WO2016171344A1 - Saengjang, which is novel aloe variety, and molecular marker for classifying same - Google Patents

Saengjang, which is novel aloe variety, and molecular marker for classifying same Download PDF

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WO2016171344A1
WO2016171344A1 PCT/KR2015/009884 KR2015009884W WO2016171344A1 WO 2016171344 A1 WO2016171344 A1 WO 2016171344A1 KR 2015009884 W KR2015009884 W KR 2015009884W WO 2016171344 A1 WO2016171344 A1 WO 2016171344A1
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aloe
seq
nos
oligonucleotide primer
primer sets
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PCT/KR2015/009884
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French (fr)
Korean (ko)
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김권희
고문경
장선일
황성수
고윤정
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김권희
고문경
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a new breed of aloe (Saengjang) and a marker for determining the same.
  • Aloe is a succulent plant native to Africa.
  • the plant is native to South Africa, Madagascar, and Arabia, mainly in the African continent and neighboring islands, and is now widely cultivated in tropical and temperate regions.
  • the leaves are thick, sword-shaped, with thorns on the edges, and have orange or pink flowers.
  • It contains a large amount of ingredients useful for the human body, such as alloin and saponin, and has been developed and used in medicine, health supplements, and cosmetics since ancient times.
  • More than 600 species of Aloe are known, but currently edible aloe is on the order of Aloe vera (Aloe vera), aloe ahbore sense (Aloe arborescens), aloe sand paper or Syrian (Aloe saponaria).
  • Aloe vera is a representative species of aloe used for commercial and medicinal purposes. It is usually vulnerable only after three years of cultivation. However, because it is a tropical crop, domestic cultivation requires a relatively high cost, and imported products from foreign countries have a problem in that the quality is not constant due to uneven supply and demand imbalance. Aloe vera also removes toxic thick skin from the leaves and separates only the inner gel, which is a cumbersome process in the production of aloe-related products. Aloe saponaria, on the other hand, can be used as a peel because it is mild in aloe used as a raw leaf, but it belongs to a small species of plants of the genus Aloe.
  • Korean Patent No. 0133344 discloses a 'tissue culture method for mass propagation of aloe'
  • US Patent No. PP023913 discloses 'Aloe plant named Tiki Tahi', but the growth of new aloe species of the present invention and discrimination thereof are disclosed. No molecular markers have been disclosed.
  • the present invention is derived from the above requirements, by mixing aloe vera and saponaria planting natural hybrids with a thin shell and mass asexual breeding to cultivate new varieties and compare the morphological and molecular genetic characteristics Compared to aloe vera, the plant height is lower, the number of leaves is large, and there are white spots in the pale green leaves. Compared to the aloe vera, the leaf of the leaf is thinner and softer than the aloe vera.
  • the new aloe varieties named 'Saegjang' to complete the present invention.
  • the present invention has the following morphological features, has a white spots in the leaves of light green, compared to the aloe vera, the leaf is thinner and softer thorns, a new breed of aloe capable of mass propagation by asexual breeding Saengjang offers:
  • Bark thickness of the leaf 0.05-0.1 mm.
  • the present invention also provides oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
  • the present invention comprises the steps of separating genomic DNA from aloe plant samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; And it provides a method for determining a new breed of aloe, comprising the step of detecting the amplification product.
  • Growth of a new aloe varieties according to the present invention is thin leaves and soft thorns can be used to grind the aloe leaves without removing the bark and drinking, 5-20 young shoots grow around the adult aloe to plant a new shoot The ability to breed in large quantities can greatly contribute to the increase in aloe farmers' income.
  • the primer set used in the present invention varieties for the determination of varieties according to the origin of aloe, purity test, breed protection and seed management system It can be used for testing.
  • 1 is a photograph showing the growth of the new year-old varieties (left) and Vera (right) as a control variety.
  • Figure 2 is a photograph showing the growth state of the new breed growth (left) and the control varieties Vera (right).
  • Figure 3 is a photograph showing a packaging foreground of new breed growth.
  • Figure 4 is a photograph showing the leaf cross section of aloe vera (left) and new varieties growth (right).
  • FIG. 5 is a photograph showing the flower form of aloe vera (left) and new breed growth (right).
  • FIG. 6 is a photograph showing aloe vera (A), aloe aborescens (B), aloe saponaria (C), and new breed growth (D).
  • FIG. 7 shows UPGMA phenograms showing genetic correlations between various aloe species, including aloe new species growth.
  • the present invention has the morphological characteristics of the following (a) to (g), has white spots in the leaves of light green, the leaf peel is thinner than the aloe vera, soft spines, asexual Providing a new breed of aloe Saengjang, which is capable of mass breeding by breeding:
  • Bark thickness of the leaf 0.05-0.1 mm.
  • New breed growth according to one embodiment of the present invention was cultivated in a mixed planting method of aloe vera and aloe saponaria to induce natural hybrids and obtain seedlings to grow lines through asexual breeding.
  • Aloe Vera (Aloe vera) belongs to the fastest growing section during 1-2 years after growing only deodiji two years, the flowers come up with a long spine, regardless of the season of the year smokes a yellow or reddish orange flowers.
  • the leaves are green, the leaves are 60 ⁇ 90cm in size, one leaf is about 0.5 ⁇ 1.5kg, and 3 ⁇ 6 leaves can be harvested when harvesting 5 ⁇ 6 year old one. Breeding is carried out by two or three young sprouts next to the mothers. When eaten, the bitter taste is strong, and when peeled and eaten, it's tasteless and odorless.
  • Aloe saponaria is a small species of aloe that grows on the ground and has light leaves with white spots. Flower beds rise long and bloom red-colored flowers. The leaf size is 30 ⁇ 60cm and the number of leaves of 1kg is 3 ⁇ 5. Aloe saponaria strikes 3-5 cubs. The bark has no bitter taste, so only the thorns are used and the bark is used a lot.
  • Aloe growth according to an embodiment of the present invention grows in the height of the body is 30 ⁇ 55cm, the stem is long and tender leaves grow in abandonment method, almost no growth after 1-2 years of growth, the leaves of growth 40cm, It has a light green background about 6cm wide with white spots, and leaves have small, weak spines.
  • aloe growth is characterized by the growth of more than 5-20 young seedlings near the root of the leaf body, the thickness of the leaf bark is thinner than the vera, and the flower stalk may not have branches.
  • New aloe varieties growth of the present invention was deposited on the microorganism resource center of Korea Research Institute of Bioscience and Biotechnology and deposited on callus KCTC 12780BP on March 30, 2015.
  • Tissue culture can regenerate a plant characterized by the present invention.
  • the regenerative cells used for such tissue culture may be immature embryos, plasma, meristem cells, callus, pollen, leaves, pollen sacs, roots, root tips or flowers, preferably callus.
  • the present invention also provides oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
  • the oligonucleotide is an oligonucleotide consisting of at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides in the sequence of SEQ ID NO: 1 and at least 17, 18 in the sequence of SEQ ID NO: 2. At least 19 and at least 20 contiguous nucleotide segments.
  • the oligonucleotide may be an oligonucleotide consisting of fragments of at least 17, at least 18, at least 19, at least 20 consecutive nucleotides in the sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the oligonucleotide is at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, and at least 26 in the sequence of SEQ ID NO.
  • the oligonucleotide is an oligonucleotide consisting of at least 17, at least 18, at least 19, at least 20 consecutive nucleotide segments in the sequence of SEQ ID NO: 7 and at least 17, at least 18, in the sequence of SEQ ID NO: 8, Oligonucleotides consisting of segments of at least 19, at least 20, at least 21, at least 22 consecutive nucleotides.
  • Oligonucleotide primer set is a combination of two primer sets, three primer set combinations, four primer set combinations of the four primer sets, most preferably four primer set combinations .
  • the oligonucleotide primer sets of the present invention are most preferably oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; It is a set of molecular label primers for the determination of new growth of aloe comprising the oligonucleotide primer set of SEQ ID NO: 7 and 8.
  • primer refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product.
  • the length and sequence of the primers should allow to start the synthesis of extension products. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.
  • the plant's chloroplast genome (cpDNA) is easier to analyze than the nuclear genome because there are hundreds of identical chloroplast genomes in a single cell. It is a marker that distinguishes a specific species or a specific strain within the same species, and thus is the most reliable and quick analysis marker.
  • cpDNA chloroplast genome
  • An "Internal Transcribed Spacer” is a site that is transcribed from nuclear ribosomal DNA but not expressed as a protein.
  • ITS sequences have been used to infer subclass levels of a diverse array of organisms, including plants, fungi, insects, and the like.
  • the ITS region is known not only to have high information at the subclass level, but also to exhibit conserved sequence patterns and high alignment of pizza plants.
  • the ITS region is easily amplified in most pizza plants because small primers exhibit a peripheral sequence that is strongly conserved from the rRNA gene.
  • the base polymorphism was investigated in the ITS region amplified by SEQ ID NO: 7 and 8.
  • the invention also comprises the steps of separating genomic DNA from aloe plant samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; And it provides a method for determining a new breed of aloe, comprising the step of detecting the amplification product.
  • the method includes separating genomic DNA from aloe plant samples.
  • a method for separating genomic DNA from the sample a method known in the art may be used.
  • the CTAB method may be used, or a wizard prep kit (Promega) may be used.
  • oligonucleotide primer sets may be used as a primer to amplify a target sequence.
  • Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification systems, There are any suitable methods for amplifying via strand displacement amplification or Q ⁇ replication or for amplifying nucleic acid molecules known in the art.
  • PCR is a method of amplifying a target nucleic acid from a pair of primers specifically binding to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.
  • the amplified target sequence may be labeled with a detectable labeling substance.
  • the labeling material may be, but is not limited to, a material emitting fluorescence, phosphorescence, or radioactivity.
  • the labeling substance is Cy-5 or Cy-3.
  • PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance.
  • the label using the radioactive material may add radioactive isotopes such as 32 P or 35 S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized.
  • the primer set used to amplify the target sequence is as described above.
  • the method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through sequencing, capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of methods for detecting amplification products, gel electrophoresis may be performed, and gel electrophoresis may use acrylamide gel electrophoresis or agarose gel electrophoresis depending on the size of the amplification product. Capillary electrophoresis can also be performed. Capillary electrophoresis can use, for example, an ABi Sequencer.
  • PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter.
  • radioactivity measuring method is to add a radioactive isotope such as 32 P or 35 S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.
  • aloe vera and aloe saponaria were artificially created in the greenhouse located in Yongjin-myeon, Wanju-gun, Jeonbuk, to artificially create environmental conditions for neglected moisture. .
  • seedlings with light green leaves with thin skins and thorny white spots were obtained in a greenhouse mixed with aloe vera and aloe saponaria, and since 1981, the lineage has been cultivated and the characteristics of the selection hybrids are maintained.
  • aloes are planted in a 600m 2 greenhouse located in Yongjin-myeon, Wanju-gun, Jeollabuk-do to avoid direct sunlight, and cultivate them at a cultivation rate of 20 ⁇ 35 °C in spring and autumn, and 10 ⁇ 15 °C in winter in banyang. Proliferated.
  • the ground part of the new breed growth of the present invention expressed the external characteristics of the vera and saponaria. Growth of new varieties was compared to morphological characteristics of Vera, the most similar variety in appearance.
  • the leaves of growth had white spots on pale green background about 40cm long and 6cm wide and small, weak spines at the edges of the leaves.
  • aloe vera showed a strong spine, dark green color, and no leaf pattern (Table 1, FIGS. 1 and 2).
  • the thickness of the leaf peel was thinner than the growth of Vera (Fig. 4).
  • the aloe vera showed a form in which the flower stalks branched, but in the case of growth, the flower stalks did not branch.
  • FIG. 6 Three Korean aloes, A. vera , Aloe arborescens , Aloe saponaria and Aloe new varieties were used as genomic DNA extraction samples (FIG. 6). 100 mg of leaves of aloe were taken and pulverized with liquid nitrogen, and DNA was extracted using a modified CTAB method. The extracted DNA was loaded on a 0.8% agarose gel to confirm the DNA state.
  • Primer sequences and annealing temperatures used in the present invention are as shown in Table 2.
  • Three chloroplast DNA regions (matK, trnL-F, rbcL) and nuclear ITS DNA regions were amplified.
  • the PCR reaction solution was 20 ⁇ l of total reaction solution, with 25ng of template DNA, 1 ⁇ PCR buffer, 2.5mM dNTP, 1U Taq DNA polymerase, and 20 pmol primer set (see Table 2 below) respectively. It was.
  • GeneAmp PCR system 2700 thermal cycler was used for PCR reaction, PCR reaction conditions were 95 °C (3 minutes), [94 °C (30 seconds), the corresponding annealing temperature of Table 2 (30 seconds), 72 °C (1 minutes), 35 cycles], 72 ° C. (10 minutes).
  • the PCR reaction product was extracted from agarose gel and analyzed for sequencing by ABI prism 3700 sequencer.
  • aloe growth was most closely collected with A. vera . Aloe growth is not yet reported in any kind of aloe It was not morphologically and genetically consistent with species in the genus. The results of the collection analysis of the investigated aloe species were in agreement with previous studies.
  • the aloe growth tissue was cut and cultured callus was deposited on March 30, 2015 with the accession number KCTC 12780BP to the Korea Institute of Bioscience and Biotechnology.
  • Aloe callus induction was performed by the following method. Only the areas where the stems including the aloe seedling roots were concentrated were cut. The cut aloe sample was subjected to surface sterilization with shaking for 10 minutes in a 10% commercial Lax solution. After the surface sterilization, the aloe sample was washed three times with sterile water. The sample was removed in a sterile workbench using a sterile filter paper to remove the remaining water, and then cut into a size of about 5 mm using a scalpel to induce the callus by incubating the medium.
  • Callus induction medium was prepared in MS (Murashige & Skoog) medium with 3% sugar, 0.4% gelrite, 0.4 mg / L thiamine-HCl, 100 mg / L myoinositol, 1 mg / L 2, Medium with 4-D and 0.3 mg / L kinetin added was used as basal medium. After about 3 weeks of incubation, a white cell mass was formed from the aloe slices in culture in the medium to which 2,4-D was added. Then, callus was proliferated through passage in the same medium.

Abstract

The present invention relates to Saengjang, which is a novel aloe variety, and a method for classifying the same. The leaf rind of the Saengjang, which is a novel aloe variety, is thin and the thorns are soft, and thus it is possible to drink ground unpeeled aloe leaves without removing the rind. In addition, since 5 to 20 young buds grow around an adult aloe, mass propagation is enabled by transplanting buds, thereby contributing largely to an increase of income for aloe cultivating farms. In addition, according to the present invention, it is possible to classify the aloe by using four types of molecular labeling primer sets, and the marker used in the present invention is expected to be applied in variety classification according to the origin of the aloe, purity testing, and variety testing for protecting variety and establishing a seed management system.

Description

알로에 신품종 생장 및 이를 판별하기 위한 분자마커Growth of new aloe varieties and molecular markers to identify them
본 발명은 알로에 신품종 생장(Saengjang) 및 이를 판별하기 위한 마커에 관한 것이다.The present invention relates to a new breed of aloe (Saengjang) and a marker for determining the same.
알로에는 아프리카 지방에 자생하는 백합과의 다육식물로, 알로에 속 식물 원산지는 아프리카 대륙 및 인근 군도를 중심으로 남아프리카, 마다카스칼섬, 아라비아 지방으로 현재는 열대와 온대지방에 폭넓게 재배하고 있다. 잎은 두꺼운 칼모양이고 가장자리에 가시가 나 있으며, 주황색 또는 분홍색의 꽃이 핀다. 알로인, 사포닌 등 인체에 유용한 성분을 대량 함유하고 있어 예로부터 의약품, 건강보조식품, 화장품 등으로 개발 이용해 왔으며, 연중 푸른 잎줄기를 감상할 수 있어 최근에는 관상용으로도 인기를 끈다. 600 여종 이상의 알로에가 알려져 있으나 이 중 현재 식용 가능한 알로에는 알로에 베라(Aloe vera), 알로에 아보레센스(Aloe arborescens), 알로에 사포나리아(Aloe saponaria) 정도이다.Aloe is a succulent plant native to Africa. The plant is native to South Africa, Madagascar, and Arabia, mainly in the African continent and neighboring islands, and is now widely cultivated in tropical and temperate regions. The leaves are thick, sword-shaped, with thorns on the edges, and have orange or pink flowers. It contains a large amount of ingredients useful for the human body, such as alloin and saponin, and has been developed and used in medicine, health supplements, and cosmetics since ancient times. More than 600 species of Aloe are known, but currently edible aloe is on the order of Aloe vera (Aloe vera), aloe ahbore sense (Aloe arborescens), aloe sand paper or Syrian (Aloe saponaria).
알로에 베라는 상업적이나 약용으로서 사용되는 대표적인 알로에 종으로, 보통 재배한 지 3년 이상이 되어야 약성이 뛰어나다. 그러나 열대성 작물이라, 국내에서의 재배는 상대적으로 많은 비용이 요구되며, 외국에서 수입하는 가공품은 작황의 불균일 및 수급 불균형에 의해 품질이 일정하지 않은 문제가 있다. 또한 알로에 베라는 잎의 독성이 있는 두꺼운 껍질을 제거하고 내부 겔만 분리하여 사용하는데, 이는 실제 알로에 관련 제품 생산 단계에서 번거로운 공정에 해당한다. 반면 알로에 사포나리아는 생잎으로 쓰는 알로에 중 약성이 순하여 껍질째 사용이 가능하나, 알로에 속 식물 중 소형종에 속한다.Aloe vera is a representative species of aloe used for commercial and medicinal purposes. It is usually vulnerable only after three years of cultivation. However, because it is a tropical crop, domestic cultivation requires a relatively high cost, and imported products from foreign countries have a problem in that the quality is not constant due to uneven supply and demand imbalance. Aloe vera also removes toxic thick skin from the leaves and separates only the inner gel, which is a cumbersome process in the production of aloe-related products. Aloe saponaria, on the other hand, can be used as a peel because it is mild in aloe used as a raw leaf, but it belongs to a small species of plants of the genus Aloe.
따라서 생육 속도가 빠르고 생육 방법이 용이한 품종, 생육 지속 기간이 긴 품종 등 각 목적에 부합되는 다양한 알로에 품종의 개발이 요구되고 있으며, 이러한 요구 조건에 부합할 뿐만 아니라 국내 토양에 가장 알맞고, 산업적으로 활용이 용이할 수 있도록 가시와 껍질이 얇고 많은 즙을 가지는 알로에 신품종에 대한 육종이 절실히 요구되고 있다.Therefore, the development of various aloe varieties for each purpose, such as varieties with fast growth rate and easy growth method, and long growing period, is required.In addition to meeting these requirements, they are most suitable for domestic soil and industrially. There is an urgent need for breeding a new breed of aloe with thin thorns, skins, and much juice to facilitate its utilization.
한편, 최근 분자 생물학이 발달하면서 특정 DNA 염기 서열의 다형성을 탐색하고 이를 분자마커로 활용하는 기술은 환경의 영향을 받지 않고 연차간 변이가 거의 없기 때문에 유전자 지도 작성, 내병성 유전자의 맵핑(mapping), 품종 식별 등 다양한 분야에 활용 가능하게 되었다. 특히, 유전적 분자마커 기술 개발은 작물 육성에서 이용가치가 증가되고 있다. On the other hand, with the recent development of molecular biology, the technology of searching for polymorphisms of specific DNA sequences and using them as molecular markers is not influenced by the environment and there is little variation between them. Therefore, gene mapping, mapping of disease-resistant genes, It can be used in various fields such as breed identification. In particular, the development of genetic molecular marker technology is increasing the useful value in crop growth.
한국등록특허 제0133344호에는 '알로에의 대량 증식방법을 위한 조직배양법'이 개시되어 있고, 미국등록특허 제PP023913에는 'Aloe plant named Tiki Tahi'가 개시되어 있으나, 본 발명의 알로에 신품종 생장 및 이를 판별하기 위한 분자마커에 대해서는 개시된 바가 없다.Korean Patent No. 0133344 discloses a 'tissue culture method for mass propagation of aloe', and US Patent No. PP023913 discloses 'Aloe plant named Tiki Tahi', but the growth of new aloe species of the present invention and discrimination thereof are disclosed. No molecular markers have been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 알로에 베라와 사포나리아를 혼합식재하여 껍질이 얇고 대량 무성번식을 하는 자연교잡종을 선발해 신품종을 육성하고 형태적 및 분자유전학적 특성을 비교하여, 알로에 베라에 비해 식물체 높이가 낮고, 잎 수가 많으며, 연녹색의 잎 내에 흰색 반점을 가지며, 알로에 베라에 비해 잎의 껍질이 얇고 가시가 부드러우며, 무성번식에 의하여 대량번식이 가능한 알로에를 확인하고 이를 알로에 신품종 '생장(Saegjang)'으로 명명하여 본 발명을 완성하였다.The present invention is derived from the above requirements, by mixing aloe vera and saponaria planting natural hybrids with a thin shell and mass asexual breeding to cultivate new varieties and compare the morphological and molecular genetic characteristics Compared to aloe vera, the plant height is lower, the number of leaves is large, and there are white spots in the pale green leaves. Compared to the aloe vera, the leaf of the leaf is thinner and softer than the aloe vera. The new aloe varieties named 'Saegjang' to complete the present invention.
상기 과제를 해결하기 위해, 본 발명은 하기의 형태적 특징을 가지며, 연녹색의 잎 내에 흰색 반점을 가지고 알로에 베라에 비해 잎의 껍질이 얇고 가시가 부드러우며, 무성번식에 의하여 대량번식이 가능한 알로에 신품종 생장(Saengjang)을 제공한다: In order to solve the above problems, the present invention has the following morphological features, has a white spots in the leaves of light green, compared to the aloe vera, the leaf is thinner and softer thorns, a new breed of aloe capable of mass propagation by asexual breeding Saengjang offers:
(a) 식물체의 높이: 30~55cm(a) Height of plant: 30 ~ 55cm
(b) 식물체의 너비: 10~30cm(b) width of plant: 10 ~ 30cm
(c) 식물체의 잎 수: 18~22매(c) Number of leaves of plant: 18-22
(d) 잎의 길이: 30~40cm(d) leaf length: 30 ~ 40cm
(e) 잎의 너비: 4~6cm (e) leaf width: 4 ~ 6cm
(f) 잎의 두께: 15~20mm 및(f) leaf thickness: 15-20mm and
(g) 잎의 껍질 두께: 0.05~0.1mm.(g) Bark thickness of the leaf: 0.05-0.1 mm.
또한, 본 발명은 서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 및 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트로 구성되는 군으로부터 선택된 하나 이상의 프라이머 세트를 포함하는, 알로에 신품종 생장 판별용 프라이머 세트를 제공한다.The present invention also provides oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
또한, 본 발명은 알로에 식물 시료에서 게놈 DNA를 분리하는 단계; 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및 상기 증폭 산물을 검출하는 단계를 포함하는, 알로에 신품종 생장의 판별 방법을 제공한다.In addition, the present invention comprises the steps of separating genomic DNA from aloe plant samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; And it provides a method for determining a new breed of aloe, comprising the step of detecting the amplification product.
본 발명에 따른 알로에 신품종인 생장은 잎의 껍질이 얇고 가시가 부드러워 껍질을 제거하지 않고 알로에 잎을 껍질째 갈아 음용할 수 있으며, 성체 알로에 주변에서 어린 새순이 5~20개가 성장하여 새순 옮겨심기로 대량 번식이 가능하게 함으로써 알로에 재배 농가 소득 증대에 크게 기여할 수 있다.Growth of a new aloe varieties according to the present invention is thin leaves and soft thorns can be used to grind the aloe leaves without removing the bark and drinking, 5-20 young shoots grow around the adult aloe to plant a new shoot The ability to breed in large quantities can greatly contribute to the increase in aloe farmers' income.
또한, 본 발명에 따르면, 4가지 프라이머 세트를 이용하여 알로에 신품종 생장 판별이 가능하였으며, 본 발명에 사용된 프라이머 세트는 알로에 원산지에 따른 품종판별, 순도 검정, 품종보호와 종자 관리체계 확립을 위한 품종 검정에 활용될 수 있을 것이다.In addition, according to the present invention, it was possible to determine the growth of new aloe varieties using the four primer sets, the primer set used in the present invention varieties for the determination of varieties according to the origin of aloe, purity test, breed protection and seed management system It can be used for testing.
도 1은 1년생 신품종 생장(좌)와 대조품종인 베라(우)의 생육 모양을 비교하여 나타내는 사진이다.1 is a photograph showing the growth of the new year-old varieties (left) and Vera (right) as a control variety.
도 2는 신품종 생장(좌)와 대조품종인 베라(우)의 생육상태를 비교하여 나타내는 사진이다. Figure 2 is a photograph showing the growth state of the new breed growth (left) and the control varieties Vera (right).
도 3은 신품종 생장의 포장 전경을 나타내는 사진이다. Figure 3 is a photograph showing a packaging foreground of new breed growth.
도 4는 알로에 베라(좌)와 신품종 생장(우)의 잎 단면을 나타내는 사진이다.Figure 4 is a photograph showing the leaf cross section of aloe vera (left) and new varieties growth (right).
도 5는 알로에 베라(좌)와 신품종 생장(우)의 꽃 형태를 나타내는 사진이다.5 is a photograph showing the flower form of aloe vera (left) and new breed growth (right).
도 6은 알로에 베라(A), 알로에 아보레센스(B), 알로에 사포나리아(C) 및 신품종 생장(D)을 나타내는 사진이다.6 is a photograph showing aloe vera (A), aloe aborescens (B), aloe saponaria (C), and new breed growth (D).
도 7은 알로에 신품종 생장을 포함한 다양한 알로에 종간 유전적 상관관계를 보여주는 UPGMA 페노그램(phenogram)을 나타낸 것이다.FIG. 7 shows UPGMA phenograms showing genetic correlations between various aloe species, including aloe new species growth.
본 발명의 목적을 달성하기 위하여, 본 발명은 하기 (a) 내지 (g)의 형태적 특징을 가지며, 연녹색의 잎 내에 흰색 반점을 가지고 알로에 베라에 비해 잎의 껍질이 얇고 가시가 부드러우며, 무성번식에 의하여 대량번식이 가능한 알로에 신품종 생장(Saengjang)을 제공한다: In order to achieve the object of the present invention, the present invention has the morphological characteristics of the following (a) to (g), has white spots in the leaves of light green, the leaf peel is thinner than the aloe vera, soft spines, asexual Providing a new breed of aloe Saengjang, which is capable of mass breeding by breeding:
(a) 식물체의 높이: 30~55cm(a) Height of plant: 30 ~ 55cm
(b) 식물체의 너비: 10~30cm(b) width of plant: 10 ~ 30cm
(c) 식물체의 잎 수: 18~22매(c) Number of leaves of plant: 18-22
(d) 잎의 길이: 30~40cm(d) leaf length: 30 ~ 40cm
(e) 잎의 너비: 4~6cm(e) leaf width: 4 ~ 6cm
(f) 잎의 두께: 15~20mm 및(f) leaf thickness: 15-20mm and
(g) 잎의 껍질 두께: 0.05~0.1mm.(g) Bark thickness of the leaf: 0.05-0.1 mm.
본 발명의 일 구현 예에 따른 신품종 생장은 알로에 베라와 알로에 사포나리아를 혼합식재하는 방식으로 재배한 것으로 자연교잡종을 유도하고 실생묘를 획득해 무성번식을 통해 계통 육성하였다.New breed growth according to one embodiment of the present invention was cultivated in a mixed planting method of aloe vera and aloe saponaria to induce natural hybrids and obtain seedlings to grow lines through asexual breeding.
알로에 베라(Aloe vera)는 1~2년 동안은 성장이 더디지만 2년이 지나면 성장이 빠른 편에 속하며, 꽃은 일년 중 계절에 관계없이 긴 꽃대가 올라와 노랑 또는 적등색 꽃을 피운다. 잎의 색은 녹색이고 잎의 크기는 60~90cm이며 잎 1개가 0.5~1.5kg 정도이며 5~6년생 한 포기를 수확할 때 3~6 잎을 수확할 수 있다. 번식은 어미포기의 옆에서 2~3개의 새끼가 돋아나 떼어심기를 한다. 껍질째 먹으면 쓴맛이 강하며 껍질을 벗기고 먹게 되면 무미(無味) 무취(無臭)의 젤리 질이 많은 품종이기에 껍질을 벗겨서 사용하는데 많이 쓰인다.Aloe Vera (Aloe vera) belongs to the fastest growing section during 1-2 years after growing only deodiji two years, the flowers come up with a long spine, regardless of the season of the year smokes a yellow or reddish orange flowers. The leaves are green, the leaves are 60 ~ 90cm in size, one leaf is about 0.5 ~ 1.5kg, and 3 ~ 6 leaves can be harvested when harvesting 5 ~ 6 year old one. Breeding is carried out by two or three young sprouts next to the mothers. When eaten, the bitter taste is strong, and when peeled and eaten, it's tasteless and odorless.
알로에 사포나리아(Aloe saponaria)는 알로에 중에서 소형종에 속하며 땅에 붙어 자라고 잎이 연하면서 하얀 점이 있다. 꽃대는 길게 위로 올라오며 적등색의 꽃을 피운다. 잎의 크기는 30~60cm, 1kg의 잎의 숫자는 3~5개 정도된다. 알로에 사포나리아는 3~5개의 새끼를 친다. 껍질에 쓴맛이 없어 가시만 따내고 껍질째 많이 사용하다. Aloe saponaria is a small species of aloe that grows on the ground and has light leaves with white spots. Flower beds rise long and bloom red-colored flowers. The leaf size is 30 ~ 60cm and the number of leaves of 1kg is 3 ~ 5. Aloe saponaria strikes 3-5 cubs. The bark has no bitter taste, so only the thorns are used and the bark is used a lot.
본 발명의 일 구현 예에 따른 알로에 신품종 생장과 알로에 베라의 생육 특성을 비교한 결과는 표 1 및 도 1 내지 5에 나타내었다. 본 발명의 일 구현 예에 따른 알로에 생장은 몸체의 높이는 30~55cm로 자라며, 줄기는 길고 부드러운 잎이 포기방식으로 성장하며, 1-2년 성장 후 거의 성장하지 않으며, 생장의 잎은 길이 40cm, 너비 6cm 정도의 엷은 녹색 바탕에 흰색 반점을 가지며, 잎의 가장자리엔 작고 약한 가시를 가진다. 또한 알로에 생장은 잎몸의 뿌리근처에 어린 묘목이 5-20개 이상 성장하고, 잎 껍질의 두께는 베라보다 얇으며, 꽃대가 가지를 치지 않는 특징을 가질 수 있다.The results of comparing the growth characteristics of the new aloe varieties growth and aloe vera according to an embodiment of the present invention are shown in Table 1 and Figures 1 to 5. Aloe growth according to an embodiment of the present invention grows in the height of the body is 30 ~ 55cm, the stem is long and tender leaves grow in abandonment method, almost no growth after 1-2 years of growth, the leaves of growth 40cm, It has a light green background about 6cm wide with white spots, and leaves have small, weak spines. In addition, aloe growth is characterized by the growth of more than 5-20 young seedlings near the root of the leaf body, the thickness of the leaf bark is thinner than the vera, and the flower stalk may not have branches.
본 발명의 알로에 신품종 생장은 캘러스를 유기하여 2015년 03월 30일자로 한국생명공학연구원 미생물자원센터에 수탁번호 KCTC 12780BP로 기탁되었다.New aloe varieties growth of the present invention was deposited on the microorganism resource center of Korea Research Institute of Bioscience and Biotechnology and deposited on callus KCTC 12780BP on March 30, 2015.
본 발명의 일 구현 예에 따른 조직 배양은 본 발명에 따른 특징이 있는 식물을 재생할 수 있다. 이러한 조직 배양에 사용되는 재생 세포는 미숙 배아, 원형질, 분열 조직 세포, 캘러스, 화분, 잎, 화분낭, 뿌리, 뿌리 끝 또는 꽃일 수 있으며, 바람직하게는 캘러스이다. Tissue culture according to an embodiment of the present invention can regenerate a plant characterized by the present invention. The regenerative cells used for such tissue culture may be immature embryos, plasma, meristem cells, callus, pollen, leaves, pollen sacs, roots, root tips or flowers, preferably callus.
본 발명은 또한, 서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 및 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트로 구성되는 군으로부터 선택된 하나 이상의 프라이머 세트를 포함하는, 알로에 신품종 생장 판별용 프라이머 세트를 제공한다.The present invention also provides oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And one or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
상기 올리고뉴클레오티드는 서열번호 1의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상, 21개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드 및 서열번호 2의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드일 수 있다.The oligonucleotide is an oligonucleotide consisting of at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides in the sequence of SEQ ID NO: 1 and at least 17, 18 in the sequence of SEQ ID NO: 2. At least 19 and at least 20 contiguous nucleotide segments.
또한, 상기 올리고뉴클레오티드는 각각 서열번호 3 및 서열번호 4의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드일 수 있다.In addition, the oligonucleotide may be an oligonucleotide consisting of fragments of at least 17, at least 18, at least 19, at least 20 consecutive nucleotides in the sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
또한, 상기 올리고뉴클레오티드는 서열번호 5의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상, 21개 이상, 22개 이상, 23개 이상, 24개 이상, 25개 이상, 26개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드 및 서열번호 6의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드일 수 있다.In addition, the oligonucleotide is at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, and at least 26 in the sequence of SEQ ID NO. Oligonucleotides consisting of fragments of at least one continuous nucleotide and oligonucleotides consisting of fragments of at least 17, at least 18, at least 19, at least 20 consecutive nucleotides in the sequence of SEQ ID NO: 6.
또한, 상기 올리고뉴클레오티드는 서열번호 7의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드 및 서열번호 8의 서열 내의 17개 이상, 18개 이상, 19개 이상, 20개 이상, 21개 이상, 22개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드일 수 있다.In addition, the oligonucleotide is an oligonucleotide consisting of at least 17, at least 18, at least 19, at least 20 consecutive nucleotide segments in the sequence of SEQ ID NO: 7 and at least 17, at least 18, in the sequence of SEQ ID NO: 8, Oligonucleotides consisting of segments of at least 19, at least 20, at least 21, at least 22 consecutive nucleotides.
본 발명의 일 구현예에 따른 올리고뉴클레오티드 프라이머 세트는 상기 4가지 프라이머 세트 중 2가지 프라이머 세트 조합, 3가지 프라이머 세트 조합, 4가지 프라이머 세트 조합이 가능하며, 가장 바람직하게는 4가지 프라이머 세트 조합이다. 따라서, 본 발명의 올리고뉴클레오티드 프라이머 세트는 가장 바람직하게는 서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트를 포함하는 알로에 신품종 생장 판별용 분자 표지 프라이머 세트이다.Oligonucleotide primer set according to an embodiment of the present invention is a combination of two primer sets, three primer set combinations, four primer set combinations of the four primer sets, most preferably four primer set combinations . Thus, the oligonucleotide primer sets of the present invention are most preferably oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; It is a set of molecular label primers for the determination of new growth of aloe comprising the oligonucleotide primer set of SEQ ID NO: 7 and 8.
본 발명에 있어서, "프라이머"는 카피하려는 핵산 가닥에 상보적인 단일 가닥 올리고뉴클레오티드 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 의존할 것이다.In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primers should allow to start the synthesis of extension products. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.
서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 및 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트는 각각 엽록체의 matK, trnL-F, rbcL cpDNA와 핵 ITS DNA 서열을 증폭하여, 이를 알로에 신품종 생장의 판별용 마커로 활용할 수 있었다(도 5). 또한 상기 마커를 이용하여 알로에 품종을 판별할 수 있음을 확인하였다.Oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And oligonucleotide primer sets of SEQ ID NOs: 7 and 8 amplify the matK, trnL-F, rbcL cpDNA and nuclear ITS DNA sequences of chloroplasts, respectively, and could be used as markers for the determination of new aloe species growth (FIG. 5). In addition, it was confirmed that the marker can be used to determine the aloe variety.
식물의 엽록체 게놈(cpDNA)은 한 개의 세포에 동일한 엽록체 게놈이 수백 개 이상 존재하기 때문에 핵 게놈에 비해 상대적으로 분석이 용이하고, 양친 유전자간의 교잡 없이 모계 유전을 통해서만 후대로 전이되어 종내 변이가 거의 없기 때문에 특정 종 혹은 동일 종내 특정 계통을 구분하는 마커로서 가장 신뢰할 수 있으면서도 신속하게 분석할 수 있는 마커이다. 본 발명에서는 matK, trnL-F, rbcL cpDNA 지역을 대상으로 염기 다형성을 조사하였다. The plant's chloroplast genome (cpDNA) is easier to analyze than the nuclear genome because there are hundreds of identical chloroplast genomes in a single cell. It is a marker that distinguishes a specific species or a specific strain within the same species, and thus is the most reliable and quick analysis marker. In the present invention, the base polymorphism of the matK, trnL-F, rbcL cpDNA region was investigated.
"ITS(Internal Transcribed Spacer)"는 핵 리보솜 DNA(nuclear ribosomal DNA)로부터 전사되나 단백질로 발현되지는 않는 부위이다. ITS 염기서열은 식물, 균류, 곤충 등을 포함한 생물의 다양한 배열의 하위 분류군 수준의 계통을 추론하기 위해 이용되어 왔다. ITS 지역은 하위 분류군 수준에서 높은 정보를 가질 뿐만 아니라 피자식물의 보존된 염기서열 패턴과 높은 정렬성을 나타내는 것으로 알려져 있다. ITS 지역은 적은 크기의 프라이머들이 rRNA 유전자로부터 강하게 보존된 주변 염기서열을 나타내기 때문에 대부분의 피자식물에서 쉽게 증폭된다. 본 발명에서는 서열번호 7 및 8로 증폭된 ITS 지역을 대상으로 염기 다형성을 조사하였다.An "Internal Transcribed Spacer" is a site that is transcribed from nuclear ribosomal DNA but not expressed as a protein. ITS sequences have been used to infer subclass levels of a diverse array of organisms, including plants, fungi, insects, and the like. The ITS region is known not only to have high information at the subclass level, but also to exhibit conserved sequence patterns and high alignment of pizza plants. The ITS region is easily amplified in most pizza plants because small primers exhibit a peripheral sequence that is strongly conserved from the rRNA gene. In the present invention, the base polymorphism was investigated in the ITS region amplified by SEQ ID NO: 7 and 8.
본 발명은 또한, 알로에 식물 시료에서 게놈 DNA를 분리하는 단계; 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및 상기 증폭 산물을 검출하는 단계를 포함하는, 알로에 신품종 생장의 판별 방법을 제공한다.The invention also comprises the steps of separating genomic DNA from aloe plant samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of the present invention; And it provides a method for determining a new breed of aloe, comprising the step of detecting the amplification product.
본 발명의 방법은 알로에 식물 시료에서 게놈 DNA를 분리하는 단계를 포함한다. 상기 시료에서 게놈 DNA를 분리하는 방법은 당업계에 공지된 방법을 이용할 수 있으며, 예를 들면, CTAB 방법을 이용할 수도 있고, wizard prep 키트(Promega 사)를 이용할 수도 있다.The method includes separating genomic DNA from aloe plant samples. As a method for separating genomic DNA from the sample, a method known in the art may be used. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used.
상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 일 실시예에 따른 하나 이상의 올리고뉴클레오티드 프라이머 세트를 프라이머로 이용하여 증폭 반응을 수행하여 표적 서열을 증폭할 수 있다. 표적 핵산을 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. 이 중에서, PCR이란 중합효소를 이용하여 표적 핵산에 특이적으로 결합하는 프라이머 쌍으로부터 표적 핵산을 증폭하는 방법이다. 이러한 PCR 방법은 당업계에 잘 알려져 있으며, 상업적으로 이용가능한 키트를 이용할 수도 있다.Using the isolated genomic DNA as a template, one or more oligonucleotide primer sets according to one embodiment of the present invention may be used as a primer to amplify a target sequence. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification systems, There are any suitable methods for amplifying via strand displacement amplification or Qβ replication or for amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a pair of primers specifically binding to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.
본 발명의 방법에 있어서, 상기 증폭된 표적 서열은 검출가능한 표지 물질로 표지될 수 있다. 일 구현 예에서, 상기 표지 물질은 형광, 인광 또는 방사성을 발하는 물질일 수 있으나, 이에 제한되지 않는다. 바람직하게는, 상기 표지 물질은 Cy-5 또는 Cy-3이다. 표적 서열의 증폭시 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지될 수 있다. 또한, 방사성 물질을 이용한 표지는 PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하면 증폭 산물이 합성되면서 방사성이 증폭 산물에 혼입되어 증폭 산물이 방사성으로 표지될 수 있다. 표적 서열을 증폭하기 위해 이용된 프라이머 세트는 상기에 기재된 바와 같다.In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material emitting fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as 32 P or 35 S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. The primer set used to amplify the target sequence is as described above.
본 발명의 방법은 상기 증폭 산물을 검출하는 단계를 포함한다. 상기 증폭 산물의 검출은 시퀀싱, 모세관 전기영동, DNA 칩, 겔 전기영동, 방사성 측정, 형광 측정 또는 인광 측정을 통해 수행될 수 있으나, 이에 제한되지 않는다. 증폭 산물을 검출하는 방법 중의 하나로서, 겔 전기영동을 수행할 수 있으며, 겔 전기영동은 증폭 산물의 크기에 따라 아크릴아미드 겔 전기영동 또는 아가로스 겔 전기영동을 이용할 수 있다. 또한, 모세관 전기영동을 수행할 수 있다. 모세관 전기영동은 예를 들면, ABi Sequencer를 이용할 수 있다. 또한, 형광 측정 방법은 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지되며, 이렇게 표지된 형광은 형광 측정기를 이용하여 측정할 수 있다. 또한, 방사성 측정 방법은 PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하여 증폭 산물을 표지한 후, 방사성 측정기구, 예를 들면, 가이거 계수기(Geiger counter) 또는 액체섬광계수기(liquid scintillation counter)를 이용하여 방사성을 측정할 수 있다.The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through sequencing, capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of methods for detecting amplification products, gel electrophoresis may be performed, and gel electrophoresis may use acrylamide gel electrophoresis or agarose gel electrophoresis depending on the size of the amplification product. Capillary electrophoresis can also be performed. Capillary electrophoresis can use, for example, an ABi Sequencer. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as 32 P or 35 S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 알로에 신품종 생장의 육성과정 및 수집Example 1 Growth and Collection of New Aloe Varieties
가시와 껍질이 연하여 잎을 껍질째 사용할 수 있는 알로에 신품종 육성을 하고자 알로에 베라와 알로에 사포나리아를 전북 완주군 용진면 지역에 소재한 온실에서 방임수분의 환경여건을 인위적으로 조성하여 자연교잡이 이루어지도록 하였다. 1981년 알로에 베라와 알로에 사포나리아가 혼합 식재된 온실에서 껍질이 얇고 가시가 약한 흰 반점의 연녹색 잎을 갖는 실생묘을 획득하여 1981년도부터 계통 육성하게 되었으며 선발교잡종의 특성이 일정하게 유지됨을 확인하였다. 이들 알로에는 전북 완주군 용진면 지역에 소재한 600m2 넓이의 온실에서 차광막을 설치하여 직사광선을 피하고 반양지 상태로 봄 내지 가을에는 20~35℃, 겨울에는 10~15℃을 유지하며 재배하여 영양번식의 방법으로 증식하였다.In order to cultivate new varieties of aloe that can be used for peeling leaves with soft thorns and husks, aloe vera and aloe saponaria were artificially created in the greenhouse located in Yongjin-myeon, Wanju-gun, Jeonbuk, to artificially create environmental conditions for neglected moisture. . In 1981, seedlings with light green leaves with thin skins and thorny white spots were obtained in a greenhouse mixed with aloe vera and aloe saponaria, and since 1981, the lineage has been cultivated and the characteristics of the selection hybrids are maintained. These aloes are planted in a 600m 2 greenhouse located in Yongjin-myeon, Wanju-gun, Jeollabuk-do to avoid direct sunlight, and cultivate them at a cultivation rate of 20 ~ 35 ℃ in spring and autumn, and 10 ~ 15 ℃ in winter in banyang. Proliferated.
1981년~2013년 육성과정에서 한 차례도 병충해에 노출되지 않았으며, 이형체가 발현되지 않고 동일체만 성장 및 번식하여 안정성을 확보하고 있고, 외관상 균일성을 유지하였다. 또한, 발육상태가 좋고 병변현상이 발현되지 않아 우수한 계통임을 확인하기에 이를 생장(saengjang)으로 명명하였다.During the 1981-2013 breeding process, it was not exposed to pests at all, and only the same body was grown and propagated to ensure stability, and uniformity was maintained. In addition, it was named saengjang because it is well developed and confirmed that it is an excellent line because no lesion phenomenon is expressed.
<실시예 2> 알로에 신품종 생장의 형태적 변이 분석 Example 2 Morphological Variation Analysis of New Aloe Variety Growth
상기 실시예 1에서 선별된 신품종 생장과 대조품종인 베라의 형태적인 특성을 포장에서 실험 관찰하였으며, 그 결과를 하기 표 1 및 도 1 내지 5에 나타내었다.Experimental observation of the morphological characteristics of the new breed growth and control varieties selected in Example 1 in the packaging, the results are shown in Table 1 and Figures 1 to 5 below.
표 1 신품종 생장과 대조품종 베라와의 형태적 특징 비교
특징 생장 대조품종(베라)
식물체: 높이(cm) 52 85
식물체: 너비(cm) 22 60
식물체: 잎 수(매) 18~20 16~18
잎: 길이(cm) 40 60
잎: 너비(cm) 6 10
잎: 두께(mm) 15~20 20~30
잎: 어린 잎의 색 연녹색 녹색
잎: 색 연녹색 진한녹색
잎: 무늬(반점) 유무 유 무
잎: 무늬 색 흰색 반점
잎: 껍질 두께(mm) 0.05~0.1 0.1~0.3
Table 1 Comparison of Morphological Characteristics between New Variety Growth and Control Vera
Characteristic growth Control variety (vera)
Plant: Height (cm) 52 85
Plants: Width (cm) 22 60
Plants: Number of leaves (hawks) 18-20 16-18
Leaf: Length (cm) 40 60
Leaf: Width (cm) 6 10
Leaf: thickness (mm) 15-20 20-30
Leaf: color of young leaf Light green green
Leaf: color Light green Dark green
Leaf: With or without pattern U radish
Leaf: Pattern Color White spots
Leaf: Peel Thickness (mm) 0.05 ~ 0.1 0.1-0.3
본 발명의 신품종 생장의 지상부는 베라와 사포나리아의 외형적 특성을 발현하였다. 신품종 생장은 외관상 제일 유사한 품종인 베라를 대상으로 형태적 특성을 대조하였다.The ground part of the new breed growth of the present invention expressed the external characteristics of the vera and saponaria. Growth of new varieties was compared to morphological characteristics of Vera, the most similar variety in appearance.
형태적 특성 조사 결과, 생장은 몸체의 높이는 30~55cm로 자라며, 줄기는 길고 부드러운 잎이 포기방식으로 성장함을 확인하였다. 생장은 1-2년 성장 후 거의 성장하지 않으나, 대조품종인 베라는 1년 성장 후에도 계속적으로 크게 성장을 하였다(도 1). As a result of morphological characteristics, the growth of the body grows to 30 ~ 55cm in height, and the stem is long and the soft leaves grow in the aeration method. Growth was hardly grown after 1-2 years of growth, but the control variety, Vera, continued to grow significantly after 1 year of growth (FIG. 1).
생장의 잎은 길이 40cm, 너비 6cm 정도의 엷은 녹색 바탕에 흰색 반점을 가지며 잎의 가장자리엔 작고 약한 가시를 가짐을 확인하였다. 반면 알로에 베라의 경우 가시가 강하고 진한 녹색을 띠며 잎에 무늬가 없는 것이 차이를 나타냈다(표 1, 도 1 및 2).The leaves of growth had white spots on pale green background about 40cm long and 6cm wide and small, weak spines at the edges of the leaves. On the other hand, aloe vera showed a strong spine, dark green color, and no leaf pattern (Table 1, FIGS. 1 and 2).
생장의 경우 잎몸의 뿌리근처에 어린 묘목이 5-20개 이상 관찰되었으나(도 3), 베라의 경우에는 어린 묘목이 2-3개 성장하였다. 이는 알로에 신품종 생장이 대량 무성번식이 가능함을 나타내었다.In the case of growth, more than 5-20 young seedlings were observed near the root of the leaf body (FIG. 3), but in the case of Vera, 2-3 young seedlings grew. This indicated that a new breed of aloe is capable of mass asexual reproduction.
또한, 잎 껍질의 두께는 생장이 베라보다 얇았다(도 4).In addition, the thickness of the leaf peel was thinner than the growth of Vera (Fig. 4).
도 5에서 보이는 바와 같이 알로에 베라는 꽃대가 가지를 치는 형태를 보이는 반면, 생장의 경우 꽃대가 가지를 치지 않는 차이를 나타냈다.As shown in FIG. 5, the aloe vera showed a form in which the flower stalks branched, but in the case of growth, the flower stalks did not branch.
<실시예 3> 알로에 게놈 DNA 추출 및 cpDNA와 ITS 염기변이에 근거한 신품종 알로에 생장의 유전적 상관관계 분석Example 3 Genetic Correlation Analysis of New Aloe Growth Based on Aloe Genomic DNA Extraction and cpDNA and ITS Base Mutations
한국산 알로에 3종인 알로에 베라(A. vera), 알로에 아보레센스(A. arborescens), 알로에 사포나리아(A. saponaria) 및 알로에 신품종 생장을 게놈 DNA 추출 샘플로 사용하였다(도 6). 알로에의 잎 100mg을 채취한 후 액체 질소로 분쇄하여 변형된 CTAB 방법을 사용하여 DNA를 추출하였다. 추출한 DNA는 0.8% 아가로스 겔에 로딩하여 DNA 상태를 확인하였다.Three Korean aloes, A. vera , Aloe arborescens , Aloe saponaria and Aloe new varieties were used as genomic DNA extraction samples (FIG. 6). 100 mg of leaves of aloe were taken and pulverized with liquid nitrogen, and DNA was extracted using a modified CTAB method. The extracted DNA was loaded on a 0.8% agarose gel to confirm the DNA state.
본 발명에 사용된 프라이머 서열 및 어닐링 온도(annealing temperature)는 표 2에서 보는 바와 같다. 3가지 엽록체 DNA 지역(matK, trnL-F, rbcL) 및 핵 ITS DNA 지역이 증폭되었다. PCR 반응액은 총 반응액 20㎕로, 주형 DNA 25ng, 1 X PCR 버퍼(buffer), 2.5mM dNTP, 1U Taq DNA 중합효소(polymerase), 각각 20 pmol의 프라이머 세트(하기 표 2 참조)를 첨가하였다. PCR 반응에는 GeneAmp PCR system 2700 thermal cycler를 사용하였으며, PCR 반응조건은 95℃(3분), [94℃(30초), 표 2의 해당 어닐링온도(30초), 72℃(1분), 35 cycle], 72℃(10분)이었다. PCR 반응 산물은 아가로스 겔에서 추출하여 ABI prism 3700 sequencer로 염기서열을 분석하였다.Primer sequences and annealing temperatures used in the present invention are as shown in Table 2. Three chloroplast DNA regions (matK, trnL-F, rbcL) and nuclear ITS DNA regions were amplified. The PCR reaction solution was 20 μl of total reaction solution, with 25ng of template DNA, 1 × PCR buffer, 2.5mM dNTP, 1U Taq DNA polymerase, and 20 pmol primer set (see Table 2 below) respectively. It was. GeneAmp PCR system 2700 thermal cycler was used for PCR reaction, PCR reaction conditions were 95 ℃ (3 minutes), [94 ℃ (30 seconds), the corresponding annealing temperature of Table 2 (30 seconds), 72 ℃ (1 minutes), 35 cycles], 72 ° C. (10 minutes). The PCR reaction product was extracted from agarose gel and analyzed for sequencing by ABI prism 3700 sequencer.
표 2 본 발명에 사용된 분자 표지, 프라이머 서열 및 어닐링 온도
분자 표지 프라이머 서열(서열번호) 길이(bp) 어닐링온도(℃)
matK 5'-TAATTTACGATCAATTCATTC-3'(서열번호 1)5'-GTTCTAGCACCAGAAAGTCG-3'(서열번호 2) 868 48
trnL-F 5'-GGTTCAAGTCCCTCTATCCC-3'(서열번호 3)5'-ATTTGAACTGGTGACACGAG-3'(서열번호 4) 481 58
rbcLa 5'-ATGTCACCACAAACAGAGACTAAAGC-3'(서열번호 5)5'-GTAAAATCAAGTCCACCRCG-3'(서열번호 6) 537 58
nrITS 5'-TCCTCCGCTTATTGATATGC-3'(서열번호 7)5'-GGAAGTAAAAGTCGTAACAAGG-3'(서열번호 8) 680 56
TABLE 2 Molecular Labels, Primer Sequences, and Annealing Temperatures Used in the Invention
Molecular label Primer Sequence (SEQ ID NO) Length (bp) Annealing Temperature (℃)
matK 5'-TAATTTACGATCAATTCATTC-3 '(SEQ ID NO: 1) 5'-GTTCTAGCACCAGAAAGTCG-3' (SEQ ID NO: 2) 868 48
trnL-F 5'-GGTTCAAGTCCCTCTATCCC-3 '(SEQ ID NO: 3) 5'-ATTTGAACTGGTGACACGAG-3' (SEQ ID NO: 4) 481 58
rbcLa 5'-ATGTCACCACAAACAGAGACTAAAGC-3 '(SEQ ID NO: 5) 5'-GTAAAATCAAGTCCACCRCG-3' (SEQ ID NO: 6) 537 58
nrITS 5'-TCCTCCGCTTATTGATATGC-3 '(SEQ ID NO: 7) 5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID NO: 8) 680 56
알로에 신품종 생장을 판별하기 위해 선발된 4 가지 분자 표지(trnL-F, rbcL, matK, ITS)에 대한 염기서열 alignment 분석 및 편집은 CLUSTAL X와 BioEdit를 사용하였으며 품종간 유전적 거리 분석은 UPGMA 트리를 만들어 수행하였다.Sequence alignment analysis and editing of four molecular markers ( trnL-F, rbcL, matK , ITS) selected to determine the growth of new aloe species were performed using CLUSTAL X and BioEdit. Made and performed.
생장과 한국산 알로에에 더하여, 기존에 알려진 다른 알로에 속 식물들의 염기서열은 Genbank 데이타베이스에서 다운로드받아서 유전적 상관관계 분석에 추가하였다(표 3).In addition to growth and Korean aloes, the genetic sequences of other known aloe plants can be downloaded from the Genbank database for genetic correlation. Added to analysis (Table 3).
표 3
Figure PCTKR2015009884-appb-T000001
 TABLE 3
Figure PCTKR2015009884-appb-T000001
3개의 엽록체 matK, trnL-F, rbcL cpDNA 염기서열과 1개의 핵 ITS DNA 염기서열을 근거로 한국산 알로에 3종인 알로에 베라(A. vera), 알로에 아보레센스(A. arborescens), 알로에 사포나리아(A. saponaria) 및 알로에 신품종 생장의 유전적 상관관계를 알고자 수행한 결과, 전체 2,420bp 서열이 증폭되었다(표 4). 두 개의 삽입-결실(indel)이 trnL 지역에서 확인되었고, 또한 여러 개의 종 특이적인 염기자위가 전체 29개의 최소변이 정보지역(parsimonious informative site)에서 확인되었다. 조사된 한국산 4종간에는 148곳 염기변이 지역이 있었으며, 세계산 알로에 종들을 포함하여 비교해서는 170개의 변이지역 중 75개의 최소변이 지역이 확인되었다. 도 7의 UPGMA를 이용한 페노그램(phenogram)에서 알로에 생장은 알로에 베라(A. vera)와 가장 가깝게 유집되었다. 알로에 생장은 아직까지 보고된 어떤 종류의 알로에 속 내의 종들과 형태적 및 유전적으로 일치하지 않았다. 조사된 알로에 종들의 유집분석 결과는 기존의 연구결과와 일치하였다.Based on three chloroplast matK, trnL-F, rbcL cpDNA sequences and one nuclear ITS DNA sequence, three Korean aloes, A. vera , A. arborescens and Aloe saponaria ( A. saponaria ) and aloe A new breed growth was performed to determine the genetic amplification, the entire 2,420bp sequence was amplified (Table 4). Two indels were identified in the trnL region, and several species-specific nucleotides were identified in all 29 parsimonious informative sites. Among the four Korean species examined, there were 148 mutant regions, and 75 of the 170 mutant regions were identified, including the world's aloe species. In the phenogram using UPGMA of FIG. 7, aloe growth was most closely collected with A. vera . Aloe growth is not yet reported in any kind of aloe It was not morphologically and genetically consistent with species in the genus. The results of the collection analysis of the investigated aloe species were in agreement with previous studies.
표 4 본 발명에서 증폭된 분자 표지의 특성
분자 표지 길이(bp) 평균GC율(%) 변이 사이트 최소변이 정보지역
trnL-F 481 29.1 45 25
rbcL 537 43.2 16 8
matK 868 30.7 39 18
ITS 680 62.7 81 27
조합(trnL-F, rbcL, matK) 1740 33.6 90 48
조합(trnL-F, rbcL, matK, ITS) 2420 41.6 170 75
Table 4 Characterization of Amplified Molecular Labels in the Present Invention
Molecular label Length (bp) Average GC Rate (%) Mutation site Minimum Variation Information Area
trnL-F 481 29.1 45 25
rbcL 537 43.2 16 8
matK 868 30.7 39 18
ITS 680 62.7 81 27
Combination ( trnL-F, rbcL, matK ) 1740 33.6 90 48
Combination ( trnL-F, rbcL, matK , ITS) 2420 41.6 170 75
<실시예 4> 알로에 신품종 생장의 조직배양에 의한 캘러스 유도 및 기탁Example 4 Callus Induction and Deposit by Tissue Culture of Aloe New Breed Growth
알로에 생장 조직을 절취하여 캘러스(callus)를 배양하여 2015년 03월 30일자로 한국생명공학연구원 미생물자원센터에 수탁번호 KCTC 12780BP로 기탁되었다.The aloe growth tissue was cut and cultured callus was deposited on March 30, 2015 with the accession number KCTC 12780BP to the Korea Institute of Bioscience and Biotechnology.
알로에 캘러스 유도는 하기 방법으로 수행하였다. 알로에 모종 뿌리를 포함한 줄기가 밀집된 부위만을 절단하였다. 절단된 알로에 시료를 10% 상업용 락스 용액에 약 20분간 흔들어주면서 표면 살균을 실시하였다. 표면 살균을 마친 알로에 시료를 멸균수로 3회 세척하였다. 상기의 시료를 무균 작업대내에서 멸균 여과지를 이용하여 남아 있는 수분을 제거한 다음 메스를 이용하여 약 5 mm 크기로 절단하여 배지에 치상함으로써 캘러스를 유도하였다. 캘러스 유도 배지는 MS (Murashige & Skoog) 배지에 3% 설탕, 0.4% 겔라이트(gelrite), 0.4mg/L 티아민(thiamine)-HCl, 100mg/L 미오이노시톨(myoinositol), 1 mg/L 2,4-D 및 0.3mg/L 키네틴(kinetin)이 첨가된 배지를 기본 배지로 사용하였다. 약 3주 배양 후 2,4-D가 첨가된 배지에 배양 중인 알로에 절편으로부터 백색의 세포괴가 형성되었으며 이후 동일한 배지에 계대배양을 통해 캘러스를 증식하였다.Aloe callus induction was performed by the following method. Only the areas where the stems including the aloe seedling roots were concentrated were cut. The cut aloe sample was subjected to surface sterilization with shaking for 10 minutes in a 10% commercial Lax solution. After the surface sterilization, the aloe sample was washed three times with sterile water. The sample was removed in a sterile workbench using a sterile filter paper to remove the remaining water, and then cut into a size of about 5 mm using a scalpel to induce the callus by incubating the medium. Callus induction medium was prepared in MS (Murashige & Skoog) medium with 3% sugar, 0.4% gelrite, 0.4 mg / L thiamine-HCl, 100 mg / L myoinositol, 1 mg / L 2, Medium with 4-D and 0.3 mg / L kinetin added was used as basal medium. After about 3 weeks of incubation, a white cell mass was formed from the aloe slices in culture in the medium to which 2,4-D was added. Then, callus was proliferated through passage in the same medium.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원Depositary: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC12780BPAccession number: KCTC12780BP
수탁일자 : 20150330Deposit date: 20150330

Claims (5)

  1. 하기 (a) 내지 (g)의 형태적 특징을 가지며, 연녹색의 잎 내에 흰색 반점을 가지고 알로에 베라에 비해 잎의 껍질이 얇고 가시가 부드러우며, 무성번식에 의하여 대량생산이 가능한 알로에 신품종 생장(Saengjang): It has the morphological characteristics of the following (a) to (g), has a white spot in the pale green leaves, compared to the aloe vera, the leaves are thinner and softer, and the mass production is possible due to asexual breeding (Saengjang ):
    (a) 식물체의 높이: 30~55cm(a) Height of plant: 30 ~ 55cm
    (b) 식물체의 너비: 10~30cm(b) width of plant: 10 ~ 30cm
    (c) 식물체의 잎 수: 18~22매(c) Number of leaves of plant: 18-22
    (d) 잎의 길이: 30~40cm(d) leaf length: 30 ~ 40cm
    (e) 잎의 너비: 4~6cm(e) leaf width: 4 ~ 6cm
    (f) 잎의 두께: 15~20mm 및(f) leaf thickness: 15-20mm and
    (g) 잎의 껍질 두께: 0.5~1mm.(g) Bark thickness of leaves: 0.5-1 mm.
  2. 제1항에 있어서, 상기 알로에 신품종 생장은 수탁번호 KCTC 12780BP로 기탁된 것을 특징으로 하는 알로에 신품종 생장.The new breed of aloe according to claim 1, wherein the new aloe variety is grown under accession number KCTC 12780BP.
  3. 서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 및 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트로 구성되는 군으로부터 선택된 하나 이상의 프라이머 세트를 포함하는, 알로에 신품종 생장 판별용 프라이머 세트.Oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And at least one primer set selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
  4. 제3항에 있어서, 서열번호 1 및 2의 올리고뉴클레오티드 프라이머 세트; 서열번호 3 및 4의 올리고뉴클레오티드 프라이머 세트; 서열번호 5 및 6의 올리고뉴클레오티드 프라이머 세트; 및 서열번호 7 및 8의 올리고뉴클레오티드 프라이머 세트를 포함하는, 알로에 신품종 생장 판별용 프라이머 세트.The method of claim 3, wherein the oligonucleotide primer set of SEQ ID NO: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And a set of primers for determining a new breed growth of aloe, including oligonucleotide primer sets of SEQ ID NOs: 7 and 8.
  5. 알로에 식물 시료에서 게놈 DNA를 분리하는 단계;Isolating genomic DNA from aloe plant samples;
    상기 분리된 게놈 DNA를 주형으로 하고, 제3항 또는 제4항의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of claim 3 or 4; And
    상기 증폭 산물을 검출하는 단계를 포함하는, 알로에 신품종 생장의 판별 방법.Detecting the amplification product, Aloe new breed growth method.
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