 13320 atctctccaa cttcagcaaa aagagggctt ccgtatcact gcaaggtcct gagtggtaaa 13380 tctacacact cgagaaaccg aagccatcat attttattcg tgcggctgaa gaaattgcaa 13440 accctaaccc taagttaggt gtgaggggag gtatcaaatg aaagtataga gccgcactta 13500 tcaaatataa gcatcacaat ataattggaa cgggttgtga taatggacca tcccgaagcc 13560 caattttctt tttataaata tcaagttgta aacagttcag tgtattatca aaagaacaaa 13620 aacattctgt aactacggaa tatttttctt gttttctgtt tttcaaatat taatcaataa 13680 aatcaagatt caaactctct caatattctt tttacgagct aaaaatatat catattttct 13740 gtgtccgaac gctgatattg ttttttactt tggactctga tacttagcta acattataca 13800 cgtgagaaat aggaaactta tatctagtga aaattaccca tgaatctgac ag 13852 <210> 10 <211> 2516 <212> DNA <213> Allium cepa <400> 10 tgtgacgccc cacctctata ctcacgctag tcaaggacac tacaggataa taccgtcaac 60 taacatggag aataaggctg attaggagcg accatctata tccttgctct aactatctat 120 tgaatacatc aataatagac ataccaagca ggtcgacata cccaattaat acttaaaaca 180 aaaccatagg tggttgaagg aactatcaga acataatata acaactcaaa tgtaatatac 240 atcacagaac agtagatcat catatacaca tatattgata cataacagaa tttacaaact 300 gatacacgtc aagtggtata gcaaaccaca ataaacaggc aaatccaaaa gaacgcatat 360 atcagagtta caacaaaaag gactacacat aaagctttaa caaataggag caagctgaat 420 agtccatgca gaagcaaaat aggctaagtc caaaagcacc taacctgaaa catctcggtc 480 tgacccgagt ataatacacg agtcagtcta gctaaacaaa tcagttagat aaactgaaca 540 acaataacct gcaaagttac aaagtataac aaatatctct aaaagagata acacaatcat 600 ctaacaaaaa gatgaatcat atcataacaa gcctcgtatc atcggcaact cagagaaaca 660 aaggatagga atatcccaaa acaagccata aagacaaata acaaatacca atgaaggtaa 720 cagaagttcc tcaaggtaat cgctatatat tgtgcggctc aaaaagacca acaccgaaga 780 gtgataccta gggaatatac atatcaatga aacatcagaa aattttaatt gcaacaaacc 840 caaatatatc atgagcggtg gtgaagaaac cactccaata tctctcgagc ggtggtgaag 900 aaaaaccact ccgaaagcaa tactaaaggt aaccgacaac cccaaatatc tctcgagccg 960 gggcaacgga ccaactccaa aagcgatatt aaaggcaaca agccagaagg agatagtctg 1020 aagacactat cccaaaggca acatgtgtca caaactccca gaaaggaatc cgaaaagtgg 1080 taacgtgagt ctcataacca cccgttcgca ttaaacggtt tactatccgt tagttaattt 1140 cacactcaaa gcagtacaaa gtttacctca tctcatcggt aaaccagaca caaaggtcaa 1200 aacgttcaaa cgaatagatt gagaattaac tgagaagatg aacgcacgtg ttcattgcac 1260 gtgttttatc taatcatttg attctcctcc cacttcgctc ggagtgcgaa accgtgtgtg 1320 ttttaaatag aatttataaa ttggagaaac gagcttttaa tgcaatttaa agggattatt 1380 ttataaaaaa cccgtggatg aagtcaaaaa ggtatcatca aggtcaaata tgcaaaaaca 1440 actaattgga atcaatgtgg atcgataaaa gttggaatca cagacaaata ggcaaagtag 1500 gaaaaacagg ggaacatgag caggtcggtt gaccggactc tgagctcggt cgaccggccc 1560 aaaaacagag aagtgctctg gtagcaggtc ggtcaccggc ctactgagtt cgatcggccg 1620 aaccctgttt ccagcacttc ccagcctatc aaaacgaggg ccaatctcga ccaaattgca 1680 atcgttcgag caacgacgcc aagggtttat gtcaatcgac tacccgatgc tatatgctcc 1740 tcgatgcatc accaaaaaca tgtttaaaca tcaaagttat gatctttaaa gatttgaacc 1800 caaaacacta aaaattggga tattcaccaa ttcaatccaa atcaagagca aatgcacatg 1860 aaaaagtgtt aggaattcat atctgacaat caaagataga tataagcata gatgtaagat 1920 aaaaatccca cctttaaccc taaaaaaaag aagaagaaga agctcaattg caaggaaaag 1980 atcaaccact ctctacttca actcgactag cttcaatcta agatcaaaac aagtatttcc 2040 taagttaggt ttaggaaact tctgaaattg aaggagaaag agttggagaa gatgaagagg 2100 aggcttacca atagcttctt ttgattttgg agcaagaatt tgagctagca agagaaacta 2160 gagcttagag tttctaattc cctttccttt ccaccatttt cagttagagc ttgagtttga 2220 ggagtgggaa tgagtgaatg gagaggagag agctgagtta gggtattaag ttagctagtg 2280 ggttgaaccc aaacccaaaa cccaactaga aaaagtagca aaattcaact tactccaaac 2340 atatcaaagc ttatactcaa aaagatcaca aagccatttt ggactcaatg gatgtggtct 2400 tgagtcccaa ttcgaccgtt tgcagaaagt agccttttgg actataatcc gacaaacgac 2460 gttatattta agtaaaatat ttaaatatac aaccgataaa tatgcgggtt gttaca 2516 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A molecular marker for selecting onion white bulb color and the use thereof <130> p10095 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 577 <212> DNA <213> Allium cepa <400> 1 caaggttcaa aattccggcg gaattccggt tattccggtg attccgttcc ggttccggtc 60 ataaccggaa cggaatccgg tcaaaattcc ggttccggtc taaattccgg atttaatttt 120 caaatattga aaaaaaatta taaaaaaata aaaaaattca acaaacatat agcaattaaa 180 aaagaaaagc aaattaaaca ggacaataaa taaattgaca aaaaatcatg tgtatgaata 240 aaaaaagttg ttatattttt gttaatttac gtcgaatcat aataaaaatt tcaaaaatcg 300 accaaaattt taaaatctta aatgagcaat acagactttt taaacaatgg ttttttaatc 360 aaagtgtatt ttacacgtaa tttaaataat gtactataaa tttatgattg tattgaatat 420 ttattagcga aatagcaaat taaagcaaaa aagtaccgga attagaaaac cggaattaaa 480 acggaacttc cgaaatttcc ggaacggaac cgaaatttta aaattaccaa tgaccgaaac 540 ggaaacggaa aacggaattt caaaccttgg ttataac 577 <210> 2 <211> 19374 <212> DNA <213> Allium cepa <400> 2 cggaccggat cgaaactgaa ccgtattaca ttcaaaccgg accgaaaccg aaccgtttaa 60 aggattggta taatggtttg tatttttgat taaccgttta atgcgatgcg gaagcggttt 120 ggactccaaa ccgcgccaaa ccgatccgcg ccacccataa gcaaaattag tttgagaatt 180 ttatcgtgtg tactacatat tataatcaaa acaaagttta gaatatgaca acctatcttt 240 attatataag gagtaaaaaa tgacgctata gtagtagatg cttttaagta gaaaggaata 300 ttagttacca ttagtgcagc atacggtctg aaagtcatat gcaattattt cccagcgatt 360 gaagatgtca aatactagat tggcagtttc tggttaaaca ttttcatgga aataattcct 420 aaatttggcc tgctatttat tacagattac cccagcattt atcacaattt ttaggcttac 480 gcaatgcatc tatcagtatc tgcagtaggt agcaagcacc aaatgacaca cccatatata 540 ctaacttttc aattgaagaa ttttgcaacg aatctggaaa tttttgtata aatttatttg 600 agaaatggga tacaaaaggt tcatggaaag atcaaatgtt tttattatga aactttgtta 660 tttgtattat cagttatatt aagtttaatg gtatttatga agagttctta ctggtgacac 720 actacaccaa ttaccaaatc ctcaattcca ttaacaaaca aataactgat tcgtgtggtt 780 ctataataaa ctataacaat ttacctaata ctctttacgt acgtattctc aatacacata 840 aagtactgac ctttattgac ataatagtaa aagagtacat gcgtttaaaa caggcatcag 900 ttcatacttc attttgttaa tacacgtgat aaatagattt gtcagtttat taccgacctg 960 tagaaccaac cattcaactc ataaacagtc catggcatgt agaatatcac tttctcattc 1020 ctattatctg tttccctcca catctattac cgaaccatat caaactgttt catcttatgt 1080 ggacaatatc acaaatgata taaaatgtag gaattcaatt aaaacgttac atgttataac 1140 ataaagcttt ggatatggca aaggactcga gtagtaatag caagcttcag gagatcctgc 1200 agctggctgt gcaaagtgtt caatggactt acggcatttt ttggcagctc tgcccaaatc 1260 aaaggtgtgt tggtttttct tgaaaagttt ccactaagcg ctcaactgac ctaattaata 1320 tggatttgaa atgggtaagg atgttaatat ggggagatgg atactacaat ggagcaataa 1380 agacaagaaa gacagtgcaa ggagttgaaa ttagcgcaga agatgtacct atacatagaa 1440 gccaacagtt aagagagctc tacgagtcac tttcaaatgc aggcgacgct gaacaaccca 1500 gtcataggtc gtgcacttct ttgtcccccg aagatttgtc tgagtatgaa tggttctacc 1560 tcatgtgttt ctcctttgct tttcctccag gtcatgggta tgtttatagt atctttgtta 1620 tttgtaattt tgaagtttta tttattttca ggtgttcaat ttatacctct aatgatttgt 1680 tttgttgccg cactacttaa ttgcaatcat gatcctcgac atcaaaacat taaaccaaag 1740 acacacatga aattttgttt tccaaagtag aaagatttga catgaaggat aggtattgat 1800 taactgttag caaccaaaat atttgaaaat attgaattgt ataaagatta tttataaaag 1860 aatcttaatt gtctgtatta aactttaatt aactacattt tctgcgaaaa gacattcttt 1920 gatgcctagg agtacgatgc attaactttt cgagatgttt aaatagtgtg atttgtttcc 1980 aggcatcttt tttccattcc tagaagtatg agatcctaat attttaactc aaaactgaag 2040 tatggattag aaagttgcca ttcaataata tgtaaatgaa tattttgcac tacgacaagg 2100 gatttggagc agtttactaa gtacttaatt aacgactgaa atcaattaga gcaagatccg 2160 tagagaagta atgtttgatg aagagagata cactcaatac taagaacgta agtgttacag 2220 tacctggtct ccaattcaag tcccagacac atatccgatt gaaaatttat caagagatga 2280 ttaatatagt accgagatct cttacagaag atacatgtta agcttttaag atatataaca 2340 tagtagtgtg tatagagaat agtcttacta cgtgatacca taataagttt cctatgttat 2400 agtgcaagag taatgtggtc gttttactgc atactgcata tgtgatacca taaatccaaa 2460 tagctccggt gtaaatgcta tataaggaag tagttggaga tgtctattac ataggaatat 2520 gtacgatgat aataatacac gttatattta caagtacagg ctaccaggaa aagcacttga 2580 actgcagcag cctgtttggc ttacaaaagc aaatggggca gacagtggaa tattcagcag 2640 ggccattctt gcaaaggtac ctcaattaaa tagaagtata gttttcttgt gggcccacaa 2700 accctacaaa ttaacagata attaagactg attttcggtg caaactattg aaacaaacat 2760 ttgaataatt ggtaatatca agtgatcaaa ttgtggaaga aactatcgca ataaagtaat 2820 gttggaaatt tcaagaggat ttttacgttc tgataccacc attgagaaat ttctatagaa 2880 agaagaaaga gaaagaagta acttctaatt tactaatgaa attgaaagaa tattttgtaa 2940 agaaggaaat atgacttatt ttctctttgt actttttttg cttgtatata attacatttt 3000 ccacatcaag tgagatagca catctcaagt tataataaag tatattatct gggcttttct 3060 atttctagac ttttttttca attttcaatt ttctaaattt ttcttgacaa accattttaa 3120 ttctccaaat tacatattat tcttaatttt aacattttct aaatataagg attaaatttg 3180 taacagaacc aatgttttct aaaaacaaag tttatttcaa acaagtaatg gtatagatgc 3240 attgcaacta tccaagtgtg ctctataaat atcacatcat aatagcgact aagcaataag 3300 tttcggtttc aaaaagtcct ttgtgccatc aaggttgatc attcagaact gagatagtaa 3360 acaaaattgt acataacaac aagaacaatc aacaaaagta tctaactatc atagaaccaa 3420 gaactccgtg atatttgtac tttttatcct tattctaact taatttctca ttcttttgtt 3480 tgtgatacat caatgtgacg aattaaaccg aattcggtat gcataatcag agcgcgcaca 3540 ttcaggttat attttttctc gaaaccaatc tccataattt tatttttttg ttcaaaagaa 3600 gaaaacgtct aaccataatg cttgatgttg cagacagtaa tctgcattcc tgtaatgcat 3660 ggggtatttg aacttggaac gctagaaaat gtgagttcaa atttgttatt tccgccaact 3720 atttttgtca ttacctattt ctgttaaagg tttaacttta gtgaaaaaga tctatcagtt 3780 atacataaag caagtaaact taagttacct tttattttca tccacggtct ctttcactta 3840 tccgcaaata taattccatt aagttattaa ctcaccaatt attctaaact aatccgcatg 3900 tgggttagct gacagaataa ttaaggtatt ctaattttca tttatttgct tactctttta 3960 ctttgctgtt tataattcta cttctattca tgaaatacaa aatctgtaca ttagagtcaa 4020 aattatgaga aaagctactc aaatagtcag ctaaaaaaca attgatacaa accaaacata 4080 ctaggtgtca taatataaga ttttagcaga aagattacac atttcttaca tatataaaat 4140 ctccaatcca tacaccattt aatgcgacta caataaaatc agaagatggt catttatctt 4200 tgtaagccta aatcattttc agatttgaac tgcagtggaa aagaaagagt gacttatatt 4260 acaagaagaa agaagtatac ttggtggata aactaatcat tttttcaaaa ttatataaaa 4320 gtttgaagac gctatttttg tgggcggatt agaagactaa aaatgttgat tcatgtaaaa 4380 cttaccgacc ttaaaaatac aacgagtatc aaatgaaagt atagagccgc agttatcgca 4440 gttatcaaat ataagcatca caatataatt gaaacgggtt gtgataatgg accatcccga 4500 agcccaattt tctttttata aatatcaagt tgtaaacagt tcactgtatt atcaaaagaa 4560 caaatacatt ctgtaactac agaatatttt tcttgttcac tgtttttcaa atattaatca 4620 ataaaatcaa gattcaaact ctctcatatt ctttcttggt atcaaagcgt gtacattgct 4680 taacagcaaa agaaaagttc atcagaaaat caacagagaa gaagagaaca gcaaaagaaa 4740 agctcatcag aaaatcaaca caaaaaggag gaaaacacaa atcgtgccat gtcattaacc 4800 tctcaaagaa gatctcatca cagagattag gctgcactta aaatcatcag aaaattaaaa 4860 ttttcatcat ctgattgtga atcatcagag aattaacatt ctcttcatct gattgtgaat 4920 atatttgtct aacctgggtt tttgtcaaat ctcctcctct ctgttcaaca tttctgatca 4980 cttcactcta ctgcctcgtc aagaaatcgg ttgtcagaga caatttcatc actcaattga 5040 tttctggctg cacttttctc taagtgtatt cattcccaca aatttttctg taaaaaattc 5100 tttaatccca taagttcttg aatatggttg aaggggatat taacaactca gcttcttctg 5160 aagcaagaac aaccaattca gcatcttaaa ctgccaatac ccaagatctc atcaaagcca 5220 tcataaagtc acaaagtcaa atagtcagtg ttcaactaga tgaaaacaat tacttgctct 5280 ggaaattcca gatagaaaca gcagtgagag gctatggcct tgaggatcat gttcttggaa 5340 cattaattac accatcaaaa tacactgcag ataaagatga caaattagtg agtaatcaag 5400 attatgttac acaccaatgg aaagacagtc taattagtgc ttggttactg tcttttataa 5460 gtgcaaacat tctaccatag ctaattgagt gcaagagtgc acatgaaata tagtcaacag 5520 tggagcagat ctttaacaca cagtctgctg caaaaatcat gtattacaaa agacaactac 5580 aaagcataag aaaagaaaac atgtcaatga gagagtacat gaccaagatc aagacactgt 5640 gtgatcaact tgcatcaatg ggtcataaaa tctcagacac tgggcaagtc ttatgcattc 5700 taggtggttt aaatgaagaa tatgaggcag tggtagcagt tatttcctcg aaggaaacaa 5760 caccaaatat acaatatgtt cactcaactc tactagccca tgaaggaaga atagaacaaa 5820 gaagatcaac aatttcatat cttattgtaa atgtgacgcc ccacctctat actcacgcta 5880 gtcaaggaca ctacaggata ataccgtcaa ctaacatgga gaataaggct gattaggagc 5940 gaccatctat atccttgctc taactatcta ttgaatacat caataataga cataccaagc 6000 aggtcgacat acccaattaa tacttaaaac aaaaccatag gtggttgaag gaactatcag 6060 aacataatat aacaactcaa atgtaatata catcacagaa cagtagatca tcatatacac 6120 atatattgat acataacaga atttacaaac tgatacacgt caagtggtat agcaaaccac 6180 aataaacagg caaatccaaa agaacgcata tatcagagtt acaacaaaaa ggactacaca 6240 taaagcttta acaaatagga gcaagctgaa tagtccatgc agaagcaaaa taggctaagt 6300 ccaaaagcac ctaacctgaa acatctcggt ctgacccgag tataatacac gagtcagtct 6360 agctaaacaa atcagttaga taaactgaac aacaataacc tgcaaagtta caaagtataa 6420 caaatatctc taaaagagat aacacaatca tctaacaaaa agatgaatca tatcataaca 6480 agcctcgtat catcggcaac tcagagaaac aaaggatagg aatatcccaa aacaagccat 6540 aaagacaaat aacaaatacc aatgaaggta acagaagttc ctcaaggtaa tcgctatata 6600 ttgtgcggct caaaaagacc aacaccgaag agtgatacct agggaatata catatcaatg 6660 aaacatcaga aaattttaat tgcaacaaac ccaaatatat catgagcggt ggtgaagaaa 6720 ccactccaat atctctcgag cggtggtgaa gaaaaaccac tccgaaagca atactaaagg 6780 taaccgacaa ccccaaatat ctctcgagcc ggggcaacgg accaactcca aaagcgatat 6840 taaaggcaac aagccagaag gagatagtct gaagacacta tcccaaaggc aacatgtgtc 6900 acaaactccc agaaaggaat ccgaaaagtg gtaacgtgag tctcataacc acccgttcgc 6960 attaaacggt ttactatccg ttagttaatt tcacactcaa agcagtacaa agtttacctc 7020 atctcatcgg taaaccagac acaaaggtca aaacgttcaa acgaatagat tgagaattaa 7080 ctgagaagat gaacgcacgt gttcattgca cgtgttttat ctaatcattt gattctcctc 7140 ccacttcgct cggagtgcga aaccgtgtgt gttttaaata gaatttataa attggagaaa 7200 cgagctttta atgcaattta aagggattat tttataaaaa acccgtggat gaagtcaaaa 7260 aggtatcatc aaggtcaaat atgcaaaaac aactaattgg aatcaatgtg gatcgataaa 7320 agttggaatc acagacaaat aggcaaagta ggaaaaacag gggaacatga gcaggtcggt 7380 tgaccggact ctgagctcgg tcgaccggcc caaaaacaga gaagtgctct ggtagcaggt 7440 cggtcaccgg cctactgagt tcgatcggcc gaaccctgtt tccagcactt cccagcctat 7500 caaaacgagg gccaatctcg accaaattgc aatcgttcga gcaacgacgc caagggttta 7560 tgtcaatcga ctacccgatg ctatatgctc ctcgatgcat caccaaaaac atgtttaaac 7620 atcaaagtta tgatctttaa agatttgaac ccaaaacact aaaaattggg atattcacca 7680 attcaatcca aatcaagagc aaatgcacat gaaaaagtgt taggaattca tatctgacaa 7740 tcaaagatag atataagcat agatgtaaga taaaaatccc acctttaacc ctaaaaaaaa 7800 gaagaagaag aagctcaatt gcaaggaaaa gatcaaccac tctctacttc aactcgacta 7860 gcttcaatct aagatcaaaa caagtatttc ctaagttagg tttaggaaac ttctgaaatt 7920 gaaggagaaa gagttggaga agatgaagag gaggcttacc aatagcttct tttgattttg 7980 gagcaagaat ttgagctagc aagagaaact agagcttaga gtttctaatt ccctttcctt 8040 tccaccattt tcagttagag cttgagtttg aggagtggga atgagtgaat ggagaggaga 8100 gagctgagtt agggtattaa gttagctagt gggttgaacc caaacccaaa acccaactag 8160 aaaaagtagc aaaattcaac ttactccaaa catatcaaag cttatactca aaaagatcac 8220 aaagccattt tggactcaat ggatgtggtc ttgagtccca attcgaccgt ttgcagaaag 8280 tagccttttg gactataatc cgacaaacga cgttatattt aagtaaaata tttaaatata 8340 caaccgataa atatgcgggt tgttacagac tcccctcctt aaaaaaattg cgtcctcgca 8400 attcttaaga agaaaataaa gagtaacaac ccaaaaacat atcaacacat aagaataaca 8460 gaataagaag ttcaccataa agtgcacgaa aggataagcc ctaacttgtt tgctcataga 8520 attttgtgga ttcctaagct caaaataatt cggaacaccg acatggcgca acaatacgtc 8580 aaatgaacca aagctcaaca aaatgaagta caacaaaacg tccgatgaac ttggtaaaaa 8640 gatcattcca ttcggacatc aacaaagaaa caatcacttg ttgcttacaa ctaaatgcat 8700 tggcaacacg atccccccttt caggaataaa aagaatgaga gtattggaaa agagttcttt 8760 taaataatca actttcccga aaagctaaaa ctttcaaaat cacaattatc tttatgtaat 8820 tatttttcta gaaaattttt cttatttcaa atcaattcac tgtgtctgat tgtctcatca 8880 aagctttagg tgattcctat cgataacgtt gattaagcgc atcgaccaga attcttaaag 8940 cacttaatgg gcaatacaat cacaaaacaa tcttctatat gataaacata tttctaaaaa 9000 cttttaaaaa ttatctttgt tttttttaag aataaaaatc ccatttatac ttataaataa 9060 atcttatgaa aaagatttgc aaaaattttg aattgctaat ttccatccaa aaccaataaa 9120 tgctaaaatg gaatcatcga gatattcaca aaatactcga ttcatacaat ggatgaaatg 9180 agctagacaa actgaatgac ataataaatc attcataaga atcacaacga gtgcggaatg 9240 cggtaagtgg agtgctaact tccgcaactc gtagccaaat caaaaccggt ggcaaggtcg 9300 agctgaaaag accttaatac cactcaaagg attgcttagg agataatcct aagactagac 9360 ccaaaatacc agaaaattct cgaactataa gaatgtgaga aacaacactt tcctcatcat 9420 gagggattaa agaaataaaa tgatgccatc aaattggaaa tcgaatcaca aagaaaagta 9480 gaaacctacc aaaatagatg gagacataac ttatccctga gaagaacctc cagaagcatt 9540 tgttacattt aacttaccag atggagaatt attcggagca atgggacaag tggaactacg 9600 atggtcttcg ttcttgcaat agcggcagaa gagaggctta ggacaagctc gaatcatatg 9660 atcaggagaa ccacaattga aacacttccg aatggaaggc gttccagcag gctgagactg 9720 agaatcagat tcaacggact ttttcttctt ggaagggaaa aattcccgct catccaatgg 9780 agactgcttt ttgttggggt tcttacgaac ctcaacatca tcaccactat cacaatcctt 9840 ctcgatggtt tcaagatgaa gtgcagaatc taccaactcc tcaaaagaaa gcttaaactg 9900 gagcaataaa gacttcttaa gatatttctt aagaccagaa acaaacttca aattcttctt 9960 ataagaagtg caaatgaatt catgaccata cttagataaa gcctcaaatg atgaaacata 10020 ctcagaaaca gaagacttcc cttacttcaa cacgaggaag cgacgggtca ataaaacttt 10080 ttcagattca ggaacaaact tgagttcgaa tcgtccccga aactgagtcc aaaaaagagt 10140 agactcatca atttcggcta caagaagctc ataccaatca agagcacgct attgtaataa 10200 gctagaagtt acaagaaccc tatcatgatc agaaagatcc atgcgagaga aaatacgctc 10260 gcacgtgcga agccaagctt ggacttcaag aacagactcg gagcctttaa acggctttcc 10320 accaagatga gaaaaatcac ttcacaggcg tgcaaaagta gacttgcgag gtggagatac 10380 acgagtagga gctggaggct gaatgctcct aaacaactcc ataaactgat cggcagacat 10440 ataaacgcca tccggatgtg agccactgga ttgcccagca tccgaacgtc gtcccctgcc 10500 tcttccagcc atacgttgaa agttaagata agaacaagat tattcacaag gcagatgatt 10560 caatatggaa tactacagga tttaaatgca ttcctagacg tattcccaca ttctatcaac 10620 caccaagaca caaaagaaat atagaggcat gatgattcaa agatttgtat cacttataca 10680 ctttgacgta ccggaatatg tgcgtctatt ctgtgtttat catattttgt tctaaacctc 10740 gctctgatac caagctgtga cgcctcacct ctatacccac gctagccaag gacactacag 10800 ggtaatgccg tcaactaaca cggagaataa ggctggttag gagcgaccat ctatagcctt 10860 gctctaacta tctattgaat acatcgacaa tagacatacc aagcaggtcg acgtacctaa 10920 tgaatactta aaacaaaacc ataggtggtt ggaggatcta tcagaacata atataacaac 10980 tcaaatgtaa tatacatcac agaacagtag atcatcatat acacatatat tgatacataa 11040 cagagtttac aaactgatac acgtcaagtg atatagcaaa ccacaataaa caggcaaatc 11100 caaaagaata catatatcag agttacaaca aaaaggacta cacataaagc tttaacaaaa 11160 aggagcaagc tgaatagtcc atgcggaagc aaaatagcta agtccaaaag cacctaacct 11220 gaaacatctc ggtctgaccc gagtataata cacgggtcag tctagctaaa caaatcagtt 11280 agataaactg aacaacaata acctgcaaag tcacaaagta taacaaatat ctctaaaaga 11340 gataacacaa tcatctaaca aaaagatgaa tcatatcata acaagcctcg tatcatcggc 11400 aactcataga aacaaaggat aggaatatcc caaaacaagc cataaaggca aataacaaat 11460 actagtgaag gtaacacaag ttccacaagg taatcgctat atatcgtgcg gctcaaaaag 11520 accaacaccg aagagcgata cctagggaat atacatatca atgaaacatc ataaaatttt 11580 aattgcaaca aacccaaata tctcatgagc ggtggtgaag aaaccagtcc aatatctctc 11640 gagcggtggt gaagaaaaac cactccgaaa gcaatactaa aggtaaccga caaccccaaa 11700 tatctctcga gccggggcaa aggaccaact ccaaaagcga tatttaaggc aacaagccag 11760 aaggagacag tccaaagaca ctatcccaaa ggcaacatgt gtcacaaact ctcagaaagg 11820 aatccgaaaa gtggtaacgt gagtctcata accacccgtt cgcactaaac ggtttcttat 11880 ccgttagtta atttcacact caaagcagta caaagtttac ctcatctcat cggtaaacca 11940 gacacaaagg tcaaaacgtt caaacgaata tattgggaat taactgagaa tatgaacgca 12000 cgtgttcatt gcacgcgttt tatctaatca tttgattctc ctcccacttc gctcggagtg 12060 cgaaaccgtg tgtgttttaa atagaattta taaattggag aaatgtgctt ttaatgcaac 12120 ttaaagggat tattttataa aaatcccgtg gatgaagtca aaaaggaatc atcaaggtca 12180 aatatgcata aacaactaat tggaatcaac gtgcatcgat aaaagttgga atcacagaca 12240 aataggcaaa gtagaaaaaa caggggaaca ggagcaggtc ggttgaccgg actttgagct 12300 cggtcgaccg gcccaaaaac agagaagtgc tctggtagca ggtcggtcga ccggcctact 12360 gagttcgatc ggccaaaccc tgtttccagc acttcccagc ctatcaaaac gagggccaat 12420 ctcgaccaaa ttgcaatcgt tcgagcaaag ccgccaaggg tttatgccaa tcgactaccc 12480 gacgctatat gctcctcgat gcatcaccaa aaacatgttt aaacatcaaa gttatgatct 12540 ttaaagattt gaacccaaaa cactaaaaat tgggatattc accaattcaa tccaaatcaa 12600 gagcaaatgc acatgaaaaa gtgttaggaa ttcatacctg acaatcaaag atagatataa 12660 gcatagatgt gtgataaaaa tctcaccttt aaccctaaaa aaagaagaag aagacgctca 12720 attgtaagga aaagatcaac cactctccac ttcaactcta ctagcttcaa tctaagatca 12780 aaacaagtat ttcttaagtt aggtttagga aacttctgaa attgaaggag aaagagttgg 12840 agaagatgaa gaggaggctt accaatagct tcttttgatt ttggagcaag aatttgagct 12900 agcaagagaa actagggctt ggagtttcta attccctttc ctttccacca ttttcagtta 12960 gagcttgagt ttgagaagtg ggaatgagtg aatggagagg agagagctga gttagggtat 13020 taagttagct agtgggctga acccaaaccc aaaacccaac tagaaaaagt agcaaaattt 13080 aacttactcc aaacatatca aagcttatat tcaaaaagat tgcaaacgcc attttggact 13140 caatggaggt ggtcttgagt cccaattcga ccgtttgcag aaagtagctt tttggactat 13200 aatccgacaa atgacgttat atttaaataa aatatttaaa tatgcaaccg ataaatatgc 13260 gggttgttac agtaaacttc gccagcaata acagattcaa gaatcaagaa agaagcaaca 13320 acaataaaaa tcagcctaat tttcaaagaa atggtcaatc ctctagatcc agaggaagaa 13380 atggaagata taacaacagc aataacagac tatggtgtca aatttgtgaa aagtttggtc 13440 atgcagccaa cgaatgttac ttcagatttg atgccaacta cattccatct cagcaaaata 13500 acaacagttc aagaaatcac aatctcaatg caaacatggt aaatacttat ccagattcta 13560 aagaacaagc agataccaaa agtacagaga ctgcaaagat tgatgatatc taggatgaaa 13620 gctagtatcc agattatgag gctaccaatc atgtaactaa taatctcaac aatctaaact 13680 tgggaagcag ggagtacaga ggtaggcaat taattcatat gggtaatggt gaatcagtaa 13740 aaattacaca tattggaaat gcaagtttca gtggcagaaa ataactcttt ctaaaaaatc 13800 tgctaagagt tccatctatt agaaaaacct tattgagtgt ttcgcatttt gcccaaggca 13860 atgatgtgta ttttatccca aatactgttt aattagagat attctaacta aggaaataat 13920 tttgcagggt aagaaattta aaggattgta caaattcgat gttgcaaaat caggaaattc 13980 cgaatttcat ggaaaatggt gtaatgttac tgaaattgaa catgaagata actataaaga 14040 ttttgatatt tgacattcca aacttggaca tccaagtgta agcattgtaa aaaaggtgtt 14 100 aagtaatgat aatatttctg ttaaagatgt tgcaatgcct tatgtatgta catattgtca 14160 aatgggcaaa agccataaac ttgcatttcc aatctcaaaa ataaactata caaaaccctt 14220 agattaattg cttgagatct atggggtcca tcttctgtta acactgatta tggctataaa 14280 tactttatat catttgtcga tgcatactct agatatgtat ggatttattt cttgaaatca 14340 aaatctgaaa cttatgatgc agtcttacag tttataactc aagctgagag gcaaacaaat 14400 tgccaattaa aagtcttgca gactgatggt ggaaccgagt ttcaaccact taaggaatac 14460 cttcaaaaga aaggtataat tcacagaaca aactgtccat acacatcaga acaaaatgga 14520 ctggttgaaa gaaaacacag acatattgtt gagactgggc ttactttact agcccaagca 14580 tctcttcctc taaagttttg accagatgcc tttgccacta ctgtgttttt aataaataga 14640 ttatccataa aagttttaca gttcaaaagt ccagtggaaa tcttatttaa agttaagcca 14700 gaatatcata gtttgaaagt ttttggatgt ttatgctttc cctatttaag gccatataat 14760 acacacaagt taagtttcag gtcatcacca tgtaccttct taggctatgc agccaataag 14820 aagggataca aatgcttgga tagcaatgaa ttttgttttt gtttctatac atgtggtgtt 14880 taatgaaaga atatttccct ttaatcaatc aaacataaaa acatcacaga aagtgtataa 14940 ttcaaaactt tctataccac cagtacctga gcaacttgat gtcataactc aagttgaaga 15000 tgaaagaatg cagcacaata taactttaga gtctcctgtc aaatccactt cacaatacac 15060 tgtgcccaga cctgagccag gtcatgaaga agatgaacaa gctggaagca caagtgaggc 15120 atctcatatg tgccccatct atcagtcaga ctgcagtcag tcattagatg attcacaatc 15180 atctaatgat caacaggaag tcacagctga agagccaagt caatcacctg cacaagctag 15240 acatcacatg ataacaaggg ccaagagtgg tatattcaca ccaaaaatat acagtgttgc 15300 tatgaacaca gaagagccag atacctatca tcaagctatg cagaatgaaa actggatgtc 15360 tgctatggaa gaagaataca atgcattgat aaaaaataaa acatgggatc taacttcatt 15420 gccaagcaat aaaaatgtca taggctgtaa acggacatat agaataaaga gaagtggtga 15480 tggttcggtt tctaaatata aggcaaggct agtagccgaa gggtattcat agcaaagtgg 15540 ctttgacttt actgaaactt tttcaccagt tgtgaaaccc tcaactatta gaattgttct 15600 tactattgcc ttacatacaa actgaaacat caagcaatta gatgtaaata atgccctttt 15660 gtggcatttt acaggaggaa gtctacatgt aacagcctct aggttttgaa caaaaaaggg 15720 gtggaaaact tgtatgcaaa ttgaacaaag cgatctatag actcaaacat gcaccaaggg 15780 tctggtttga aaagctgaag tcaatactaa tcagacttgg ctacagctca acaaaatcat 15840 acaattcttt atttacaaaa tttaaaaaca atgcaaccat gtatgtacta atatatgttg 15900 atgattttat tattacaagc agcagtgaac cagagatgaa taaactcaca gacctcttaa 15960 ataaagaatt ttcaatcaaa gacttgggga acttaaatta ctttcttggc attgaggtta 16020 aaagactttc acacatagaa atgcttctaa gtcaaaggaa atacatctca gaaatcttac 16080 agaaagcaaa aacggataaa gcaaatgcaa tatctactcc tatggcaagc aattcacatc 16140 tgtcaacgta taaaggagaa tccatatcac atggcaagca gtacagaagt attgtaggag 16200 cattacagta tgtaacaatc actaagccag aaatttcatt tagtatcaat aaagtgagtc 16260 aatatatgca aatttcactg gatgaacatt ggaaagcagt taagagaatt ctaagatatc 16320 tcaaaggaac tataaatcat ggattaaaga tgcaatcatg caaatctatc catataaaag 16380 ggtatgcaga tgcagattgg gccacagaca tggatgacag aagatctacc actggttatt 16440 gcatatttct aggcaacaat ccagtttcat ggtgctcaaa aaaacagcct acagtatctc 16500 ggtcaagtac tgaagccgag tacagaaacg ttgctaatgc aacagctgaa ataatatgga 16560 taatgtcctt gctcagtgaa cttcatatca agtttacaga aaaaccgaca atttggtgcg 16620 acaatctgag caccgttctg ccctcagcaa atctagttct acatgctaga accaagcaca 16680 tagaactgga tctgtatttt gtcagagaac aagtaacaga ggaaaaaatt aacataagtc 16740 atctaccatc atcttaccaa atcgctgatg ttatgataaa acctctatca aagaagaact 16800 ttgtttgatt taagcaagaa ttgaaggtta ctgagttcga cagcaacgaa aaaggcaaga 16860 tctatgataa ctcaacaaat tgaagcttga cagcatcgtc tactccaatt gaagagaaat 16920 tccacttgac aatcaagtga aatttcccct gatgaagaaa gcaagtctcg atatacggca 16980 atttaaccaa aatcgaaaca cgacagctta atctctccaa cttcagcaaa aagagggctt 17040 ccgtatcact gcaaggtcct gagtggtaaa tctacacact cgagaaaccg aagccatcat 17100 attttattcg tgcggctgaa gaaattgcaa accctaaccc taagttaggt gtgaggggag 17160 gtatcaaatg aaagtataga gccgcactta tcaaatataa gcatcacaat ataattggaa 17220 cgggttgtga taatggacca tcccgaagcc caattttctt tttataaata tcaagttgta 17280 aacagttcag tgtattatca aaagaacaaa aacattctgt aactacggaa tatttttctt 17340 gttttctgtt tttcaaatat taatcaataa aatcaagatt caaactctct caatattctt 17400 tttacgagct aaaaatatat catattttct gtgtccgaac gctgatattg ttttttactt 17460 tggactctga tacttagcta acattataca cgtgagaaat aggaaactta tatctagtga 17520 aaattaccca tgaatctgac aggttgctga agatataaat ttaatcagac atataaagag 17580 ccttttcgtg gatcattatg attgccatcc aaagcctgcc ctctcagaac agtcttcgtc 17640 taattcaaaa gcacatgcac agcatggccc ctttgatatt caagaaaaca taacaataat 17700 caagccaact cgtaacaacg caaataaaga aaacgaagat gaatgcatag gtgatgaaga 17760 aaatgggaat gatgaaacag aaggagaaac tgatactgaa acatatagca cacccataac 17820 gccagcagat attcaaccaa gcgagcatat gcaaatcgat acatgcgaaa atattccatt 17880 tggttcatct catgagtact attcaaacaa tttggataat gaaccacaga tgttagatag 17940 agtggatgat tcttccagag aatggcacca tttgaatgat gatacatgtg gtggtctacc 18000 agagctatcc ggtacgtacc aactgtgtta taacacataa taattgaagt gtctttcgga 18060 ccctaaccgt gcagggataa atcaataatt tcatatgttt gtacgtagct cctcggccag 18120 ttatatgtag cttgcaataa attcatcttt tacattcacc agttgcataa ttttctcctt 18180 aggttcaagt ttacttcaac agttatctgc agaggattcc cagtactcag aaacagtttc 18240 tacaattttg cataagacct caagtcaatg gactggacct attactcgaa atcatctggt 18300 ttgttctcag caatcagcat tctcaaaatg gaatgatact gatcatatac ttcacatttc 18360 atctgaggat acatcacaga aaactctgaa atatatcttg ctcagtgttc ccaagctata 18420 cagcaaggac aaaacaaaaa agttttcagc atctaaagat agaattactt ctcaagagga 18480 actatgtgct aaccatgtac ttgctgaacg taaacgtagg gagaagctta atgaaaagtt 18540 cattattctt cgatcgttgg ttccttttgt taccaaggta ttgatgactt gtagacatga 18600 cacaagttta aagtaagaaa atacatagag ctcagagtca aaacaacgca tgtttccata 18660 ataatttaat tattatttaa tctgtaatgt ggatcacgat ataagtatat aactgctgaa 18720 taatatatgg tcattgacgc aggtggataa agcttctata ttgggtgaca caattgagta 18780 tataaagcaa ttaacgtgga aaatacaaga actcggatca caaaacaagt ctactgaaag 18840 tgaaaacagt ttcagaccag gcaaagttca aagacaaact actactgcaa aaagcaaggt 18900 taaatcaaat gtgactgcga aatcaataga aaatcatcgc gttcatttgc aagtttcaat 18960 tattgaggca gacgcacttc tcgaaatgca gtgcccatac aaagaagggt tgatactgca 19020 aattatacag acactagata aacttgggct agaaattact tcgattcaat caacatcaaa 19080 caatggatta atgaaggcag aatttagggc taaggcaagt gtattccttc ttatatgcat 19140 ttttggttca atttatgcaa ttaatatgac tacatttgca acttatatgc aggttaaaga 19200 catcggagga agaaaggcca caattgtcaa agtgaagaaa gcaatttacc atattctctc 19260 acagtaattt ataatacata ctgatataac agtgacatta taatatatat atatatatat 19320 atatatatat atatatatat atatatatat atatatatat atatatatat atat 19374 <210> 3 <211> 19915 <212> DNA <213> Allium cepa <400> 3 cggaccggat cgaaactgaa ccgtattaca ttcaaaccga accgaaaccg aaccgtttaa 60 agaattggta taatagtttg tatttttgat taaccgttta atgcggtgcg gaagcggttt 120 ggactccaaa ccgcaccaaa ccgatccgcg ccacccataa gcaaaattag tttgagaatt 180 ttatcgtgtg tactacatat tataatcaaa acaaagttta gaatatgaca acctatcttt 240 attatataag gagtaaaaaa tgacgctata gtagtagatg cttttaagta gaaaggaata 300 ttagttacca ttagtgcagc atacggtctg aaagtcatat gcaattattt cccagcgatt 360 gaagatgtca aatactagat tggcagtttc tggttaaaca ttttcatgga aataattcct 420 aaatttggcc tgctatttat tacagattac cccagcattt atcacaattt ttaggcttac 480 gcaatgcatc tatcagtatc tgcagtaggt agcaagcacc aaatgacaca cccatatata 540 ctaacttttc aattgaagaa ttttgcaacg aatctggaaa tttttgtata aatttatttg 600 agaaatggga tacaaaaggt tcatggaaag atcaaatgtt tttattatga aactttgtta 660 tttgtattat cagttatgtt aagtttaatg gtatttatga agagttctta ctggtgacac 720 actacaccaa ttaccaaatc ctcaatttca ttaacaaaca aataactgat tcgtgtggtt 780 ttataataaa ctataacaat ttacctaata ctctttacgc acgtattctc aatacacata 840 aagtactgag ctttagtgac aaaatagtaa aagagtacat gcgtttaaaa caggcgtcag 900 ttcatacttc atattgttaa tacatgtgat aaatagattt gtcagtttat taccgacctg 960 tagaaccaac cattcaactc ataaacaatc catggcatgt agaatatcac tttctcattc 1020 ctattatttg tttccctcca cgtctattac cgaaccatat caaactgttt catcttatgt 1080 ggacaatatc acaaatgata taaaatgtag gaattcaatt aaaacgttac atgttataac 1140 caaggttcaa aattccggcg gaattccggt tattccggtg attccgttcc ggttccggtc 1200 ataaccggaa cggaatccgg tcaaaattcc ggttccggtc taaattccgg atttaatttt 1260 caaatattga aaaaaaatta taaaaaaata aaaaaattca acaaacatat agcaattaaa 1320 aaagaaaagc aaattaaaca ggacaataaa taaattgaca aaaaatcatg tgtatgaata 1380 aaaaaagttg ttatattttt gttaatttac gtcgaatcat aataaaaatt tcaaaaatcg 1440 accaaaattt taaaatctta aatgagcaat acagactttt taaacaatgg ttttttaatc 1500 aaagtgtatt ttacacgtaa tttaaataat gtactataaa tttatgattg tattgaatat 1560 ttattagcga aatagcaaat taaagcaaaa aagtaccgga attagaaaac cggaattaaa 1620 acggaacttc cgaaatttcc ggaacggaac cgaaatttta aaattaccaa tgaccgaaac 1680 ggaaacggaa aacggaattt caaaccttgg ttataacata aagctttgga tatggcaaag 1740 gactcgagta gtaatagcaa gcttcaggag atcctgcagc tggctgtgca aagtgttcaa 1800 tggacttacg gcattttttg gcagctctgc ccaaatcaaa ggtgtgttgg tttttcttga 1860 aaagtttcca ctaagcgctc aactgaacta attaatatgg atttgaaatg ggtaaggatg 1920 ttaatatggg gagatggata ctacaatgga gcaataaaga caagaaagac agtgcaagga 1980 gttgaaatta gcgcagaaga tgtacctata catagaagcc aacagttaag agagctctac 2040 gagtcacttt caaatgcagg cgacgctgaa caacccagtc ataggtcgtg cacttctttg 2100 tcccccgaag atttgtctga gtatgaatgg ttctacctca tgtgtttctc ctttgctttt 2160 cctccaggtc atgggtatgt ttatagtatc tttgttattt gtaattttga agttttattt 2220 attttcaggt gttcaattta tacctctaat gatttgtttt gttgccgcac tacttaattg 2280 caatcatgat cctcgacatc aaaacattaa accaaagaca cacatgaaat tttgttttcc 2340 aaagtagaaa gatttgacat gaaggatagg tattgattaa ctgttagcaa ccaaaatatt 2400 tgaaaatatt gaattgtata aagattattt ataaaagaat cttaattgtc tgtattaaac 2460 tttaattaac tacattttct gcgaaaagac attctttgat gcctaggagt acgatgcatt 2520 aacttttcga gatgtttaaa tagtgtgatt tgtttccagg catctttttt ccattcctag 2580 aagtatgaga tcctaatatt ttaactcaaa actgaagtat ggattagaaa gttgccattc 2640 aataatatgt aaatgaatat tttgcactac gacaagggat ttggagcagt ttactaagta 2700 cttaattaac gactgaaatc aattagagca agatccgtag agaagtaatg tttgatgaag 2760 agagatacac tcaatactaa gaacgtaagt gttacagtac ctggtctcca attcaagtcc 2820 cagacacata tccgattgaa aatttatcaa gagatgatta atatagtacc gagatctctt 2880 acagaagata catgttaagc ttttaagata tataacatag tagtgtgtat agagaatagt 2940 cttactacgt gataccataa taagtttcct atgttatagt gcaagagtaa tgtggtcgtt 3000 ttactgcata ctgcatatgt gataccataa atccaaatag ctccggtgta aatgctatat 3060 aaggaagtag ttggagatgt ctattacata ggaatatgta cgatgataat aatacacgtt 3120 atatttacaa gtacaggcta ccaggaaaag cacttgaact gcagcagcct gtttggctta 3180 caaaagcaaa tggggcagac agtggaatat tcagcagggc cattcttgca aaggtacctc 3240 aattaaatag aagtatagtt ttcttgtggg cccacaaacc ctacaaatta acagataatt 3300 aagactgatt ttcggtgcaa actattgaaa caaacatttg aataattggt aatatcaagt 3360 gatcaaattg tggaagaaac tatcgcaata aagtaatgtt ggaaatttca agaggatttt 3420 tacgttctga taccaccatt gagaaatttc tatagaaaga agaaagagaa agaagtaact 3480 tctaatttac taatgaaatt gaaagaatat tttgtaaaga aggaaatatg acttattttc 3540 tctttgtact ttttttgctt gtatataatt acattttcca catcaagtga gatagcacat 3600 ctcaagttat aataaagtat attatctggg cttttctatt tctagacttt tttttcaatt 3660 ttcaattttc taaatttttc ttgacaaacc attttaattc tccaaattac atattattct 3720 taattttaac attttctaaa tataaggatt aaatttgtaa cagaaccaat gttttctaaa 3780 aacaaagttt atttcaaaca agtaatggta tagatgcatt gcaactatcc aagtgtgctc 3840 tataaatatc acatcataat agcgactaag caataagttt cggtttcaaa aagtcctttg 3900 tgccatcaag gttgatcatt cagaactgag atagtaaaca aaattgtaca taacaacaag 3960 aacaatcaac aaaagtatct aactatcata gaaccaagaa ctccgtgata tttgtacttt 4020 ttatccttat tctaacttaa tttctcattc ttttgtttgt gatacatcaa tgtgacgaat 4080 taaaccgaat tcggtatgca taatcagagc gcgcacattc aggttatatt ttttctcgaa 4140 accaatctcc ataattttat ttttttgttc aaaagaagaa aacgtctaac cataatgctt 4200 gatgttgcag acagtaatct gcattcctgt aatgcatggg gtatttgaac ttggaacgct 4260 agaaaatgtg agttcaaatt tgttatttcc gccaactatt tttgtcatta cctatttctg 4320 ttaaaggttt aactttagtg aaaaagatct atcagttata cataaagcaa gtaaacttaa 4380 gttacctttt attttcatcc acggtctctt tcacttatcc gcaaatataa ttccattaag 4440 ttattaactc accaattatt ctaaactaat ccgcatgtgg gttagctgac agaataatta 4500 aggtattcta attttcattt atttgcttac tcttttactt tgctgtttat aattctactt 4560 ctattcatga aatacaaaat ctgtacatta gagtcaaaat tatgagaaaa gctactcaaa 4620 tagtcagcta aaaaacaatt gatacaaacc aaacatacta ggtgtcataa tataagattt 4680 tagcagaaag attacacatt tcttacatat ataaaatctc caatccatac accatttaat 4740 gcgactacaa taaaatcaga agatggtcat ttatctttgt aagcctaaat cattttcaga 4800 tttgaactgc agtggaaaag aaagagtgac ttatattaca agaagaaaga agtatacttg 4860 gtggataaac taatcatttt ttcaaaatta tataaaagtt tgaagacgct atttttgtgg 4920 gcggattaga agactaaaaa tgttgattca tgtaaaactt accgacctta aaaatacaac 4980 gagtatcaaa tgaaagtata gagccgcagt tatcgcagtt atcaaatata agcatcacaa 5040 tataattgaa acgggttgtg ataatggacc atcccgaagc ccaattttct ttttataaat 5100 atcaagttgt aaacagttca ctgtattatc aaaagaacaa atacattctg taactacaga 5160 atatttttct tgttcactgt ttttcaaata ttaatcaata aaatcaagat tcaaactctc 5220 tcatattctt tcttggtatc aaagcgtgta cattgcttaa cagcaaaaga aaagttcatc 5280 agaaaatcaa cagagaagaa gagaacagca aaagaaaagc tcatcagaaa atcaacacaa 5340 aaaggaggaa aacacaaatc gtgccatgtc attaacctct caaagaagat ctcatcacag 5400 agattaggct gcacttaaaa tcatcagaaa attaaaattt tcatcatctg attgtgaatc 5460 atcagagaat taacattctc ttcatctgat tgtgaatata tttgtctaac ctgggttttt 5520 gtcaaatctc ctcctctctg ttcaacattt ctgatcactt cactctactg cctcgtcaag 5580 aaatcggttg tcagagacaa tttcatcact caattgattt ctagctgcac ttttctctaa 5640 gtgtattcat tcccacaaat ttttctgtaa aaaattcttt aatcccataa gttcttgaat 5700 atggttgaag gggatattaa caactcagct tcttctgaag caagaacaac caattcagca 5760 tcttaaactg ccaataccca agatctcatc aaagccatca taaagtcaca aagtcaaata 5820 gtcagtgttc aactagatga aaacaattac ttgctctgga aattccagat agaaacagca 5880 gtgagaggct atggccttga ggatcatgtt cttggaacat taattacacc atcaaaatac 5940 actgcagata aagatgacaa attagtgagt aatcaagatt atgttacaca ccaatggaaa 6000 gacagtctaa ttagtgcttg gttactgtct tttataagtg caaacattct accatagcta 6060 attgagtgca agagtgcaca tgaaatatag tcaacagtgg agcagatctt taacacacag 6120 tctgctgcaa aaatcatgta ttacaaaaga caactacaaa gcataagaaa agaaaacatg 6180 tcaatgagag agtacatgac caagatcaag acactgtgtg atcaacttgc atcaatgggt 6240 cataaaatct cagacactgg gcaagtctta tgcattctag gtggtttaaa tgaagaatat 6300 gaggcagtgg tagcagttat ttcctcgaag gaaacaacac caaatataca atatgttcac 6360 tcaactctac tagcccatga aggaagaata gaacaaagaa gatcaacaat ttcatatctt 6420 attgtaaatg tgacgcccca cctctatact cacgctagtc aaggacacta caggataata 6480 ccgtcaacta acatggagaa taaggctgat taggagcgac catctatatc cttgctctaa 6540 ctatctattg aatacatcaa taatagacat accaagcagg tcgacatacc caattaatac 6600 ttaaaacaaa accataggtg gttgaaggaa ctatcagaac ataatataac aactcaaatg 6660 taatatacat cacagaacag tagatcatca tatacacata tattgataca taacagaatt 6720 tacaaactga tacacgtcaa gtggtatagc aaaccacaat aaacaggcaa atccaaaaga 6780 acgcatatat cagagttaca acaaaaagga ctacacataa agctttaaca aataggagca 6840 agctgaatag tccatgcaga agcaaaatag gctaagtcca aaagcaccta acctgaaaca 6900 tctcggtctg acccgagtat aatacacgag tcagtctagc taaacaaatc agttagataa 6960 actgaacaac aataacctgc aaagttacaa agtataacaa atatctctaa aagagataac 7020 acaatcatct aacaaaaaga tgaatcatat cataacaagc ctcgtatcat cggcaactca 7080 gagaaacaaa ggataggaat atcccaaaac aagccataaa gacaaataac aaataccaat 7140 gaaggtaaca gaagttcctc aaggtaatcg ctatatattg tgcggctcaa aaagaccaac 7200 accgaagagt gatacctagg gaatatacat atcaatgaaa catcagaaaa ttttaattgc 7260 aacaaaccca aatatatcat gagcggtggt gaagaaacca ctccaatatc tctcgagcgg 7320 tggtgaagaa aaaccactcc gaaagcaata ctaaaggtaa ccgacaaccc caaatatctc 7380 tcgagccggg gcaacggacc aactccaaaa gcgatattaa aggcaacaag ccagaaggag 7440 atagtctgaa gacactatcc caaaggcaac atgtgtcaca aactcccaga aaggaatccg 7500 aaaagtggta acgtgagtct cataaccacc cgttcgcatt aaacggttta ctatccgtta 7560 gttaatttca cactcaaagc agtacaaagt ttacctcatc tcatcggtaa accagacaca 7620 aaggtcaaaa cgttcaaacg aatagattga gaattaactg agaagatgaa cgcacgtgtt 7680 cattgcacgt gttttatcta atcatttgat tctcctccca cttcgctcgg agtgcgaaac 7740 cgtgtgtgtt ttaaatagaa tttataaatt ggagaaacga gcttttaatg caatttaaag 7800 ggattatttt ataaaaaacc cgtggatgaa gtcaaaaagg tatcatcaag gtcaaatatg 7860 caaaaacaac taattggaat caatgtggat cgataaaagt tggaatcaca gacaaatagg 7920 caaagtagga aaaacagggg aacatgagca ggtcggttga ccggactctg agctcggtcg 7980 accggcccaa aaacagagaa gtgctctggt agcaggtcgg tcaccggcct actgagttcg 8040 atcggccgaa ccctgtttcc agcacttccc agcctatcaa aacgagggcc aatctcgacc 8100 aaattgcaat cgttcgagca acgacgccaa gggtttatgt caatcgacta cccgatgcta 8160 tatgctcctc gatgcatcac caaaaacatg tttaaacatc aaagttatga tctttaaaga 8220 tttgaaccca aaacactaaa aattgggata ttcaccaatt caatccaaat caagagcaaa 8280 tgcacatgaa aaagtgttag gaattcatat ctgacaatca aagatagata taagcataga 8340 tgtaagataa aaatcccacc tttaacccta aaaaaaagaa gaagaagaag ctcaattgca 8400 aggaaaagat caaccactct ctacttcaac tcgactagct tcaatctaag atcaaaacaa 8460 gtatttccta agttaggttt aggaaacttc tgaaattgaa ggagaaagag ttggagaaga 8520 tgaagaggag gcttaccaat agcttctttt gattttggag caagaatttg agctagcaag 8580 agaaactaga gcttagagtt tctaattccc tttcctttcc accattttca gttagagctt 8640 gagtttgagg agtgggaatg agtgaatgga gaggagagag ctgagttagg gtattaagtt 8700 agctagtggg ttgaacccaa acccaaaacc caactagaaa aagtagcaaa attcaactta 8760 ctccaaacat atcaaagctt atactcaaaa agatcacaaa gccattttgg actcaatgga 8820 tgtggtcttg agtcccaatt cgaccgtttg cagaaagtag ccttttggac tataatccga 8880 caaacgacgt tatatttaag taaaatattt aaatatacaa ccgataaata tgcgggttgt 8940 tacagactcc cctccttaaa aaaattgcgt cctcgcaatt cttaagaaga aaataaagag 9000 taacaaccca aaaacatatc aacacataag aataacagaa taagaagttc accataaagt 9060 gcacgaaagg ataagcccta acttgtttgc tcatagaatt ttgtggattc ctaagctcaa 9120 aataattcgg aacaccgaca tggcgcaaca atacgtcaaa tgaaccaaag ctcaacaaaa 9180 tgaagtacaa caaaacgtcc gatgaacttg gtaaaaagat cattccattc ggacatcaac 9240 aaagaaacaa tcacttgttg cttacaacta aatgcattgg caacacgatc cccctttcag 9300 gaataaaaag aatgagagta ttggaaaaga gttcttttaa ataatcaact ttcccgaaaa 9360 gctaaaactt tcaaaatcac aattatcttt atgtaattat ttttctagaa aatttttctt 9420 atttcaaatc aattcactgt gtctgattgt ctcatcaaag ctttaggtga ttcctatcga 9480 taacgttgat taagcgcatc gaccagaatt cttaaagcac ttaatgggca atacaatcac 9540 aaaacaatct tctatatgat aaacatattt ctaaaaactt ttaaaaatta tctttgtttt 9600 ttttaagaat aaaaatccca tttatactta taaataaatc ttatgaaaaa gatttgcaaa 9660 aattttgaat tgctaatttc catccaaaac caataaatgc taaaatggaa tcatcgagat 9720 attcacaaaa tactcgattc atacaatgga tgaaatgagc tagacaaact gaatgacata 9780 ataaatcatt cataagaatc acaacgagtg cggaatgcgg taagtggagt gctaacttcc 9840 gcaactcgta gccaaatcaa aaccggtggc aaggtcgagc tgaaaagacc ttaataccac 9900 tcaaaggatt gcttaggaga taatcctaag actagaccca aaataccaga aaattctcga 9960 actataagaa tgtgagaaac aacactttcc tcatcatgag ggattaaaga aataaaatga 10020 tgccatcaaa ttggaaatcg aatcacaaag aaaagtagaa acctaccaaa atagatggag 10080 acataactta tccctgagaa gaacctccag aagcatttgt tacatttaac ttaccagatg 10140 gagaattatt cggagcaatg ggacaagtgg aactacgatg gtcttcgttc ttgcaatagc 10200 ggcagaagag aggcttagga caagctcgaa tcatatgatc aggagaacca caattgaaac 10260 acttccgaat ggaaggcgtt ccagcaggct gagactgaga atcagattca acggactttt 10320 tcttcttgga agggaaaaat tcccgctcat ccaatggaga ctgctttttg ttggggttct 10380 tacgaacctc aacatcatca ccactatcac aatccttctc gatggtttca agatgaagtg 10440 cagaatctac caactcctca aaagaaagct taaactggag caataaagac ttcttaagat 10500 atttcttaag accagaaaca aacttcaaat tcttcttata agaagtgcaa atgaattcat 10560 gaccatactt agataaagcc tcaaatgatg aaacatactc agaaacagaa gacttccctt 10620 acttcaacac gaggaagcga cgggtcaata aaactttttc agattcagga acaaacttga 10680 gttcgaatcg tccccgaaac tgagtccaaa aaagagtaga ctcatcaatt tcggctacaa 10740 gaagctcata ccaatcaaga gcacgctatt gtaataagct agaagttaca agaaccctat 10800 catgatcaga aagatccatg cgagagaaaa tacgctcgca cgtgcgaagc caagcttgga 10860 cttcaagaac agactcggag cctttaaacg gctttccacc aagatgagaa aaatcacttc 10920 acaggcgtgc aaaagtagac ttgcgaggtg gagatacacg agtaggagct ggaggctgaa 10980 tgctcctaaa caactccata aactgatcgg cagacatata aacgccatcc ggatgtgagc 11040 cactggattg cccagcatcc gaacgtcgtc ccctgcctct tccagccata cgttgaaagt 11100 taagataaga acaagattat tcacaaggca gatgattcaa tatggaatac tacaggattt 11160 aaatgcattc ctagacgtat tcccacattc tatcaaccac caagacacaa aagaaatata 11220 gaggcatgat gattcaaaga tttgtatcac ttatacactt tgacgtaccg gaatatgtgc 11280 gtctattctg tgtttatcat attttgttct aaacctcgct ctgataccaa gctgtgacgc 11340 ctcacctcta tacccacgct agccaaggac actacagggt aatgccgtca actaacacgg 11400 agaataaggc tggttaggag cgaccatcta tagccttgct ctaactatct attgaataca 11460 tcgacaatag acataccaag caggtcgacg tacctaatga atacttaaaa caaaaccata 11520 ggtggttgga ggatctatca gaacataata taacaactca aatgtaatat acatcacaga 11580 acagtagatc atcatataca catatattga tacataacag agtttacaaa ctgatacacg 11640 tcaagtgata tagcaaacca caataaacag gcaaatccaa aagaatacat atatcagagt 11700 tacaacaaaa aggactacac ataaagcttt aacaaaaagg agcaagctga atagtccatg 11760 cggaagcaaa atagctaagt ccaaaagcac ctaacctgaa acatctcggt ctgacccgag 11820 tataatacac gggtcagtct agctaaacaa atcagttaga taaactgaac aacaataacc 11880 tgcaaagtca caaagtataa caaatatctc taaaagagat aacacaatca tctaacaaaa 11940 agatgaatca tatcataaca agcctcgtat catcggcaac tcatagaaac aaaggatagg 12000 aatatcccaa aacaagccat aaaggcaaat aacaaatact agtgaaggta acacaagttc 12060 cacaaggtaa tcgctatata tcgtgcggct caaaaagacc aacaccgaag agcgatacct 12120 agggaatata catatcaatg aaacatcata aaattttaat tgcaacaaac ccaaatatct 12180 catgagcggt ggtgaagaaa ccagtccaat atctctcgag cggtggtgaa gaaaaaccac 12240 tccgaaagca atactaaagg taaccgacaa ccccaaatat ctctcgagcc ggggcaaagg 12300 accaactcca aaagcgatat ttaaggcaac aagccagaag gagacagtcc aaagacacta 12360 tcccaaaggc aacatgtgtc acaaactctc agaaaggaat ccgaaaagtg gtaacgtgag 12420 tctcataacc acccgttcgc actaaacggt ttcttatccg ttagttaatt tcacactcaa 12480 agcagtacaa agtttacctc atctcatcgg taaaccagac acaaaggtca aaacgttcaa 12540 acgaatatat tgggaattaa ctgagaatat gaacgcacgt gttcattgca cgcgttttat 12600 ctaatcattt gattctcctc ccacttcgct cggagtgcga aaccgtgtgt gttttaaata 12660 gaatttataa attggagaaa tgtgctttta atgcaactta aagggattat tttataaaaa 12720 tcccgtggat gaagtcaaaa aggaatcatc aaggtcaaat atgcataaac aactaattgg 12780 aatcaacgtg catcgataaa agttggaatc acagacaaat aggcaaagta gaaaaaacag 12840 gggaacagga gcaggtcggt tgaccggact ttgagctcgg tcgaccggcc caaaaacaga 12900 gaagtgctct ggtagcaggt cggtcgaccg gcctactgag ttcgatcggc caaaccctgt 12960 ttccagcact tcccagccta tcaaaacgag ggccaatctc gaccaaattg caatcgttcg 13020 agcaaagccg ccaagggttt atgccaatcg actacccgac gctatatgct cctcgatgca 13080 tcaccaaaaa catgtttaaa catcaaagtt atgatcttta aagatttgaa cccaaaacac 13 140 taaaaattgg gatattcacc aattcaatcc aaatcaagag caaatgcaca tgaaaaagtg 13200 ttaggaattc atacctgaca atcaaagata gatataagca tagatgtgtg ataaaaatct 13260 cacctttaac cctaaaaaaa gaagaagaag acgctcaatt gtaaggaaaa gatcaaccac 13320 tctccacttc aactctacta gcttcaatct aagatcaaaa caagtatttc ttaagttagg 13380 tttaggaaac ttctgaaatt gaaggagaaa gagttggaga agatgaagag gaggcttacc 13440 aatagcttct tttgattttg gagcaagaat ttgagctagc aagagaaact agggcttgga 13500 gtttctaatt ccctttcctt tccaccattt tcagttagag cttgagtttg agaagtggga 13560 atgagtgaat ggagaggaga gagctgagtt agggtattaa gttagctagt gggctgaacc 13620 caaacccaaa acccaactag aaaaagtagc aaaatttaac ttactccaaa catatcaaag 13680 cttatattca aaaagattgc aaacgccatt ttggactcaa tggaggtggt cttgagtccc 13740 aattcgaccg tttgcagaaa gtagcttttt ggactataat ccgacaaatg acgttatatt 13800 taaataaaat atttaaatat gcaaccgata aatatgcggg ttgttacagt aaacttcgcc 13860 agcaataaca gattcaagaa tcaagaaaga agcaacaaca ataaaaatca gcctaatttt 13920 caaagaaatg gtcaatcctc tagatccaga ggaagaaatg gaagatataa caacagcaat 13980 aacagactat ggtgtcaaat ttgtgaaaag tttggtcatg cagccaacga atgttacttc 14040 agatttgatg ccaactacat tccatctcag caaaataaca acagttcaag aaatcacaat 14100 ctcaatgcaa acatggtaaa tacttatcca gattctaaag aacaagcaga taccaaaagt 14160 acagagactg caaagattga tgatatctag gatgaaagct agtatccaga ttatgaggct 14220 accaatcatg taactaataa tctcaacaat ctaaacttgg gaagcaggga gtacagaggt 14280 aggcaattaa ttcatatggg taatggtgaa tcagtaaaaa ttacacatat tggaaatgca 14340 agtttcagtg gcagaaaata actctttcta aaaaatctgc taagagttcc atctattaga 14400 aaaaccttat tgagtgtttc gcattttgcc caaggcaatg atgtgtattt tatcccaaat 14460 actgtttaat tagagatatt ctaactaagg aaataatttt gcagggtaag aaatttaaag 14520 gattgtacaa attcgatgtt gcaaaatcag gaaattccga atttcatgga aaatggtgta 14580 atgttactga aattgaacat gaagataact ataaagattt tgatatttga cattccaaac 14640 ttggacatcc aagtgtaagc attgtaaaaa aggtgttaag taatgataat atttctgtta 14700 aagatgttgc aatgccttat gtatgtacat attgtcaaat gggcaaaagc cataaacttg 14760 catttccaat ctcaaaaata aactatacaa aacccttaga ttaattgctt gagatctatg 14820 gggtccatct tctgttaaca ctgattatgg ctataaatac tttatatcat ttgtcgatgc 14880 atactctaga tatgtatgga tttatttctt gaaatcaaaa tctgaaactt atgatgcagt 14940 cttacagttt ataactcaag ctgagaggca aacaaattgc caattaaaag tcttgcagac 15000 tgatggtgga accgagtttc aaccacttaa ggaatacctt caaaagaaag gtataattca 15060 cagaacaaac tgtccataca catcagaaca aaatggactg gttgaaagaa aacacagaca 15120 tattgttgag actgggctta ctttactagc ccaagcatct cttcctctaa agttttgacc 15180 agatgccttt gccactactg tgtttttaat aaatagatta tccataaaag ttttacagtt 15240 caaaagtcca gtggaaatct tatttaaagt taagccagaa tatcatagtt tgaaagtttt 15300 tggatgttta tgctttccct atttaaggcc atataataca cacaagttaa gtttcaggtc 15360 atcaccatgt accttcttag gctatgcagc caataagaag ggatacaaat gcttggatag 15420 caatgaattt tgtttttgtt tctatacatg tggtgtttaa tgaaagaata tttcccttta 15480 atcaatcaaa cataaaaaca tcacagaaag tgtataattc aaaactttct ataccaccag 15540 tacctgagca acttgatgtc ataactcaag ttgaagatga aagaatgcag cacaatataa 15600 ctttagagtc tcctgtcaaa tccacttcac aatacactgt gcccagacct gagccaggtc 15660 atgaagaaga tgaacaagct ggaagcacaa gtgaggcatc tcatatgtgc cccatctatc 15720 agtcagactg cagtcagtca ttagatgatt cacaatcatc taatgatcaa caggaagtca 15780 cagctgaaga gccaagtcaa tcacctgcac aagctagaca tcacatgata acaagggcca 15840 agagtggtat attcacacca aaaatataca gtgttgctat gaacacagaa gagccagata 15900 cctatcatca agctatgcag aatgaaaact ggatgtctgc tatggaagaa gaatacaatg 15960 cattgataaa aaataaaaca tgggatctaa cttcattgcc aagcaataaa aatgtcatag 16020 gctgtaaacg gacatataga ataaagagaa gtggtgatgg ttcggtttct aaatataagg 16080 caaggctagt agccgaaggg tattcatagc aaagtggctt tgactttact gaaacttttt 16140 caccagttgt gaaaccctca actattagaa ttgttcttac tattgcctta catacaaact 16200 gaaacatcaa gcaattagat gtaaataatg cccttttgtg gcattttaca ggaggaagtc 16260 tacatgtaac agcctctagg ttttgaacaa aaaaggggtg gaaaacttgt atgcaaattg 16320 aacaaagcga tctatagact caaacatgca ccaagggtct ggtttgaaaa gctgaagtca 16380 atactaatca gacttggcta cagctcaaca aaatcataca attctttatt tacaaaattt 16440 aaaaacaatg caaccatgta tgtactaata tatgttgatg attttattat tacaagcagc 16500 agtgaaccag agatgaataa actcacagac ctcttaaata aagaattttc aatcaaagac 16560 ttggggaact taaattactt tcttggcatt gaggttaaaa gactttcaca catagaaatg 16620 cttctaagtc aaaggaaata catctcagaa atcttacaga aagcaaaaac ggataaagca 16680 aatgcaatat ctactcctat ggcaagcaat tcacatctgt caacgtataa aggagaatcc 16740 atatcacatg gcaagcagta cagaagtatt gtaggagcat tacagtatgt aacaatcact 16800 aagccagaaa tttcatttag tatcaataaa gtgagtcaat atatgcaaat ttcactggat 16860 gaacattgga aagcagttaa gagaattcta agatatctca aaggaactat aaatcatgga 16920 ttaaagatgc aatcatgcaa atctatccat ataaaagggt atgcagatgc agattgggcc 16980 acagacatgg atgacagaag atctaccact ggttattgca tatttctagg caacaatcca 17040 gtttcatggt gctcaaaaaa acagcctaca gtatctcggt caagtactga agccgagtac 17100 agaaacgttg ctaatgcaac agctgaaata atatggataa tgtccttgct cagtgaactt 17160 catatcaagt ttacagaaaa accgacaatt tggtgcgaca atctgagcac cgttctgccc 17220 tcagcaaatc tagttctaca tgctagaacc aagcacatag aactggatct gtattttgtc 17280 agagaacaag taacagagga aaaaattaac ataagtcatc taccatcatc ttaccaaatc 17340 gctgatgtta tgataaaacc tctatcaaag aagaactttg tttgatttaa gcaagaattg 17400 aaggttactg agttcgacag caacgaaaaa ggcaagatct atgataactc aacaaattga 17460 agcttgacag catcgtctac tccaattgaa gagaaattcc acttgacaat caagtgaaat 17520 ttcccctgat gaagaaagca agtctcgata tacggcaatt taaccaaaat cgaaacacga 17580 cagcttaatc tctccaactt cagcaaaaag agggcttccg tatcactgca aggtcctgag 17640 tggtaaatct acacactcga gaaaccgaag ccatcatatt ttattcgtgc ggctgaagaa 17700 attgcaaacc ctaaccctaa gttaggtgtg aggggaggta tcaaatgaaa gtatagagcc 17760 gcacttatca aatataagca tcacaatata attggaacgg gttgtgataa tggaccatcc 17820 cgaagcccaa ttttcttttt ataaatatca agttgtaaac agttcagtgt attatcaaaa 17880 gaacaaaaac attctgtaac tacggaatat ttttcttgtt ttctgttttt caaatattaa 17940 tcaataaaat caagattcaa actctctcaa tattcttttt acgagctaaa aatatatcat 18000 attttctgtg tccgaacgct gatattgttt tttactttgg actctgatac ttagctaaca 18060 ttatacacgt gagaaatagg aaacttatat ctagtgaaaa ttacccatga atctgacagg 18120 ttgctgaaga tataaattta atcagacata taaagagcct tttcgtggat cattatgatt 18180 gccatccaaa gcctgccctc tcagaacagt cttcgtctaa ttcaaaagca catgcacagc 18240 atggcccctt tgatattcaa gaaaacataa caataatcaa gccaactcgt aacaacgcaa 18300 ataaagaaaa cgaagatgaa tgcataggtg atgaagaaaa tgggaatgat gaaacagaag 18360 gagaaactga tactgaaaca tatagcacac ccataacgcc agcagatatt caaccaagcg 18420 agcatatgca aatcgataca tgcgaaaata ttccatttgg ttcatctcat gagtactatt 18480 caaacaattt ggataatgaa ccacagatgt tagatagagt ggatgattct tccagagaat 18540 ggcaccattt gaatgatgat acatgtggtg gtctaccaga gctatccggt acgtaccaac 18600 tgtgttataa cacataataa ttgaagtgtc tttcggaccc taaccgtgca gggataaatc 18660 aataatttca tatgtttgta cgtagctcct cggccagtta tatgtagctt gcaataaatt 18720 catcttttac attcaccagt tgcataattt tctccttagg ttcaagttta cttcaacagt 18780 tatctgcaga ggattcccag tactcagaaa cagtttctac aattttgcat aagacctcaa 18840 gtcaatggac tggacctatt actcgaaatc atctggtttg ttctcagcaa tcagcattct 18900 caaaatggaa tgatactgat catatacttc acatttcatc tgaggataca tcacagaaaa 18960 ctctgaaata tatcttgctc agtgttccca agctatacag caaggacaaa acaaaaaagt 19020 tttcagcatc taaagataga attacttctc aagaggaact atgtgctaac catgtacttg 19080 ctgaacgtaa acgtagggag aagcttaatg aaaagttcat tattcttcga tcgttggttc 19140 cttttgttac caaggtattg atgacttgta gacatgacac aagtttaaag taagaaaata 19200 catagagctc agagtcaaaa caacgcatgt ttccataata atttaattat tatttaatct 19260 gtaatgtgga tcacgatata agtatataac tgctgaataa tatatggtca ttgacgcagg 19320 tggataaagc ttctatattg ggtgacacaa ttgagtatat aaagcaatta acgtggaaaa 19380 tacaagaact cggatcacaa aacaagtcta ctgaaagtga aaacagtttc agaccaggca 19440 aagttcaaag acaaactact actgcaaaaa gcaaggttaa atcaaatgtg actgcgaaat 19500 caatagaaaa tcatcgcgtt catttgcaag tttcaattat tgaggcagac gcacttctcg 19560 aaatgcagtg cccatacaaa gaagggttga tactgcaaat tatacagaca ctagataaac 19620 ttgggctaga aattacttcg attcaatcaa catcaaacaa tggattaatg aaggcagaat 19680 ttagggctaa ggcaagtgta ttccttctta tatgcatttt tggttcaatt tatgcaatta 19740 atatgactac atttgcaact tatatgcagg ttaaagacat cggaggaaga aaggccacaa 19800 ttgtcaaagt gaagaaagca atttaccata ttctctcaca gtaatttata atacatactg 19860 atataacagt gacattataa tatatatata tatatatata tatatatata tatat 19915 <210> 4 <211> 713 <212> DNA <213> Allium cepa <400> 4 acatgttata accaaggttc aaaattccgg cggaattccg gttattccgg tgattccgtt 60 ccggttccgg tcataaccgg aacggaatcc ggtcaaaatt ccggttccgg tctaaattcc 120 ggatttaatt ttcaaatatt gaaaaaaaat tataaaaaaa taaaaaaatt caacaaacat 180 atagcaatta aaaaagaaaa gcaaattaaa caggacaata aataaattga caaaaaatca 240 tgtgtatgaa taaaaaaagt tgttatattt ttgttaattt acgtcgaatc ataataaaaa 300 tttcaaaaat cgaccaaaat tttaaaatct taaatgagca atacagactt tttaaacaat 360 ggttttttaa tcaaagtgta ttttacacgt aatttaaata atgtactata aatttatgat 420 tgtattgaat atttattagc gaaatagcaa attaaagcaa aaaagtaccg gaattagaaa 480 accggaatta aaacggaact tccgaaattt ccggaacgga accgaaattt taaaattacc 540 aatgaccgaa acggaaacgg aaaacggaat ttcaaacctt ggttataaca taaagctttg 600 gatatggcaa aggactcgag tagtaatagc aagcttcagg agatcctgca gctggctgtg 660 caaagtgttc aatggactta cggcattttt tggcagctct gcccaaatca aag 713 <210> 5 <211> 21 <212> DNA <213> Allium cepa <400> 5 tgggatacaa aaggttcatg g 21 <210> 6 <211> 21 <212> DNA <213> Allium cepa <400> 6 tgggatagaa aaggttcatg g 21 <210> 7 <211> 23 <212> DNA <213> Allium cepa <400> 7 aaaatttcaa aaatcgacca aaa 23 <210> 8 <211> 20 <212> DNA <213> Allium cepa <400> 8 tgccgtaagt ccattgaaca 20 <210> 9 <211> 13852 <212> DNA <213> Allium cepa <400> 9 gtgagttcaa atttgttatt tccgccaact atttttgtca ttacctattt ctgttaaagg 60 tttaacttta gtgaaaaaga tctatcagtt atacataaag caagtaaact taagttacct 120 tttattttca tccacggtct ctttcactta tccgcaaata taattccatt aagttattaa 180 ctcaccaatt attctaaact aatccgcatg tgggttagct gacagaataa ttaaggtatt 240 ctaattttca tttatttgct tactctttta ctttgctgtt tataattcta cttctattca 300 tgaaatacaa aatctgtaca ttagagtcaa aattatgaga aaagctactc aaatagtcag 360 ctaaaaaaca attgatacaa accaaacata ctaggtgtca taatataaga ttttagcaga 420 aagattacac atttcttaca tatataaaat ctccaatcca tacaccattt aatgcgacta 480 caataaaatc agaagatggt catttatctt tgtaagccta aatcattttc agatttgaac 540 tgcagtggaa aagaaagagt gacttatatt acaagaagaa agaagtatac ttggtggata 600 aactaatcat tttttcaaaa ttatataaaa gtttgaagac gctatttttg tgggcggatt 660 agaagactaa aaatgttgat tcatgtaaaa cttaccgacc ttaaaaatac aacgagtatc 720 aaatgaaagt atagagccgc agttatcgca gttatcaaat ataagcatca caatataatt 780 gaaacgggtt gtgataatgg accatcccga agcccaattt tctttttata aatatcaagt 840 tgtaaacagt tcactgtatt atcaaaagaa caaatacatt ctgtaactac agaatatttt 900 tcttgttcac tgtttttcaa atattaatca ataaaatcaa gattcaaact ctctcatatt 960 ctttcttggt atcaaagcgt gtacattgct taacagcaaa agaaaagttc atcagaaaat 1020 caacagagaa gaagagaaca gcaaaagaaa agctcatcag aaaatcaaca caaaaaggag 1080 gaaaacacaa atcgtgccat gtcattaacc tctcaaagaa gatctcatca cagagattag 1140 gctgcactta aaatcatcag aaaattaaaa ttttcatcat ctgattgtga atcatcagag 1200 aattaacatt ctcttcatct gattgtgaat atatttgtct aacctgggtt tttgtcaaat 1260 ctcctcctct ctgttcaaca tttctgatca cttcactcta ctgcctcgtc aagaaatcgg 1320 ttgtcagaga caatttcatc actcaattga tttctggctg cacttttctc taagtgtatt 1380 cattcccaca aatttttctg taaaaaattc tttaatccca taagttcttg aatatggttg 1440 aaggggatat taacaactca gcttcttctg aagcaagaac aaccaattca gcatcttaaa 1500 ctgccaatac ccaagatctc atcaaagcca tcataaagtc acaaagtcaa atagtcagtg 1560 ttcaactaga tgaaaacaat tacttgctct ggaaattcca gatagaaaca gcagtgagag 1620 gctatggcct tgaggatcat gttcttggaa cattaattac accatcaaaa tacactgcag 1680 ataaagatga caaattagtg agtaatcaag attatgttac acaccaatgg aaagacagtc 1740 taattagtgc ttggttactg tcttttataa gtgcaaacat tctaccatag ctaattgagt 1800 gcaagagtgc acatgaaata tagtcaacag tggagcagat ctttaacaca cagtctgctg 1860 caaaaatcat gtattacaaa agacaactac aaagcataag aaaagaaaac atgtcaatga 1920 gagagtacat gaccaagatc aagacactgt gtgatcaact tgcatcaatg ggtcataaaa 1980 tctcagacac tgggcaagtc ttatgcattc taggtggttt aaatgaagaa tatgaggcag 2040 tggtagcagt tatttcctcg aaggaaacaa caccaaatat acaatatgtt cactcaactc 2100 tactagccca tgaaggaaga atagaacaaa gaagatcaac aatttcatat cttattgtaa 2160 atgtgacgcc ccacctctat actcacgcta gtcaaggaca ctacaggata ataccgtcaa 2220 ctaacatgga gaataaggct gattaggagc gaccatctat atccttgctc taactatcta 2280 ttgaatacat caataataga cataccaagc aggtcgacat acccaattaa tacttaaaac 2340 aaaaccatag gtggttgaag gaactatcag aacataatat aacaactcaa atgtaatata 2400 catcacagaa cagtagatca tcatatacac atatattgat acataacaga atttacaaac 2460 tgatacacgt caagtggtat agcaaaccac aataaacagg caaatccaaa agaacgcata 2520 tatcagagtt acaacaaaaa ggactacaca taaagcttta acaaatagga gcaagctgaa 2580 tagtccatgc agaagcaaaa taggctaagt ccaaaagcac ctaacctgaa acatctcggt 2640 ctgacccgag tataatacac gagtcagtct agctaaacaa atcagttaga taaactgaac 2700 aacaataacc tgcaaagtta caaagtataa caaatatctc taaaagagat aacacaatca 2760 tctaacaaaa agatgaatca tatcataaca agcctcgtat catcggcaac tcagagaaac 2820 aaaggatagg aatatcccaa aacaagccat aaagacaaat aacaaatacc aatgaaggta 2880 acagaagttc ctcaaggtaa tcgctatata ttgtgcggct caaaaagacc aacaccgaag 2940 agtgatacct agggaatata catatcaatg aaacatcaga aaattttaat tgcaacaaac 3000 ccaaatatat catgagcggt ggtgaagaaa ccactccaat atctctcgag cggtggtgaa 3060 gaaaaaccac tccgaaagca atactaaagg taaccgacaa ccccaaatat ctctcgagcc 3120 ggggcaacgg accaactcca aaagcgatat taaaggcaac aagccagaag gagatagtct 3180 gaagacacta tcccaaaggc aacatgtgtc acaaactccc agaaaggaat ccgaaaagtg 3240 gtaacgtgag tctcataacc acccgttcgc attaaacggt ttactatccg ttagttaatt 3300 tcacactcaa agcagtacaa agtttacctc atctcatcgg taaaccagac acaaaggtca 3360 aaacgttcaa acgaatagat tgagaattaa ctgagaagat gaacgcacgt gttcattgca 3420 cgtgttttat ctaatcattt gattctcctc ccacttcgct cggagtgcga aaccgtgtgt 3480 gttttaaata gaatttataa attggagaaa cgagctttta atgcaattta aagggattat 3540 tttataaaaa acccgtggat gaagtcaaaa aggtatcatc aaggtcaaat atgcaaaaac 3600 aactaattgg aatcaatgtg gatcgataaa agttggaatc acagacaaat aggcaaagta 3660 ggaaaaacag gggaacatga gcaggtcggt tgaccggact ctgagctcgg tcgaccggcc 3720 caaaaacaga gaagtgctct ggtagcaggt cggtcaccgg cctactgagt tcgatcggcc 3780 gaaccctgtt tccagcactt cccagcctat caaaacgagg gccaatctcg accaaattgc 3840 aatcgttcga gcaacgacgc caagggttta tgtcaatcga ctacccgatg ctatatgctc 3900 ctcgatgcat caccaaaaac atgtttaaac atcaaagtta tgatctttaa agatttgaac 3960 ccaaaacact aaaaattggg atattcacca attcaatcca aatcaagagc aaatgcacat 4020 gaaaaagtgt taggaattca tatctgacaa tcaaagatag atataagcat agatgtaaga 4080 taaaaatccc acctttaacc ctaaaaaaaa gaagaagaag aagctcaatt gcaaggaaaa 4140 gatcaaccac tctctacttc aactcgacta gcttcaatct aagatcaaaa caagtatttc 4200 ctaagttagg tttaggaaac ttctgaaatt gaaggagaaa gagttggaga agatgaagag 4260 gaggcttacc aatagcttct tttgattttg gagcaagaat ttgagctagc aagagaaact 4320 agagcttaga gtttctaatt ccctttcctt tccaccattt tcagttagag cttgagtttg 4380 aggagtggga atgagtgaat ggagaggaga gagctgagtt agggtattaa gttagctagt 4440 gggttgaacc caaacccaaa acccaactag aaaaagtagc aaaattcaac ttactccaaa 4500 catatcaaag cttatactca aaaagatcac aaagccattt tggactcaat ggatgtggtc 4560 ttgagtccca attcgaccgt ttgcagaaag tagccttttg gactataatc cgacaaacga 4620 cgttatattt aagtaaaata tttaaatata caaccgataa atatgcgggt tgttacagac 4680 tcccctcctt aaaaaaattg cgtcctcgca attcttaaga agaaaataaa gagtaacaac 4740 ccaaaaacat atcaacacat aagaataaca gaataagaag ttcaccataa agtgcacgaa 4800 aggataagcc ctaacttgtt tgctcataga attttgtgga ttcctaagct caaaataatt 4860 cggaacaccg acatggcgca acaatacgtc aaatgaacca aagctcaaca aaatgaagta 4920 caacaaaacg tccgatgaac ttggtaaaaa gatcattcca ttcggacatc aacaaagaaa 4980 caatcacttg ttgcttacaa ctaaatgcat tggcaacacg atcccccttt caggaataaa 5040 aagaatgaga gtattggaaa agagttcttt taaataatca actttcccga aaagctaaaa 5100 ctttcaaaat cacaattatc tttatgtaat tatttttcta gaaaattttt cttatttcaa 5160 atcaattcac tgtgtctgat tgtctcatca aagctttagg tgattcctat cgataacgtt 5220 gattaagcgc atcgaccaga attcttaaag cacttaatgg gcaatacaat cacaaaacaa 5280 tcttctatat gataaacata tttctaaaaa cttttaaaaa ttatctttgt tttttttaag 5340 aataaaaatc ccatttatac ttataaataa atcttatgaa aaagatttgc aaaaattttg 5400 aattgctaat ttccatccaa aaccaataaa tgctaaaatg gaatcatcga gatattcaca 5460 aaatactcga ttcatacaat ggatgaaatg agctagacaa actgaatgac ataataaatc 5520 attcataaga atcacaacga gtgcggaatg cggtaagtgg agtgctaact tccgcaactc 5580 gtagccaaat caaaaccggt ggcaaggtcg agctgaaaag accttaatac cactcaaagg 5640 attgcttagg agataatcct aagactagac ccaaaatacc agaaaattct cgaactataa 5700 gaatgtgaga aacaacactt tcctcatcat gagggattaa agaaataaaa tgatgccatc 5760 aaattggaaa tcgaatcaca aagaaaagta gaaacctacc aaaatagatg gagacataac 5820 ttatccctga gaagaacctc cagaagcatt tgttacattt aacttaccag atggagaatt 5880 attcggagca atgggacaag tggaactacg atggtcttcg ttcttgcaat agcggcagaa 5940 gagaggctta ggacaagctc gaatcatatg atcaggagaa ccacaattga aacacttccg 6000 aatggaaggc gttccagcag gctgagactg agaatcagat tcaacggact ttttcttctt 6060 ggaagggaaa aattcccgct catccaatgg agactgcttt ttgttggggt tcttacgaac 6120 ctcaacatca tcaccactat cacaatcctt ctcgatggtt tcaagatgaa gtgcagaatc 6180 taccaactcc tcaaaagaaa gcttaaactg gagcaataaa gacttcttaa gatatttctt 6240 aagaccagaa acaaacttca aattcttctt ataagaagtg caaatgaatt catgaccata 6300 cttagataaa gcctcaaatg atgaaacata ctcagaaaca gaagacttcc cttacttcaa 6360 cacgaggaag cgacgggtca ataaaacttt ttcagattca ggaacaaact tgagttcgaa 6420 tcgtccccga aactgagtcc aaaaaagagt agactcatca atttcggcta caagaagctc 6480 ataccaatca agagcacgct attgtaataa gctagaagtt acaagaaccc tatcatgatc 6540 agaaagatcc atgcgagaga aaatacgctc gcacgtgcga agccaagctt ggacttcaag 6600 aacagactcg gagcctttaa acggctttcc accaagatga gaaaaatcac ttcacaggcg 6660 tgcaaaagta gacttgcgag gtggagatac acgagtagga gctggaggct gaatgctcct 6720 aaacaactcc ataaactgat cggcagacat ataaacgcca tccggatgtg agccactgga 6780 ttgcccagca tccgaacgtc gtcccctgcc tcttccagcc atacgttgaa agttaagata 6840 agaacaagat tattcacaag gcagatgatt caatatggaa tactacagga tttaaatgca 6900 ttcctagacg tattcccaca ttctatcaac caccaagaca caaaagaaat atagaggcat 6960 gatgattcaa agatttgtat cacttataca ctttgacgta ccggaatatg tgcgtctatt 7020 ctgtgtttat catattttgt tctaaacctc gctctgatac caagctgtga cgcctcacct 7080 ctatacccac gctagccaag gacactacag ggtaatgccg tcaactaaca cggagaataa 7140 ggctggttag gagcgaccat ctatagcctt gctctaacta tctattgaat acatcgacaa 7200 tagacatacc aagcaggtcg acgtacctaa tgaatactta aaacaaaacc ataggtggtt 7260 ggaggatcta tcagaacata atataacaac tcaaatgtaa tatacatcac agaacagtag 7320 atcatcatat acacatatat tgatacataa cagagtttac aaactgatac acgtcaagtg 7380 atatagcaaa ccacaataaa caggcaaatc caaaagaata catatatcag agttacaaca 7440 aaaaggacta cacataaagc tttaacaaaa aggagcaagc tgaatagtcc atgcggaagc 7500 aaaatagcta agtccaaaag cacctaacct gaaacatctc ggtctgaccc gagtataata 7560 cacgggtcag tctagctaaa caaatcagtt agataaactg aacaacaata acctgcaaag 7620 tcacaaagta taacaaatat ctctaaaaga gataacacaa tcatctaaca aaaagatgaa 7680 tcatatcata acaagcctcg tatcatcggc aactcataga aacaaaggat aggaatatcc 7740 caaaacaagc cataaaggca aataacaaat actagtgaag gtaacacaag ttccacaagg 7800 taatcgctat atatcgtgcg gctcaaaaag accaacaccg aagagcgata cctagggaat 7860 atacatatca atgaaacatc ataaaatttt aattgcaaca aacccaaata tctcatgagc 7920 ggtggtgaag aaaccagtcc aatatctctc gagcggtggt gaagaaaaac cactccgaaa 7980 gcaatactaa aggtaaccga caaccccaaa tatctctcga gccggggcaa aggaccaact 8040 ccaaaagcga tatttaaggc aacaagccag aaggagacag tccaaagaca ctatcccaaa 8100 ggcaacatgt gtcacaaact ctcagaaagg aatccgaaaa gtggtaacgt gagtctcata 8160 accacccgtt cgcactaaac ggtttcttat ccgttagtta atttcacact caaagcagta 8220 caaagtttac ctcatctcat cggtaaacca gacacaaagg tcaaaacgtt caaacgaata 8280 tattgggaat taactgagaa tatgaacgca cgtgttcatt gcacgcgttt tatctaatca 8340 tttgattctc ctcccacttc gctcggagtg cgaaaccgtg tgtgttttaa atagaattta 8400 taaattggag aaatgtgctt ttaatgcaac ttaaagggat tattttataa aaatcccgtg 8460 gatgaagtca aaaaggaatc atcaaggtca aatatgcata aacaactaat tggaatcaac 8520 gtgcatcgat aaaagttgga atcacagaca aataggcaaa gtagaaaaaa caggggaaca 8580 ggagcaggtc ggttgaccgg actttgagct cggtcgaccg gcccaaaaac agagaagtgc 8640 tctggtagca ggtcggtcga ccggcctact gagttcgatc ggccaaaccc tgtttccagc 8700 acttcccagc ctatcaaaac gagggccaat ctcgaccaaa ttgcaatcgt tcgagcaaag 8760 ccgccaaggg tttatgccaa tcgactaccc gacgctatat gctcctcgat gcatcaccaa 8820 aaacatgttt aaacatcaaa gttatgatct ttaaagattt gaacccaaaa cactaaaaat 8880 tgggatattc accaattcaa tccaaatcaa gagcaaatgc acatgaaaaa gtgttaggaa 8940 ttcatacctg acaatcaaag atagatataa gcatagatgt gtgataaaaa tctcaccttt 9000 aaccctaaaa aaagaagaag aagacgctca attgtaagga aaagatcaac cactctccac 9060 ttcaactcta ctagcttcaa tctaagatca aaacaagtat ttcttaagtt aggtttagga 9120 aacttctgaa attgaaggag aaagagttgg agaagatgaa gaggaggctt accaatagct 9180 tcttttgatt ttggagcaag aatttgagct agcaagagaa actagggctt ggagtttcta 9240 attccctttc ctttccacca ttttcagtta gagcttgagt ttgagaagtg ggaatgagtg 9300 aatggagagg agagagctga gttagggtat taagttagct agtgggctga acccaaaccc 9360 aaaacccaac tagaaaaagt agcaaaattt aacttactcc aaacatatca aagcttatat 9420 tcaaaaagat tgcaaacgcc attttggact caatggaggt ggtcttgagt cccaattcga 9480 ccgtttgcag aaagtagctt tttggactat aatccgacaa atgacgttat atttaaataa 9540 aatatttaaa tatgcaaccg ataaatatgc gggttgttac agtaaacttc gccagcaata 9600 acagattcaa gaatcaagaa agaagcaaca acaataaaaa tcagcctaat tttcaaagaa 9660 atggtcaatc ctctagatcc agaggaagaa atggaagata taacaacagc aataacagac 9720 tatggtgtca aatttgtgaa aagtttggtc atgcagccaa cgaatgttac ttcagatttg 9780 atgccaacta cattccatct cagcaaaata acaacagttc aagaaatcac aatctcaatg 9840 caaacatggt aaatacttat ccagattcta aagaacaagc agataccaaa agtacagaga 9900 ctgcaaagat tgatgatatc taggatgaaa gctagtatcc agattatgag gctaccaatc 9960 atgtaactaa taatctcaac aatctaaact tgggaagcag ggagtacaga ggtaggcaat 10020 taattcatat gggtaatggt gaatcagtaa aaattacaca tattggaaat gcaagtttca 10080 gtggcagaaa ataactcttt ctaaaaaatc tgctaagagt tccatctatt agaaaaacct 10140 tattgagtgt ttcgcatttt gcccaaggca atgatgtgta ttttatccca aatactgttt 10200 aattagagat attctaacta aggaaataat tttgcagggt aagaaattta aaggattgta 10260 caaattcgat gttgcaaaat caggaaattc cgaatttcat ggaaaatggt gtaatgttac 10320 tgaaattgaa catgaagata actataaaga ttttgatatt tgacattcca aacttggaca 10380 tccaagtgta agcattgtaa aaaaggtgtt aagtaatgat aatatttctg ttaaagatgt 10440 tgcaatgcct tatgtatgta catattgtca aatgggcaaa agccataaac ttgcatttcc 10500 aatctcaaaa ataaactata caaaaccctt agattaattg cttgagatct atggggtcca 10560 tcttctgtta acactgatta tggctataaa tactttatat catttgtcga tgcatactct 10620 agatatgtat ggatttattt cttgaaatca aaatctgaaa cttatgatgc agtcttacag 10680 tttataactc aagctgagag gcaaacaaat tgccaattaa aagtcttgca gactgatggt 10740 ggaaccgagt ttcaaccact taaggaatac cttcaaaaga aaggtataat tcacagaaca 10800 aactgtccat acacatcaga acaaaatgga ctggttgaaa gaaaacacag acatattgtt 10860 gagactgggc ttactttact agcccaagca tctcttcctc taaagttttg accagatgcc 10920 tttgccacta ctgtgttttt aataaataga ttatccataa aagttttaca gttcaaaagt 10980 ccagtggaaa tcttatttaa agttaagcca gaatatcata gtttgaaagt ttttggatgt 11040 ttatgctttc cctatttaag gccatataat acacacaagt taagtttcag gtcatcacca 11100 tgtaccttct taggctatgc agccaataag aagggataca aatgcttgga tagcaatgaa 11160 ttttgttttt gtttctatac atgtggtgtt taatgaaaga atatttccct ttaatcaatc 11220 aaacataaaa acatcacaga aagtgtataa ttcaaaactt tctataccac cagtacctga 11280 gcaacttgat gtcataactc aagttgaaga tgaaagaatg cagcacaata taactttaga 11340 gtctcctgtc aaatccactt cacaatacac tgtgcccaga cctgagccag gtcatgaaga 11400 agatgaacaa gctggaagca caagtgaggc atctcatatg tgccccatct atcagtcaga 11460 ctgcagtcag tcattagatg attcacaatc atctaatgat caacaggaag tcacagctga 11520 agagccaagt caatcacctg cacaagctag acatcacatg ataacaaggg ccaagagtgg 11580 tatattcaca ccaaaaatat acagtgttgc tatgaacaca gaagagccag atacctatca 11640 tcaagctatg cagaatgaaa actggatgtc tgctatggaa gaagaataca atgcattgat 11700 aaaaaataaa acatgggatc taacttcatt gccaagcaat aaaaatgtca taggctgtaa 11760 acggacatat agaataaaga gaagtggtga tggttcggtt tctaaatata aggcaaggct 11820 agtagccgaa gggtattcat agcaaagtgg ctttgacttt actgaaactt tttcaccagt 11880 tgtgaaaccc tcaactatta gaattgttct tactattgcc ttacatacaa actgaaacat 11940 caagcaatta gatgtaaata atgccctttt gtggcatttt acaggaggaa gtctacatgt 12000 aacagcctct aggttttgaa caaaaaaggg gtggaaaact tgtatgcaaa ttgaacaaag 12060 cgatctatag actcaaacat gcaccaaggg tctggtttga aaagctgaag tcaatactaa 12120 tcagacttgg ctacagctca acaaaatcat acaattcttt atttacaaaa tttaaaaaca 12180 atgcaaccat gtatgtacta atatatgttg atgattttat tattacaagc agcagtgaac 12240 cagagatgaa taaactcaca gacctcttaa ataaagaatt ttcaatcaaa gacttgggga 12300 acttaaatta ctttcttggc attgaggtta aaagactttc acacatagaa atgcttctaa 12360 gtcaaaggaa atacatctca gaaatcttac agaaagcaaa aacggataaa gcaaatgcaa 12420 tatctactcc tatggcaagc aattcacatc tgtcaacgta taaaggagaa tccatatcac 12480 atggcaagca gtacagaagt attgtaggag cattacagta tgtaacaatc actaagccag 12540 aaatttcatt tagtatcaat aaagtgagtc aatatatgca aatttcactg gatgaacatt 12600 ggaaagcagt taagagaatt ctaagatatc tcaaaggaac tataaatcat ggattaaaga 12660 tgcaatcatg caaatctatc catataaaag ggtatgcaga tgcagattgg gccacagaca 12720 tggatgacag aagatctacc actggttatt gcatatttct aggcaacaat ccagtttcat 12780 ggtgctcaaa aaaacagcct acagtatctc ggtcaagtac tgaagccgag tacagaaacg 12840 ttgctaatgc aacagctgaa ataatatgga taatgtcctt gctcagtgaa cttcatatca 12900 agtttacaga aaaaccgaca atttggtgcg acaatctgag caccgttctg ccctcagcaa 12960 atctagttct acatgctaga accaagcaca tagaactgga tctgtatttt gtcagagaac 13020 aagtaacaga ggaaaaaatt aacataagtc atctaccatc atcttaccaa atcgctgatg 13080 ttatgataaa acctctatca aagaagaact ttgtttgatt taagcaagaa ttgaaggtta 13140 ctgagttcga cagcaacgaa aaaggcaaga tctatgataa ctcaacaaat tgaagcttga 13200 cagcatcgtc tactccaatt gaagagaaat tccacttgac aatcaagtga aatttcccct 13260 gatgaagaaa gcaagtctcg atatacggca atttaaccaa aatcgaaaca cgacagctta 13320 atctctccaa cttcagcaaa aagagggctt ccgtatcact gcaaggtcct gagtggtaaa 13380 tctacacact cgagaaaccg aagccatcat attttattcg tgcggctgaa gaaattgcaa 13440 accctaaccc taagttaggt gtgaggggag gtatcaaatg aaagtataga gccgcactta 13500 tcaaatataa gcatcacaat ataattggaa cgggttgtga taatggacca tcccgaagcc 13560 caattttctt tttataaata tcaagttgta aacagttcag tgtattatca aaagaacaaa 13620 aacattctgt aactacggaa tatttttctt gttttctgtt tttcaaatat taatcaataa 13680 aatcaagatt caaactctct caatattctt tttacgagct aaaaatatat catattttct 13740 gtgtccgaac gctgatattg ttttttactt tggactctga tacttagcta acattataca 13800 cgtgagaaat aggaaactta tatctagtga aaattaccca tgaatctgac ag 13852 <210> 10 <211> 2516 <212> DNA <213> Allium cepa <400> 10 tgtgacgccc cacctctata ctcacgctag tcaaggacac tacaggataa taccgtcaac 60 taacatggag aataaggctg attaggagcg accatctata tccttgctct aactatctat 120 tgaatacatc aataatagac ataccaagca ggtcgacata cccaattaat acttaaaaca 180 aaaccatagg tggttgaagg aactatcaga acataatata acaactcaaa tgtaatatac 240 atcacagaac agtagatcat catatacaca tatattgata cataacagaa tttacaaact 300 gatacacgtc aagtggtata gcaaaccaca ataaacaggc aaatccaaaa gaacgcatat 360 atcagagtta caacaaaaag gactacacat aaagctttaa caaataggag caagctgaat 420 agtccatgca gaagcaaaat aggctaagtc caaaagcacc taacctgaaa catctcggtc 480 tgacccgagt ataatacacg agtcagtcta gctaaacaaa tcagttagat aaactgaaca 540 acaataacct gcaaagttac aaagtataac aaatatctct aaaagagata acacaatcat 600 ctaacaaaaa gatgaatcat atcataacaa gcctcgtatc atcggcaact cagagaaaca 660 aaggatagga atatcccaaa acaagccata aagacaaata acaaatacca atgaaggtaa 720 cagaagttcc tcaaggtaat cgctatatat tgtgcggctc aaaaagacca acaccgaaga 780 gtgataccta gggaatatac atatcaatga aacatcagaa aattttaatt gcaacaaacc 840 caaatatatc atgagcggtg gtgaagaaac cactccaata tctctcgagc ggtggtgaag 900 aaaaaccact ccgaaagcaa tactaaaggt aaccgacaac cccaaatatc tctcgagccg 960 gggcaacgga ccaactccaa aagcgatatt aaaggcaaca agccagaagg agatagtctg 1020 aagacactat cccaaaggca acatgtgtca caaactccca gaaaggaatc cgaaaagtgg 1080 taacgtgagt ctcataacca cccgttcgca ttaaacggtt tactatccgt tagttaattt 1140 cacactcaaa gcagtacaaa gtttacctca tctcatcggt aaaccagaca caaaggtcaa 1200 aacgttcaaa cgaatagatt gagaattaac tgagaagatg aacgcacgtg ttcattgcac 1260 gtgttttatc taatcatttg attctcctcc cacttcgctc ggagtgcgaa accgtgtgtg 1320 ttttaaatag aatttataaa ttggagaaac gagcttttaa tgcaatttaa agggattatt 1380 ttataaaaaa cccgtggatg aagtcaaaaa ggtatcatca aggtcaaata tgcaaaaaca 1440 actaattgga atcaatgtgg atcgataaaa gttggaatca cagacaaata ggcaaagtag 1500 gaaaaacagg ggaacatgag caggtcggtt gaccggactc tgagctcggt cgaccggccc 1560 aaaaacagag aagtgctctg gtagcaggtc ggtcaccggc ctactgagtt cgatcggccg 1620 aaccctgttt ccagcacttc ccagcctatc aaaacgaggg ccaatctcga ccaaattgca 1680 atcgttcgag caacgacgcc aagggtttat gtcaatcgac tacccgatgc tatatgctcc 1740 tcgatgcatc accaaaaaca tgtttaaaca tcaaagttat gatctttaaa gatttgaacc 1800 caaaacacta aaaattggga tattcaccaa ttcaatccaa atcaagagca aatgcacatg 1860 aaaaagtgtt aggaattcat atctgacaat caaagataga tataagcata gatgtaagat 1920 aaaaatccca cctttaaccc taaaaaaaag aagaagaaga agctcaattg caaggaaaag 1980 atcaaccact ctctacttca actcgactag cttcaatcta agatcaaaac aagtatttcc 2040 taagttaggt ttaggaaact tctgaaattg aaggagaaag agttggagaa gatgaagagg 2100 aggcttacca atagcttctt ttgattttgg agcaagaatt tgagctagca agagaaacta 2160 gagcttagag tttctaattc cctttccttt ccaccatttt cagttagagc ttgagtttga 2220 ggagtgggaa tgagtgaatg gagaggagag agctgagtta gggtattaag ttagctagtg 2280 ggttgaaccc aaacccaaaa cccaactaga aaaagtagca aaattcaact tactccaaac 2340 atatcaaagc ttatactcaa aaagatcaca aagccatttt ggactcaatg gatgtggtct 2400 tgagtcccaa ttcgaccgtt tgcagaaagt agccttttgg actataatcc gacaaacgac 2460 gttatattta agtaaaatat ttaaatatac aaccgataaa tatgcgggtt gttaca 2516
Claims (15)
A gene consisting of the nucleotide sequence of SEQ ID NO: 1 (AcWHITE).
A composition for selecting white guppy-colored onions comprising a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
The composition of claim 2, wherein the primer comprises a nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8.
A kit for selecting white guppy-colored onions, including a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1.
The kit of claim 4, wherein the primer comprises a nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8.
A method for selecting white guppy-colored onions comprising the step of analyzing whether the nucleotide sequence of SEQ ID NO: 1 (AcWHITE) is present in the onion gDNA.
The method of claim 6, wherein the analyzing comprises binding a primer capable of detecting at least a part of the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 to the gDNA.
The method of claim 6, wherein the analyzing comprises amplifying a gene by polymerase chain reaction (PCR).
The method of claim 7 or 8, wherein the primer comprises a nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 8.
Recombinant vector for producing white guppy-colored onions comprising the nucleotide sequence of SEQ ID NO: 1 (AcWHITE).
A method for producing a white guppy-colored onion comprising the step of transforming the nucleotide sequence (AcWHITE) of SEQ ID NO: 1 into onions.
The method of claim 11, wherein the transformation step is performed using the recombinant vector of claim 10.
The method of claim 11, wherein the transformation step is transformed using CRISPR/Cas9.
A transformed onion transformed with the recombinant vector of claim 10.
Transformed seeds of onion according to claim 14.
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KR20230025578A (en) * | 2021-08-12 | 2023-02-22 | 전북대학교산학협력단 | Molecular marker for validating the anthocyanin content of onion and use thereof |
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KR20230083164A (en) | 2021-12-02 | 2023-06-09 | 전남대학교산학협력단 | Composition for discriminating chartreuse bulb color onion and method using the same |
CN114525361A (en) * | 2022-03-08 | 2022-05-24 | 山东省农业科学院 | cDNA molecular marker for distinguishing purple-skin onions from yellow-skin onions and application of cDNA molecular marker |
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