KR102448347B1 - Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility - Google Patents

Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility Download PDF

Info

Publication number
KR102448347B1
KR102448347B1 KR1020200188644A KR20200188644A KR102448347B1 KR 102448347 B1 KR102448347 B1 KR 102448347B1 KR 1020200188644 A KR1020200188644 A KR 1020200188644A KR 20200188644 A KR20200188644 A KR 20200188644A KR 102448347 B1 KR102448347 B1 KR 102448347B1
Authority
KR
South Korea
Prior art keywords
gene
male infertility
male
seq
radish
Prior art date
Application number
KR1020200188644A
Other languages
Korean (ko)
Other versions
KR20220056074A (en
Inventor
김성길
Original Assignee
전남대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 전남대학교 산학협력단 filed Critical 전남대학교 산학협력단
Publication of KR20220056074A publication Critical patent/KR20220056074A/en
Application granted granted Critical
Publication of KR102448347B1 publication Critical patent/KR102448347B1/en

Links

Images

Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

본 발명은 무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역에 대응하는 검출제제를 포함하는 무 웅성불임 판별용 조성물에 관한 것이다.The present invention relates to a composition for discriminating male infertility comprising a detection agent corresponding to a region of 7 kb to 8 kb upstream of the Rs295350 gene of radish.

Description

무 웅성불임 판별하는 방법 및 무 웅성불임 식물체{Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility}Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility

본 발명은 무 웅성불임 판별하는 방법 및 무 웅성불임 식물체에 관한 것이다.The present invention relates to a method for determining non-male infertility and a non-male infertility plant.

꽃 피는 식물에서, 화분의 형성과 이후의 수분 및 수정은 성적 생식을 위한 결정적인 단계이다. 소포자(microspore)의 화분 발달은 일련의 조정된 세포 사건을 포함하며, 성숙한 화분은 빠르게 발아하고 영양 세포에서 유래된 극 성장 화분관을 생산하며 두개의 정자 세포를 배아 주머니에 전달하는 특수 기능을 가지고 있다. 주요 작물의 생식에 있어서, 환경 스트레스에 가장 민감한 경우, 예를 들어 온도가 낮거나 높으면 화분의 발달, 발아 및 관의 성장에 결함이 생길 수 있으며, 임실률(spikelet fertility)과 곡물 수확량에 영향을 미친다.In flowering plants, pollen formation and subsequent pollination and fertilization are critical steps for sexual reproduction. Pollen development in microspores involves a series of coordinated cellular events, and mature pollen germinates rapidly, produces vegetatively-derived vegetatively grown pollen tubes, and has the specialized function of delivering two sperm cells to the embryonic sac. . In the reproduction of key crops, the most sensitive to environmental stresses, e.g. low or high temperatures, can lead to defects in pollen development, germination and tube growth, affecting spikelet fertility and crop yield. crazy

이에 따라, 무에서는 자가불화합성을 이용한 일대잡종의 채종이 일반화되어 있다. 그러나 자가불화합성을 이용할 경우에는 내혼계를 줄이는데 한계가 있다. 비록 자가불화합성이 잘 고정된 양친계를 사용하여도 노화수분에 의하여 얼마의 내혼계가 섞여 나오기 때문이다. 일대잡종 품종에 내혼계가 섞이면 종자의 품위가 떨어지고, 심한 경우는 소비자로부터 보상요구가 오게 되어 종자분규의 원인이 될 수 있다. 이에 대한 해결방안으로서 웅성불임성의 도입이 시도되고 있다. Accordingly, in radish, the hybridization using self-incompatibility is common. However, when self-incompatibility is used, there is a limit to reducing the internal hybrid system. This is because even if a parent system with well-fixed self-incompatibility is used, some of the inner hybrid system is mixed with aging moisture. If a hybrid breed is mixed with a hybrid breed, the quality of the seeds decreases, and in severe cases, compensation requests may come from consumers, which may cause seed disputes. As a solution to this, the introduction of male sterility is being attempted.

한편, 광범위하게 연구된 CMS (cytoplasmic male sterility)(한국 등록특허 제 10-2157801호)에 비해 무 GMS (genic male sterility)에 대해서는 알려진 바가 거의 없다. 무에서 두 가지 GMS 표현형이 보고되어 있다. 이 두 GMS의 웅성불임 표현형은 각각 꽃가루 (Wang et al. 2012)와 불개화 수술 (Duan et al. 2020)의 완전한 퇴화로 인해 발생하였다. 본 발명에서는 개약의 억제를 보여주는 신규한 GMS를 개시하고 있으며, 웅성불임에 대한 원인 유전자는 발견하여 응용한 발명이다. 본 발명은 무 작물 육성 프로그램에서 웅성불임 유도 및 신규한 GMS 적용의 기본 메커니즘을 제시하고 있다.On the other hand, little is known about non-GMS (genic male sterility) compared to extensively studied CMS (cytoplasmic male sterility) (Korean Patent No. 10-2157801). Two GMS phenotypes have been reported in radish. The male infertility phenotype of these two GMS was caused by complete regression of pollen (Wang et al. 2012) and non-flowering stamens (Duan et al. 2020), respectively. The present invention discloses a novel GMS showing inhibition of gynecomastia, and the causative gene for male infertility was discovered and applied. The present invention presents the basic mechanism of male infertility induction and novel GMS application in a radish crop cultivation program.

본 발명의 목적은 무 웅성불임 판별용 조성물 및 이를 포함하는 무 웅성불임 판별용 키트를 제공하는 것이다. 또한, 본 발명의 목적은 신규하게 발견된 유전자좌에 대한 조작을 통해 웅성불임을 유발하여 형질전환된 무 식물체를 제조하는 방법을 제공하는 것이다.It is an object of the present invention to provide a composition for determining non-male infertility and a kit for determining non-male infertility comprising the same. Another object of the present invention is to provide a method for producing a transformed radish plant by inducing male infertility through manipulation of a newly discovered locus.

그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

이하, 본원에 기재된 다양한 구현예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구현예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구현예" 또는 "구현예"에 대한 본 명세서 전체를 통한 참조는 구현예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구현예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구현예에서" 또는 "구현예"의 상황은 반드시 본 발명의 동일한 구현예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구현예에서 어떠한 적합한 방법으로 조합될 수 있다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

본 발명 내 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당 업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다.Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

용어 "식물" 또는 "식물체"는 전체 식물, 식물 기관(예를 들어, 잎, 줄기, 뿌리 등), 식물 조직, 식물 세포, 식물 부분, 종자, 육아, 배아 및 이들의 자손을 지칭한다. 식물 세포는 분화 또는 미분화 세포(예를 들어, 캘러스, 미분화 캘러스, 미숙배, 성숙배, 미숙 접합배, 미숙 자엽, 배축, 현탁 배양 세포, 원형질체, 잎, 잎 세포, 뿌리 세포, 체관부 세포 및 꽃가루)일 수 있다. 식물 세포는 종자 세포, 현탁 배양물, 외식편, 미숙배, 배아, 접합배, 체세포배, 배발생 캘러스, 분열조직, 체세포 분열조직, 기관형성 캘러스, 원형질체, 성숙 이삭-유래 종자로부터 유도된 배아, 엽저, 성숙 식물의 잎, 잎끝, 미숙 꽃, 수술, 미숙 이삭, 수염, 자엽, 미숙 자엽, 배축, 분열부, 캘러스 조직, 잎 세포, 줄기 세포, 뿌리 세포, 싹 세포, 배우체, 포자체, 꽃가루 및 미포자를 제한 없이 포함한다. 식물 부분은 분화 및 미분화 조직, 예를 들어 비제한적인 예로서 뿌리, 줄기, 싹, 잎, 꽃가루, 종자, 종양 조직, 및 배양물의 다양한 형태의 세포(예를 들어, 단일 세포, 원형질체, 배아 및 캘러스 조직)를 포함한다. 식물 조직은 식물, 또는 식물 기관, 조직 또는 세포 배양물에 있을 수 있다. 낟알은 종의 성장 또는 생식 이외의 목적을 위해 상업적 재배업자에 의해 생산된 성숙 종자를 의미하는 것이다. 재분화된 식물의 자손, 변이체 및 돌연변이체가 도입된 폴리뉴클레오타이드를 포함하는 한 이들 자손, 변이체 및 돌연변이체도 본 발명의 범위 내에 포함된다.The term "plant" or "plant body" refers to whole plants, plant organs (eg, leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, granules, embryos and their progeny. Plant cells include differentiated or undifferentiated cells (e.g., callus, undifferentiated callus, immature embryo, mature embryo, immature zygote, immature cotyledon, hypocotyl, suspension cultured cell, protoplast, leaf, leaf cell, root cell, phloem cell and pollen) ) can be Plant cells include seed cells, suspension cultures, explants, immature embryos, embryos, zygotes, somatic embryos, embryogenic callus, meristems, mitotic tissues, organogenic callus, protoplasts, embryos derived from mature ear-derived seeds. , leaf base, mature plant leaf, leaf tip, immature flower, stamen, immature ear, whisker, cotyledon, immature cotyledon, hypocotyl, meristem, callus tissue, leaf cell, stem cell, root cell, bud cell, gametophyte, sporophyte, pollen and microspores. Plant parts include differentiated and undifferentiated tissues such as, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissues, and cells of various types (eg, single cells, protoplasts, embryos and callus tissue). The plant tissue may be in a plant or plant organ, tissue or cell culture. Kernel means mature seed produced by commercial growers for purposes other than growth or reproduction of the species. As long as the progeny, variants and mutants of the redifferentiated plant include the introduced polynucleotide, these progeny, variants and mutants are also included within the scope of the present invention.

본 명세서에 있어서, "웅성불임"이란, 식물이 정상적으로 기능을 하는 수술, 꽃가루, 혹은 웅성 생식세포를 생산을 할 수 없는 것으로 정의된다. 자가수분 식물에서 수량성이나 저항성이 뛰어난 잡종종자 생산을 위해서 웅성불임 식물체의 사용은 물리적으로 수술을 없애는 과정(제웅과정)을 생략함으로써 매우 중요한 가치를 가지고 있다. 웅성불임은 1)유전적 웅성불임, 2)세포질 웅성불임, 3)세포질 유전적 웅성불임, 4)환경감수성 유전적 웅성불임, 5)화학적으로 유도 된 웅성 불임이 있다. 유전적 웅성불임은 핵 유전자에 의한 꽃가루가 불임이 된는 것이다. 이것은 일반적으로 하나의 열성형질에 의해서 지배를 받는다. 웅성불임 대립유전자는 자연적으로 발생되거나 인위적으로 유도될 수 있다. 보리, 면화, 토마토 등 대부분의 작물에서 유전적 웅성불임이 관찰되고 있다. 세포질 웅성불임 (cytoplasmic male sterility, CMS)은 핵 이외의 유전적 요인 즉 세포질에 존재하는 미토콘드리아나 엽록체 유전자에 의해 조절되는 웅성불임 현상이다. 따라서 정상적인 멘델의 법칙을 따르지 않고 웅성불임 형질이 모계를 통해서 유전되는 특성을 가지고 있다. 이러한 모계 유래의 세포질은 정상적인 임성을 가진 것과 불임인 것으로 나누어 질 수 있다. 세포질 유전적 웅성불임 (cytoplasmic genetic male sterility, CGMS)은 핵과 세포질 유전자의 상호작용으로 불임이 조절되는 현상이다. 세포질 웅성불임이 핵외 세포질 인자에 의해 제어되는 동안 핵 유전자는 CMS의 불임성 세포질의 임성을 회복하는 것에 관여한다. 환경감수성 유전적 웅성불임 (environmental sensitive genetic male sterility, EGMS)은 조건에 따라서 유전적 웅성불임이 결정되는 현상이다. 이러한 현상은 대부분 핵 유전자에 의해서 조절을 받는다. 크게 두 가지 환경 조건에 따라서 이러한 웅성불임이 결정된다. 하나는 낮의 길이가 긴 장일조건에서는 임성을 가지지만 낮의 길이가 짧은 단일조건에서 불임이 유도되는데 이를 일장감수성 유전적 웅성불임(photoperiod sensitive genetic male sterility, PGMS)이라고 한다. 다른 하나는 온도조건에 따라서 웅성불임이 결정되는데 낮은 온도에서는 임성을 유지하다가 일정 온도 이상에서 불임이 유도된다. 이러한 현상을 온도감수성 유전적 웅성불임(temperature sensitive genetic male sterility, TGMS)이라고 한다. 화학적으로 유도된 웅성불임은 수컷배우체를 죽이는 화학물질을 이용하여 웅성불임을 유도하는 비유전적인 방법이다.As used herein, "male infertility" is defined as the inability of a plant to produce normally functioning stamens, pollen, or male germ cells. For the production of hybrid seeds with excellent yield and resistance in self-pollinated plants, the use of male sterile plants has a very important value by omitting the process of physically removing the operation (removal process). Male infertility includes 1) genetic male infertility, 2) cytoplasmic male infertility, 3) cytoplasmic genetic male infertility, 4) environmental susceptibility genetic male infertility, and 5) chemically induced male infertility. Genetic male infertility is infertility caused by pollen caused by nuclear genes. It is generally governed by one thermoform. Male infertility alleles may be naturally occurring or artificially induced. Genetic male infertility has been observed in most crops such as barley, cotton, and tomatoes. Cytoplasmic male sterility (CMS) is a male infertility phenomenon regulated by genetic factors other than the nucleus, that is, mitochondrial or chloroplast genes present in the cytoplasm. Therefore, it does not follow normal Mendel's law and has the characteristic that male infertility traits are inherited through the maternal line. The maternally-derived cytoplasm can be divided into those with normal fertility and those with infertility. Cytoplasmic genetic male sterility (CGMS) is a phenomenon in which infertility is regulated by the interaction of nuclear and cytoplasmic genes. While cytoplasmic male sterility is controlled by extranuclear cytoplasmic factors, nuclear genes are involved in restoring sterile cytoplasmic fertility in CMS. Environmental sensitive genetic male sterility (EGMS) is a phenomenon in which genetic male sterility is determined according to conditions. Most of these phenomena are regulated by nuclear genes. This male infertility is largely determined by two environmental conditions. One is fertile under long day conditions, but infertility is induced under single conditions with short day lengths, which is called photoperiod sensitive genetic male sterility (PGMS). The other is that male infertility is determined according to temperature conditions. At a low temperature, fertility is maintained, but at a certain temperature or higher, infertility is induced. This phenomenon is called temperature sensitive genetic male sterility (TGMS). Chemically induced male infertility is a non-genetic method of inducing male infertility using chemicals that kill male gametes.

본 발명에서, "무(Raphanus sativus)"는 십자화과에 속하는 초본식물이다. 같은 흰무에 속하는 왜무보다 둥글고 단단하며 윗부분이 푸르다. 뿌리는 원형·원통형·세장형 등 여러 종류가 있고 뿌리의 빛깔도 흰색·검정색·붉은색 등 다양하다. 원산지는 지중해 연안으로 알려져 있으며 실크로드를 통하여 중국에 전래되었다고도 하고 아프가니스탄에서 전래되었다고도 한다. In the present invention, "radish (Raphanus sativus)" is a herbaceous plant belonging to the cruciferous family. It is rounder and harder than Japanese radish belonging to the same white radish, and the upper part is blue. There are several types of roots, such as round, cylindrical, and elongated, and the color of the root is also varied, such as white, black, and red. The place of origin is known as the Mediterranean coast, and it is said to have been brought to China through the Silk Road or from Afghanistan.

본 명세서에 있어서, "프라이머(primer)"란 짧은 서열의 뉴클레오티드인 올리고뉴클레오티드(oligonucleotide)로서 목적하는 DNA 또는 RNA의 반대편 가닥의 상보적 위치에 특이적으로 부착되어 유전자의 증폭(amplification)을 개시하는 역할을 하는 올리고뉴클레오티드로서, 바람직하게는 구제역 바이러스의 3D 유전자에 특이적으로 결합하는 올리고뉴클레오티드일 수 있으며, 더욱 바람직하게는 서열번호 4, 서열번호 5, 및/또는 서열번호 6의 염기서열을 포함하는 올리고뉴클레오티드로서, 상기 올리고뉴클레오티드는 서열번호 4, 서열번호 5, 및/또는 서열번호 6의 염기서열과 80% 이상, 90% 이상, 95% 이상, 또는 99% 이상 유사한 서열일 수 있다.In the present specification, the term "primer" refers to an oligonucleotide that is a nucleotide of a short sequence and is specifically attached to the complementary position of the opposite strand of the target DNA or RNA to initiate amplification of the gene. As an oligonucleotide serving as an oligonucleotide, it may preferably be an oligonucleotide that specifically binds to the 3D gene of foot-and-mouth disease virus, and more preferably includes the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6. As an oligonucleotide, the oligonucleotide may be 80% or more, 90% or more, 95% or more, or 99% or more similar to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6.

본 명세서에 있어서, "프로브(probe)"란, 목적하는 유전자에 특이적으로 부착되어 목적 유전자를 확인 및/또는 검출하는 탐침자를 포괄적으로 포함하는 광의의 개념이며, 바람직하게는 본 발명에 따른 무 웅성불임 유발 돌연변이 유전자에 특이적으로 결합하는 화합물, 항체, 단백질, 올리고뉴클레오티드 등 일 수 있으며, 더욱 바람직하게는 서열번호 2 또는 서열번호 3의 염기서열을 포함하는 올리고뉴클레오티드로서, 상기 올리고뉴클레오티드는 서열번호 2 또는 서열번호 3의 염기서열과 80% 이상, 90% 이상, 95% 이상, 또는 99% 이상 유사한 서열일 수 있다. 또한, 상기 프로브는 용이한 검출을 위한 표지를 하나 이상 포함할 수 있다.As used herein, the term "probe" is a broad concept comprehensively including a probe that is specifically attached to a target gene to identify and/or detect the target gene, preferably It may be a compound, antibody, protein, oligonucleotide, etc. that specifically binds to a male infertility-inducing mutant gene, and more preferably an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the oligonucleotide is the sequence It may be a sequence similar to the nucleotide sequence of No. 2 or SEQ ID NO: 3 by at least 80%, at least 90%, at least 95%, or at least 99%. In addition, the probe may include one or more labels for easy detection.

본 명세서에 있어서, "키트(kit)"란 식물체의 웅성불임 여부를 판단할 수 있는 판별용 기기를 의미하며, 웅성불임 여부를 판단할 식물체로부터 분리된 생물학적 시료로부터 웅성불임 여부를 판단할 수 있는 형태라면 제한이 없다. 바람직하게는 본 발명에 따른 무 웅성불임 유발 돌연변이 유전자에 대하여 상보적인 서열을 가지는 프로브(probe) 또는 프라이머(primer) 세트를 포함할 수 있으며, 더욱 바람직하게는 본 발명의 프라이머 세트를 포함할 수 있으며, 또는 무 웅성불임 단백질에 특이적으로 결합하는 항체(antibody) 또는 앱타머(aptamer)를 포함하는 형태일 수 있으나, 무 웅성불임 단백질에 대한 RNA를 증폭 및/또는 검출하여 무 웅성불임 여부를 판별할 수 있는 형태라면 이에 제한되지 않는다.In the present specification, "kit" means a device for determining whether a plant is male infertility, and can determine whether male infertility from a biological sample separated from a plant to determine whether male infertility is There is no limit on the form. Preferably, it may include a probe or primer set having a sequence complementary to the non-male infertility-inducing mutant gene according to the present invention, and more preferably, it may include the primer set of the present invention. , or may be in the form of an antibody or aptamer that specifically binds to non-male sterility protein, but determines whether non-male sterility by amplifying and/or detecting RNA for non-male sterility protein It is not limited thereto as long as it can be done.

본원에 개시된 서열 및 유전자, 및 이의 변이체 및 단편은 예를 들어 관심 표현형을 발현시키기 위해 형질전환된 또는 형질전환 식물을 제조하기 위해 식물의 유전자 조작에 유용하다. 본원에 사용된 바대로, 용어 "형질전환된 식물" 및 "형질전환 식물"은 게놈 내에 이종성 폴리뉴클레오타이드를 포함하는 식물을 지칭한다. 일반적으로, 이종성 폴리뉴클레오타이드는 폴리뉴클레오타이드가 다음 세대로 전달되도록 형질전환 또는 형질전환된 식물의 게놈 내에 안정적으로 통합된다. 이종성 폴리뉴클레오타이드는 단독으로 또는 재조합 DNA 작제물의 일부로서 게놈 내에 통합될 수 있다. 본원에 사용된 바대로 용어 "형질전환"은 초기에 그렇게 변경된 형질전환뿐만 아니라 초기 형질전환으로부터 유성 교배 또는 무성 번식에 의해 생성된 것을 포함하여 이종성 핵산의 존재에 의해 유전형이 변경된 임의의 세포, 세포주, 캘러스, 조직, 식물 부분 또는 식물을 포함하는 것으로 이해되어야 한다. 형질전환 사건은 관심 유전자를 포함하는 핵산 발현 카세트를 포함하는 이종성 DNA 작제물을 이용한 식물 세포의 형질전환, 전달된 유전자를 식물의 게놈에 삽입하여 생긴 식물 집단의 재분화, 및 특정 게놈 위치에 삽입되는 것을 특징으로 하는 식물의 선택에 의해 생성된다.The sequences and genes disclosed herein, and variants and fragments thereof, are useful in genetic manipulation of plants, for example, to produce transformed or transgenic plants to express a phenotype of interest. As used herein, the terms “transformed plant” and “transgenic plant” refer to a plant comprising a heterologous polynucleotide in its genome. In general, a heterologous polynucleotide is stably integrated within the genome of a transformed or transformed plant such that the polynucleotide is passed on to the next generation. Heterologous polynucleotides can be integrated into the genome either alone or as part of a recombinant DNA construct. As used herein, the term “transformation” refers to any cell, cell line in which the genotype has been altered by the presence of a heterologous nucleic acid, including those initially so altered, as well as those produced by sexual or asexual reproduction from the initial transformation. , callus, tissue, plant part or plant. Transformation events include transformation of a plant cell using a heterologous DNA construct comprising a nucleic acid expression cassette comprising a gene of interest, redifferentiation of a plant population resulting from insertion of the transferred gene into the genome of the plant, and insertion at a specific genomic location. It is produced by the selection of plants characterized in that.

본 발명에서 사용하는 용어 "벡터"는 식물에서 유전자 침묵을 유도하거나, 유전자의 발현을 유도하기 위한 뉴클레오티드 단편을 식물로 운반하기 위한 역할을 수행하는 것으로, 본 발명의 벡터는 스핑고모나스 어드헤시바(Sphingomonas adhaesiva) 또는 애그로박테리움 에스피(Agrobacterium sp.)로부터 유래된 서열을 포함한다. 여기서 본 발명의 벡터는 서열번호 2 또는 서열번호 3의 뉴클레오티드 서열이 클로닝될 수 있는 하나 이상의 제한효소 인식 뉴클레오티드 서열인 다중클로닝자리(multiple cloning site, MCS)이 포함된다. 상기 제한효소로는 Eag I, EcoR I, EcoR Ⅱ, BamH Ⅰ, Bgl Ⅱ, BstB Ⅰ, Hind Ⅲ, Taq Ⅰ, Not Ⅰ, Hinf Ⅰ, Sau3A, Pac I, Pov Ⅱ, Sma I, Hae Ⅲ, Hga I, Alu I, EcoR V, EcoP15 I, Kpn I, Pst I, Sac I, Sal I, Sca I, Spe I, Sph I, Stu I, Xba I, Xho I 중에 적어도 하나 이상을 포함할 수 있으며, 본 발명에서는 Kpn I 및 BamH Ⅰ인 것이 바람직하다. 본 발명의 벡터는 돌연변이를 통한 웅성불임화하기 위해 다른 서열과 추가적으로 융합될 수 있으며, 또 세포 내에서 복제 가능한 것이면 특별히 한정되지 않고 당업계에 알려진 임의의 벡터를 이용할 수 있다. 따라서, 본 발명에 사용될 수 있는 벡터로 다양한 클로닝 부위를 가지고 있는 대장균 유래 플라스미드(pBR322, pBR325, pUC118 및 pUC119 등), 바실러스 서브틸러스 유래 플라스미드(pUB110 및 pTP5 등), 효모 유래 플라스미드(YEp13, YEp24 및 YCp50 등) 및 Ti 플라스미드 등의 플라스미드가 있고, 식물 바이러스 벡터가 있으며, pCHF3, pPZP, pGA 및 pCAMBIA 계열과 같은 바이너리 벡터를 사용할 수 있다. 그러나 당업자라면 본 발명의 목적 단백질을 코딩하는 염기서열을 숙주 세포 내로 도입할 수 있는 벡터라면 어떠한 벡터라도 모두 사용할 수 있다. 본 발명에서는 pET21b나 pCAMBIA1300PT와, 35S CaMV 프로모터를 이용하여 과발현용으로 활용될 수 있다.The term "vector" used in the present invention serves to induce gene silencing in plants or to transport nucleotide fragments for inducing gene expression to plants, and the vector of the present invention is Sphingomonas adhesiva. (Sphingomonas adhaesiva) or Agrobacterium sp. Here, the vector of the present invention includes a multiple cloning site (MCS), which is one or more restriction enzyme recognition nucleotide sequences into which the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 can be cloned. The restriction enzymes include Eag I, EcoRI, EcoR II, BamH I, Bgl II, BstB I, Hind III, Taq I, Not I, Hinf I, Sau3A, Pac I, Pov II, Sma I, Hae III, Hga I, Alu I, EcoR V, EcoP15 I, Kpn I, Pst I, Sac I, Sal I, Sca I, Spe I, Sph I, Stu I, Xba I, may include at least one or more of Xho I, In the present invention, Kpn I and BamH I are preferred. The vector of the present invention may be additionally fused with other sequences for male sterilization through mutation, and is not particularly limited as long as it is capable of replication in a cell, and any vector known in the art may be used. Therefore, as vectors that can be used in the present invention, E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, etc.) having various cloning sites, Bacillus subtilus-derived plasmids (pUB110 and pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, etc.) and plasmids such as YCp50) and Ti plasmid, and plant virus vectors, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA families can be used. However, those skilled in the art may use any vector as long as it is a vector capable of introducing the nucleotide sequence encoding the target protein of the present invention into a host cell. In the present invention, pET21b or pCAMBIA1300PT and 35S CaMV promoter can be used for overexpression.

상기 목적을 달성하기 위하여, 무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역에 대응하는 검출제제를 포함하는 무 웅성불임 판별용 조성물을 제공한다.In order to achieve the above object, there is provided a composition for determining male infertility comprising a detection agent corresponding to the upstream 7kb to 8kb region of the Rs295350 gene of radish.

본 발명의 일 구체예에서, 상기 조성물은 무의 Rs295350 유전자의 상류 7.1kb 내지 7.9kb 영역에 대응하는 검출제제를 포함할 수 있다. 본 발명의 다른 구체예에서, 상기 조성물은 무의 Rs295350 유전자의 상류 7.2kb 내지 7.8kb 영역에 대응하는 검출제제를 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 조성물은 무의 Rs295350 유전자의 상류 7.3kb 내지 7.7kb 영역에 대응하는 검출제제를 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 조성물은 무의 Rs295350 유전자의 상류 7.4kb 내지 7.6kb 영역에 대응하는 검출제제를 포함할 수 있다. 본 발명의 또 다른 구체예에서, Rs295350 유전자는 서열번호 1이다. 본 발명의 또 다른 구체예에서, 상류 7kb 내지 8kb 영역은 서열번호 2이다. 본 발명의 또 다른 구체예에서, 상기 검출 제제는 상기 유전자에 특이적으로 결합하는 프라이머, 프로브 및 안티센스 뉴클레오티드로 이루어진 군에서 선택된 1종 이상을 포함한다. 본 발명의 또 다른 구체예에서, 상기 검출제제는 서열번호 3의 서열로 이루어진 제 1 프라이머, 서열번호 4의 서열로 이루어진 제 2 프라이머, 또는 서열번호 5의 서열로 이루어진 제 3 프라이머 중 어느 2개 이상을 포함하는 프라이머 세트이다.In one embodiment of the present invention, the composition may include a detection agent corresponding to a region of 7.1 kb to 7.9 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the composition may include a detection agent corresponding to a region of 7.2 kb to 7.8 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the composition may include a detection agent corresponding to a region of 7.3 kb to 7.7 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the composition may include a detection agent corresponding to the 7.4kb to 7.6kb region upstream of the radish Rs295350 gene. In another embodiment of the present invention, the Rs295350 gene is SEQ ID NO: 1. In another embodiment of the invention, the upstream 7kb to 8kb region is SEQ ID NO:2. In another embodiment of the present invention, the detection agent includes at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene. In another embodiment of the present invention, the detection agent is any two of the first primer consisting of the sequence of SEQ ID NO: 3, the second primer consisting of the sequence of SEQ ID NO: 4, or the third primer consisting of the sequence of SEQ ID NO: 5 It is a primer set including the above.

상기 목적을 달성하기 위하여, 상기한 조성물을 포함하는 무 웅성불임 판별용 키트를 제공한다.In order to achieve the above object, there is provided a kit for determining non-male infertility comprising the above composition.

본 발명의 일 구체예에서, 상기 키트는 서열번호 2와 비교하여 결손, 삽입 또는 치환 돌연변이를 검출하여 무의 웅성불임을 판별한다.In one embodiment of the present invention, the kit determines male infertility of radish by detecting a deletion, insertion or substitution mutation in comparison with SEQ ID NO: 2.

상기 목적을 달성하기 위하여, (a) 식물체로부터 유래된 시료로부터 게놈 DNA를 분리하는 단계; (b) 단계 (a)에서 분리한 게놈 DNA에 제 1항 내지 제 4항 중 어느 하나의 조성물을 첨가하여 돌연변이 여부를 결정하는 단계; 및 (c) 단계 (b)에서 검출된 돌연변이 부위를 확인하는 단계를 포함하는, 무의 웅성불임 판별 방법을 제공한다.In order to achieve the above object, (a) isolating genomic DNA from a sample derived from a plant; (b) determining whether a mutation is present by adding the composition of any one of claims 1 to 4 to the genomic DNA isolated in step (a); And (c) provides a method for determining male infertility of radish, comprising the step of identifying the mutation site detected in step (b).

상기 목적을 달성하기 위하여, 무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역에 대응되는 서열을 포함하는 벡터를 무 식물체에 형질전환시켜서, 웅성불임을 유도하는 방법을 제공한다.In order to achieve the above object, there is provided a method for inducing male infertility by transforming a radish plant with a vector containing a sequence corresponding to the region of 7 kb to 8 kb upstream of the Rs295350 gene of radish.

본 발명의 일 구체예에서, 상기 벡터는 무의 Rs295350 유전자의 상류 7.1kb 내지 7.9kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 다른 구체예에서, 상기 벡터는 무의 Rs295350 유전자의 상류 7.2kb 내지 7.8kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 방법은 무의 Rs295350 유전자의 상류 7.3kb 내지 7.7kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 방법은 무의 Rs295350 유전자의 상류 7.4kb 내지 7.6kb 영역에 대응하는 서열을 포함할 수 있다. In one embodiment of the present invention, the vector may include a sequence corresponding to a region of 7.1 kb to 7.9 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the vector may include a sequence corresponding to a region of 7.2 kb to 7.8 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the method may include a sequence corresponding to a region of 7.3 kb to 7.7 kb upstream of the radish Rs295350 gene. In another embodiment of the present invention, the method may include a sequence corresponding to a region of 7.4kb to 7.6kb upstream of the Rs295350 gene of radish.

상기 목적을 달성하기 위하여, 무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역에 대응되는 서열을 포함하는 벡터를 포함하는 형질전환 식물을 제공한다.In order to achieve the above object, there is provided a transgenic plant comprising a vector comprising a sequence corresponding to a region of 7 kb to 8 kb upstream of the Rs295350 gene of radish.

본 발명의 일 구체예에서, 상기 벡터는 무의 Rs295350 유전자의 상류 7.1kb 내지 7.9kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 다른 구체예에서, 상기 벡터는 무의 Rs295350 유전자의 상류 7.2kb 내지 7.8kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 방법은 무의 Rs295350 유전자의 상류 7.3kb 내지 7.7kb 영역에 대응하는 서열을 포함할 수 있다. 본 발명의 또 다른 구체예에서, 상기 방법은 무의 Rs295350 유전자의 상류 7.4kb 내지 7.6kb 영역에 대응하는 서열을 포함할 수 있다.In one embodiment of the present invention, the vector may include a sequence corresponding to a region of 7.1 kb to 7.9 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the vector may include a sequence corresponding to a region of 7.2 kb to 7.8 kb upstream of the Rs295350 gene of radish. In another embodiment of the present invention, the method may include a sequence corresponding to a region of 7.3 kb to 7.7 kb upstream of the radish Rs295350 gene. In another embodiment of the present invention, the method may include a sequence corresponding to a region of 7.4kb to 7.6kb upstream of the Rs295350 gene of radish.

본 발명에 따른 무 웅성불임 판별용 조성물 및 이를 포함하는 무 웅성불임 판별용 키트는 무의 웅성불임을 판별하여 내혼계가 섞이는 것을 방지하여 종자의 품위를 유지하며 양호한 품종의 무 작물을 선별하여 육종할 수 있다.The composition for discriminating male infertility according to the present invention and the kit for discriminating male infertility comprising the same discriminates male infertility of radishes and prevents the mixing of internal hybrids to maintain the dignity of the seeds and select and breed radish crops of good varieties. can

또한, 본 발명에 따른 웅성불임이 유발된 형질전환 무 식물체의 제조방법에 의해 웅성불임인 무를 육종할 수 있다.In addition, male infertility radish can be bred by the method for producing a transgenic radish plant induced in male infertility according to the present invention.

추가로, 본 발명에서는 개약의 억제를 보여주는 신규한 GMS를 개시하고 있다.Additionally, the present invention discloses a novel GMS that shows inhibition of drug overgrowth.

도 1은 무의 웅성임성 식물(도 1의 A, D, G, J), 본 발명에 따른 웅성불임 식물(도 1의 B, E, H, K), 및 DCGMS 세포질에 의해 부여된 CMS 표현형을 보여주는 식물(도 1의 C, F, I, L)의 사진이다. 도 1의 A, B, 및 C는 가시성을 높이기 위해 꽃잎을 떼어 낸 열린 꽃이다. 막대는 1mm를 나타낸다. 도 1의 D, E, 및 F는 열개된 약이다. 막대는 1mm를 나타낸다. 도 1의 G, H, 및 I는 꽃가루 알갱이의 전자 현미경 이미지이다. 막대는 10μm를 나타낸다. 도 1의 J, K, 및 L은 꽃가루 알갱이의 접사이다. 막대는 10μm를 나타낸다.
도 2는 특정 유전자의 상대적 발현 수준을 나타낸 그래프이다. MF는 웅성임성(male-fertile), MS는 웅성불임(male-sterile)을 의미한다. 튜불린 단백질을 코딩하는 유전자를 내부 대조군으로 사용하였다.
도 3의 A는 분자표지의 프라이머 위치 및 955-bp 삽입의 카피 수를 나타내는 개략도이다. 도 3의 B는 프라이머 조합의 PCR 산물을 나타낸다. RsMs-F1m은 미스매치된 뉴클레오티드를 포함하는 변형된 프라이머이다. A는 동형접합 우성, H는 이형접합, B는 동형접합 열성 개체를 나타낸다.1 is a male sterile plant of radish (A, D, G, J in FIG. 1), a male sterile plant according to the present invention (B, E, H, K in FIG. 1), and a CMS phenotype conferred by DCGMS cytoplasm. It is a photograph of a plant showing (C, F, I, L in FIG. 1). 1A, B, and C are open flowers with petals removed to increase visibility. Bars represent 1 mm. D, E, and F in Figure 1 are the opened drugs. Bars represent 1 mm. 1G, H, and I are electron microscope images of pollen grains. Bars represent 10 μm. 1, J, K, and L are affixes of pollen grains. Bars represent 10 μm.
2 is a graph showing the relative expression level of a specific gene. MF stands for male-fertile and MS stands for male-sterile. The gene encoding the tubulin protein was used as an internal control.
3A is a schematic diagram showing the position of the primer of the molecular marker and the copy number of the 955-bp insertion. 3B shows the PCR product of the primer combination. RsMs-F1m is a modified primer comprising mismatched nucleotides. A is homozygous dominant, H is heterozygous, and B is homozygous recessive individual.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예Example

재료 및 방법Materials and Methods

식물 재료plant material

새로운 세포질 웅성불임 (CMS, cytoplasmic male sterility) (DCGMS, Dongbu cytoplasmic and genic male sterility)을 확인하고 CMS 식물은 이전 연구에서 다양한 육종 계통으로 교차 수분시켰다. 이러한 F1 집단을 기반으로 교차 집단 [(CMS × R109) × (CMS × R121)]에서 분리 집단을 생성하였다. CMS와 새로운 웅성불임 표현형을 모두 이 집단에서 확인하였다. 각 F2 및 BC1F1 개체군 중 3개는 이전 연구에서 보고된 분리 개체군에서 선택된 3개의 이형 접합 웅성가임 식물과 3개의 웅성불임 식물에서 생산되었다. 격리된 온실에서 이형 접합 식물의 자가 수분을 통해 대규모 분리 집단을 얻었다. 이형 접합 식물의 유전형은 연관된 분자표지 (ILP14010)를 사용하여 확인되었다. 총 7,511개의 식물을 분석하여 미세 매핑을 위한 재조합체를 확인하였다.A novel cytoplasmic male sterility (CMS) (DCGMS, Dongbu cytoplasmic and genic male sterility) was identified and CMS plants were cross-pollinated into various breeding lines in previous studies. Based on this F1 population, a segregation population was generated from the crossover population [(CMS × R109) × (CMS × R121)]. Both CMS and a novel male infertility phenotype were identified in this population. Three of each F2 and BC1F1 populations were produced from three heterozygous male sterile plants and three male sterile plants selected from the segregated populations reported in previous studies. Large isolated populations were obtained through self-pollination of heterozygous plants in isolated greenhouses. The genotype of heterozygous plants was identified using an associated molecular marker (ILP14010). A total of 7,511 plants were analyzed to identify recombinants for micromapping.

신규한 종류의 웅성불임성 발견A novel type of male infertility was discovered.

이전 연구에서 DCGMS 세포질에 의해서 발생하는 웅성불임의 임성을 회복시켜주는 유전자를 찾아 Rfd1이라고 명명하였다. 그리고 Rfd1 유전자를 탐색하기 위하여 분리집단을 만들어 정밀한 유전자지도를 작성하였다. 그러나 분리집단에서 관찰된 웅성불임의 표현형이 종래의 DCGMS(Dongbu cytoplasmic and genic male sterility) 웅성불임의 표현형과 차이가 있음을 발견하였다. 표현형의 차이가 다른 종류의 웅성불임성이 존재하는 것은 아닌지 확인하기 위해서 다양한 육종계통을 가진 분리집단들을 분석하였다. 또한, 한 분리집단에서 DCGMS에서 의해서 발생하는 종래의 웅성불임과 동일한 표현형과 최근에 관찰된 다른 형태의 표현형이 혼재하는 집단을 발견하였다. 이 집단에서 관찰된 두 종류의 웅성불임성을 보다 자세히 관찰한 결과 약(anther)의 형태에서 큰 차이를 보였다. 새로 발견된 웅성불임성의 약의 경우 정상적인 개체의 약과 그 크기가 거의 같았고 약 표면 또한 매끈한 형태로 정상처럼 부풀어 있었지만, DCGMS의 경우 약의 크기가 정상에 비해서 크기가 작고 또한 약이 찌그러져 있는 형태이었다 (도 1 참고). 가장 두드러진 표현형의 차이는 새로운 형태의 웅성불임의 경우 개약이 이루어진 이후에도 꽃가루가 전혀 관찰되지 않았다. 그러나, 약을 칼로 잘랐을 경우 약 내부에서 많은 양의 꽃가루가 있음을 확인하였다. 또한 전자현미경으로 꽃가루의 형태를 관찰한 결과 새로운 형태의 웅성불임의 경우 꽃가루의 모양이 정상적인 꽃가루와 구분할 수 없을 정도로 정상적인 형태를 보였다. 그러나 DCGMS의 경우 꽃가루의 크기가 정상 개체와 비교할 때 눈에 띄게 크기가 작았으며 꽃가루 모양 또한 기형적인 것이 많았다. 이러한 결과를 종합해 볼 때 새롭게 관찰된 웅성불임의 경우 꽃가루는 정상이지만 개약 (anther dehiscence)이 되지 않아 발생한 웅성불임으로 확인되었다.In a previous study, we found a gene that restores fertility in male infertility caused by DCGMS cytoplasm and named it Rfd1 . In order to search for the Rfd1 gene, a segregation group was created and a precise genetic map was prepared. However, it was found that the phenotype of male infertility observed in the isolated group was different from that of the conventional DCGMS (Dongbu cytoplasmic and genic male sterility) male infertility. Separation groups with various breeding lines were analyzed to determine whether phenotypic differences exist in different types of male infertility. In addition, in one isolate group, a group in which the same phenotype as conventional male infertility caused by DCGMS and another recently observed phenotype were found was found. A more detailed observation of the two types of male infertility observed in this group showed a significant difference in the form of anther. In the case of the newly discovered drug for male infertility, the size of the drug was almost the same as that of a normal individual, and the drug surface was also smooth and swollen like normal. see Fig. 1). The most notable phenotypic difference was that, in the new form of male infertility, no pollen was observed even after drug reconstitution was made. However, when the medicine was cut with a knife, it was confirmed that there was a large amount of pollen inside the medicine. Also, as a result of observing the shape of pollen with an electron microscope, the shape of pollen in the new type of male infertility showed a normal shape to the extent that it was indistinguishable from normal pollen. However, in the case of DCGMS, the size of pollen was noticeably smaller than that of normal individuals, and the shape of pollen was also deformed in many cases. Considering these results, the newly observed male infertility was confirmed to be male infertility caused by normal pollen but not anther dehiscence.

신규한 종류의 웅성불임성의 유전양상 분석Analysis of the genetic pattern of a novel type of male infertility

신규한 형태의 웅성불임성을 발견한 다음 이전 연구에서 분석했던 F3 집단을 다시 재배하여 웅성불임의 형태를 관찰한 결과 DCGMS 표현형 보다는 신규한 형태의 개약이 되지 않는 웅성불임으로 확인되었다. 추가적으로 신규한 형태의 웅성불임성의 유전양상을 분석하기 위해서 F3 집단에서 3개의 이형접합 개체를 연관된 분자표지를 활용하여 선발한 다음 자가수정 시켰고 또한 동시에 3개의 웅성불임 개체와 교배하여 BC1F1 집단을 만들었다. 이들 6개의 집단을 재배하여 임성을 관찰한 결과 모든 집단에서 유의성 있게 1개의 유전자가 관여하며 새로운 형태의 웅성불임성은 열성 유전함을 확인할 수 있었다. 본 발명의 연구원들은 신규한 형태의 웅성불임성을 유기하는 핵내 유전자좌를 RsMs1 (Raphanus sativus Male sterility 1)이라고 명명하였다.After discovering a new form of male infertility, the F 3 group analyzed in the previous study was re-cultivated to observe the form of male infertility. In order to additionally analyze the genetic pattern of a novel form of male infertility, F 3 Three heterozygous individuals from the population were selected using associated molecular markers, then self-fertilized, and simultaneously crossed with three male infertile individuals to create a BC 1 F 1 population. As a result of observing fertility by cultivating these six groups, it was confirmed that one gene was significantly involved in all groups and that the new form of male sterility was recessive. The researchers of the present invention named the nuclear locus that induces a novel form of male sterility RsMs1 (Raphanus sativus Male sterility 1).

RsMs1 유전자 연관 지도 작성RsMs1 gene association mapping

이전 연구에서 생산된 F3 집단에서 개발된 분자표지와 이들 분자표지들이 위치하는 유전체 지역을 기반으로 9종류의 분자표지를 추가로 개발하였다. 이들 9종의 분자표지는 최근 한국과 일본에서 보고된 무 전장 유전체 서열 초안 정보를 활용하였다 (표 1).F 3 produced in previous studies Based on the molecular markers developed in the group and the genomic regions where these molecular markers are located, 9 types of molecular markers were additionally developed. For these nine molecular markers, information on the draft genome-free genome sequence recently reported in Korea and Japan was used (Table 1).

Figure 112020143805085-pat00001
Figure 112020143805085-pat00001

신규하게 개발된 9종의 분자표지 중에서 ILP14010 분자표지의 경우 앞서 생산된 6개의 F2 및 BC1F1 집단에서 웅성불임 표현형과 완벽하게 유전자형이 일치하였다. 그러나 흥미롭게도 이들 집단에서 4개체의 표현형이 가임으로 관찰되었지만 ILP14010 마커의 유전자형은 열성동형접합으로 확인되었고 더 나아가 주변에 위치한 9개의 분자표지들 모두 열성동형접합으로 확인되었다. 이러한 결과는 이들 4개체가 재조합체(recombinant)이기 보다는 임성이 불임에서 가임으로 전환된 복귀돌연변이체(revertant)임을 의미하는 결과이다. 새롭게 발견된 웅성불임 결정 RsMs1 유전자좌 유전자를 클로닝하기 위하여 이형접합 개체를 bulk로 격리된 온실에서 재배하여 크기가 큰 분리집단을 생산하였다. 대량의 분리집단에서 1,911 개체를 재배하여 ILP8045와 ILP14205 마커 사이의 재조합체를 선발하였다. 근접하게 연관된 분자표지들 중에서 ILP13570과 ILP14010 마커의 경우 표현형과 유전자형이 완벽하게 일치하였다. 재조합체 분석을 통하여 RsMs1 유전자좌는 ILP13570과 CAPS14075 마커 사이로 위치를 좁혀졌다. 재조합체 분석을 통하여 좁힌 유전체 영역의 범위는 무 유전체 서열 초안 (draft genome sequence)에서 약 200-250kb에 해당되는 지역이었다. 좁혀진 범위에 존재하는 유전자들을 분석한 결과, 많은 유전자 숫자 중에서 후보유전자를 도출하였다. 추가로 5,600개의 분리집단 개체를 재배하여 재조합체를 선발하였다. ILP13570과 ILP14205마커 사이에서 교차가 일어난 재조합체를 48 개체 선발하였다. 추가로 유전체 서열 초안을 활용하여 4개의 분자표지를 개발하였는데 SNP21814와 SNP21812 마커의 유전자형과 표현형은 완벽하게 일치하였다. 이들 48개체의 재조합체를 분석하여 최종적으로 RsMs1 유전자좌를 IND21816과 CAPS13940 마커 사이로 그 범위를 좁혔다.Among the nine newly developed molecular markers, the ILP14010 molecular marker perfectly matched the genotype with the male infertility phenotype in the six F 2 and BC 1 F 1 populations previously produced. Interestingly, however, the phenotype of 4 individuals in these populations was observed to be fertile, but the genotype of the ILP14010 marker was confirmed to be recessive, and furthermore, all 9 molecular markers located nearby were confirmed to be recessive. These results imply that these four individuals are not recombinants, but revertants that are converted from infertility to fertility. In order to clone the newly discovered male sterility determination RsMs1 locus gene, heterozygous individuals were grown in bulk isolated greenhouses to produce large isolates. Recombinants between ILP8045 and ILP14205 markers were selected by culturing 1,911 individuals in a large isolate. Among the closely related molecular markers, the phenotype and genotype were perfectly matched for the ILP13570 and ILP14010 markers. Through recombinant analysis, the RsMs1 locus was narrowed down between the ILP13570 and CAPS14075 markers. The range of the genomic region narrowed through recombinant analysis was a region corresponding to about 200-250 kb in the draft genome sequence. As a result of analyzing the genes existing in the narrowed range, candidate genes were derived from a large number of genes. In addition, 5,600 isolates were grown to select recombinants. Forty-eight recombinants with crossover between the markers ILP13570 and ILP14205 were selected. In addition, four molecular markers were developed using the draft genome sequence, and the genotypes and phenotypes of the SNP21814 and SNP21812 markers were perfectly matched. By analyzing the recombinants of these 48 individuals, the RsMs1 locus was finally narrowed down to between the IND21816 and CAPS13940 markers.

새로운 웅성불임을 결정짓는 후보 유전자 도출 Derivation of candidate genes for determining new male infertility

IND21816과 CAPS13940 마커로 좁힌 유전체 영역은 약 27kb 크기였다. 따라서 우성 동형접합 개체와 열성 동형접합 개체 DNA를 대상으로 27kb 유전체 영역에 해당되는 부분의 염기서열을 밝혔다. 총 119개의 SNP 또는 InDel이 탐색되었고 이들 SNP 또는 InDel은 대부분 3'말단 영역에 밀집되어 분포하였다. 염기서열이 밝혀진 영역에서 추가로 388-bp InDel이 발견되어 이 돌연변이를 기반으로 IND295320 마커를 개발하였다. 그리고 이 마커와 RsMs1 유전자좌 사이에서 2개체의 재조합체를 발견하였다. 따라서 RsMs1 유전자좌가 있는 지역은 추가로 24-kb 영역으로 좁힐 수 있었다. 6개의 유전자가 좁혀진 영역에서 발견되었다. 그 중에 Rs295360 유전자의 경우 그 기능이 대부분의 임성 회복유전자로 알려진 펜타트리코펩타이드 반복 (PPR, Pentatricopeptide Repeat) 단백질을 생성하였다. 그리고 이 유전자의 신호 펩티드를 분석한 결과 3종류의 프로그램이 모두 이 PPR 단백질이 미토콘드리아로 이동한다는 결과가 나왔다. 그러나 이 PPR 유전자의 경우 새로운 웅성불임의 후보 유전자가 아니라고 판단되었는데 그 이유는 코딩 서열과 추정 프로모터 영역(Putative promoter region)에 어떠한 다형성도 발견되지 않았기 때문이다. 또한, 이 PPR 유전자의 경우 다른 알려진 임성 회복유전자 기능을 하는 PPR과 Arabidopsis에서 연구된 Rf-like PPR 유전자들과 근연관계를 분석한 결과 이들과 유전적 거리가 멀기 때문에 임성 회복유전자 기능은 하지 않는 것으로 판단되었다. 그리고 이 PPR 유전자는 꽃 조직에서 발현양이 거의 관찰되지 않았다. 도 2에 나타나 바와 같이, 4개의 유전자 (RSG21816, Rs295350, Rs295340, Rs295330)가 꽃 조직에서 발현됨을 확인하였다. 그러나 이들 중에서 Rs295350 유전자만이 F2 분리집단에서 가임 개체와 불임 개체 사이에서 현저한 발현양 차이를 보였다. Rs295350 유전자(서열번호 1)의 경우 RNA-Seq 결과에서도 가임 bulked RNA에서 불임 bulked RNA에 비해서 8배 많은 발현양을 보였다 (표 2).The genomic region narrowed by the IND21816 and CAPS13940 markers was about 27 kb in size. Therefore, the nucleotide sequence of the 27kb genomic region was revealed for the DNA of the dominant and recessive homozygous individuals. A total of 119 SNPs or InDels were searched, and most of these SNPs or InDels were densely distributed in the 3'-end region. An additional 388-bp InDel was found in the sequenced region, and the IND295320 marker was developed based on this mutation. And two recombinants were found between this marker and the RsMs1 locus. Thus, the region with the RsMs1 locus could be further narrowed down to a 24-kb region. Six genes were found in the narrowed region. Among them, in the case of the Rs295360 gene, a pentatricopeptide repeat (PPR, Pentatricopeptide Repeat) protein whose function is known as most fertility recovery genes was generated. And as a result of analyzing the signal peptide of this gene, all three programs showed that this PPR protein was moved to the mitochondria. However, it was determined that this PPR gene was not a new candidate gene for male infertility because no polymorphism was found in the coding sequence and the putative promoter region. In addition, in the case of this PPR gene, as a result of analyzing the close relationship between PPR, which functions as another known fertility restoration gene, and Rf-like PPR genes studied in Arabidopsis, it was found that the fertility restoration gene does not function because of its long genetic distance. was judged And the expression level of this PPR gene was hardly observed in flower tissue. As shown in FIG. 2 , it was confirmed that four genes (RSG21816, Rs295350, Rs295340, Rs295330) were expressed in flower tissues. However, among them, only the Rs295350 gene showed a significant difference in expression level between fertile and infertile individuals in the F2 isolate group. In the case of the Rs295350 gene (SEQ ID NO: 1), the expression level of the fertile bulked RNA was 8 times higher than that of the infertile bulked RNA in the RNA-Seq results (Table 2).

Figure 112020143805085-pat00002
Figure 112020143805085-pat00002

1개의 SNP가 Rs295350 유전자 3번 엑손에서 발견되었지만 이 SNP의 경우 아미노산 서열에 영향을 주지 않았다. 따라서 이 SNP의 경우 발현양에 영향을 주지 않는 것을 확인하였다. 그러나, 열성 동협접합 유전자형의 경우 약 7.5kb 상류 영역(서열번호 2에 포함된 부분)에서 955-bp 삽입(서열번호 8)을 발견하였다. 이 유전자의 경우 프로모터 영역에서도 돌연변이가 발견되지 않았기 때문에 이 955-bp 삽입에 의해서 (삽입된 서열은 서열번호 6으로 나타내었다) Rs295350 유전자의 발현이 감소된 것을 확인하였다. 7.5kb 영역은 프로모터보다는 인핸서 영역에 가깝기 때문에 이 영역 주변에 다른 전사 인자가 결합할 수 있는 보존된 모티프(conserved motif)를 포함할 수 있다. 따라서, 보존된 모티프를 탐색하기 위하여 이 영역에 해당하는 유전체 영역을 배추 (Brassica rapa), 양배추 (B. oleracea), 및 애기장대 (Arabidopsis) 전장 유전체 서열로부터 탐색하여 이들을 무 영역의 염기서열과 Blast2를 통하여 분석하였다. 그 결과 235-bp의 매우 보존된 모티프가 삽입의 249 bp 하류에 위치하고 있었다. 보존된 모티프의 보존된 정도는 Rs295350 유전자의 코딩 영역의 서열만큼 보존되어 있었다 (표 3). 따라서 이 보존된 모티프는 Rs295350 유전자의 발현에 큰 영향을 줄 것으로 판단하였다.One SNP was found in exon 3 of the Rs295350 gene, but this SNP did not affect the amino acid sequence. Therefore, it was confirmed that this SNP did not affect the expression level. However, in the case of the recessive homozygous genotype, a 955-bp insertion (SEQ ID NO: 8) was found in the region upstream of about 7.5 kb (the portion included in SEQ ID NO: 2). In the case of this gene, since no mutation was found in the promoter region, it was confirmed that the expression of the Rs295350 gene was reduced by this 955-bp insertion (the inserted sequence is shown in SEQ ID NO: 6). Since the 7.5 kb region is closer to the enhancer region than the promoter region, it may include a conserved motif to which other transcription factors can bind around this region. Therefore, in order to search for conserved motifs, the genomic region corresponding to this region was searched from the full-length genome sequences of Chinese cabbage (Brassica rapa), cabbage (B. oleracea), and Arabidopsis, and these were searched for with the base sequence of the non-region and Blast2. was analyzed through As a result, a highly conserved motif of 235-bp was located 249 bp downstream of the insertion. The degree of conservation of the conserved motif was as conserved as the sequence of the coding region of the Rs295350 gene (Table 3). Therefore, it was determined that this conserved motif would have a significant effect on the expression of the Rs295350 gene.

Figure 112020143805085-pat00003
Figure 112020143805085-pat00003

탐색된 955-bp 삽입의 경우 코딩 서열이 전혀 발견되지 않았고 또한 어떠한 전이인자의 특징도 가지고 있지 않았다. 따라서 단순 반복서열로 판단하였다. 그러나 이들 반복서열과 유사한 염기서열을 무 유전체 서열 초안에서 탐색한 결과 100 카피 이상의 유사서열이 유전체 전반에서 걸쳐 분포하였다 (도 3). 955-bp 삽입이 반복서열이기 때문에 이러한 반복서열이 인핸서 영역에 삽입됨으로서 인핸서 영역을 깨거나 또는 DNA 메틸화를 유도하여 Rs295350 유전자의 발현을 감소시킨 것으로 판단하였다. Rs295350 유전자 기능은 MYB26-유사 전사 인자를 만드는 것이다. MYB26 유전자는 개약(anther dehiscence)에 결정적인 역할을 하였다. Rs295350 유전자는 Arabidopsis MYB26 유전자가 위치한 3번 염색체에서 주변 유전자들이 신터닉(syntenic)한 관계를 보였기 때문에 Rs295350 유전자는 Arabidopsis MYB26 유전자와 이종상동성(orthologous) 관계에 있었다. 따라서 이 유전자를 RsMYB26(Raphanus sativus L. MYB26)으로 명명하였다. 이와 같은 결과와 신규한 웅성불임의 표현형을 종합적으로 판단할 때 RsMYB26 유전자가 새로운 웅성불임의 원인 유전자임을 발견하였다. 추가로 RsMYB26 유전자가 원인 유전자일 것으로 판단할 수 있는 데이터로 Arabidopsis에서 MYB26 유전자의 조절을 받는 하위에 관여하는 유전자들과 상동성이 높은 이종상동성 유전자들을 무 유전체 서열 초안으로부터 탐색하였다(표 4). 이들 유전자들의 발현양을 확인하였는데 Arabidopsis의 MYB26 돌연변이처럼 이들 하위에 관여하는 유전자들의 발현양이 전반적으로 감소하였다.In the case of the searched 955-bp insertion, no coding sequence was found and it did not possess any transposable characteristics. Therefore, it was judged as a simple repeating sequence. However, as a result of searching for nucleotide sequences similar to these repetitive sequences in the draft non-genomic sequence, more than 100 copies of the similar sequences were distributed throughout the genome (FIG. 3). Since the 955-bp insertion is a repeat sequence, it was determined that the expression of the Rs295350 gene was reduced by breaking the enhancer region or inducing DNA methylation by inserting the repeat sequence into the enhancer region. The function of the Rs295350 gene is to make a MYB26-like transcription factor. The MYB26 gene played a crucial role in further dehiscence. The Rs295350 gene was in an orthologous relationship with the Arabidopsis MYB26 gene because the neighboring genes showed a syntenic relationship on chromosome 3 where the Arabidopsis MYB26 gene is located. Therefore, this gene was named RsMYB26 (Raphanus sativus L. MYB26). When comprehensively judging these results and the novel male infertility phenotype, the RsMYB26 gene was found to be the new causative gene for male infertility. In addition, as data that can determine that the RsMYB26 gene is the causative gene, orthologous genes with high homology to the genes involved in the lower MYB26 gene in Arabidopsis were searched from the draft non-genomic sequence (Table 4). The expression levels of these genes were confirmed, and the expression levels of genes involved in these subordinates, such as the MYB26 mutation in Arabidopsis, were generally reduced.

Figure 112020143805085-pat00004
Figure 112020143805085-pat00004

신규한 웅성불임을 결정짓는 유전자 선발용 분자표지 개발 Development of molecular markers for gene selection determining novel male infertility

신규한 웅성불임을 결정하는 RsMs1 유전자좌의 유전자형을 손쉽게 판별하기 위해서 955-bp 삽입에 기초해서 3종류의 프라이머로 이루어진 분자표지를 디자인하였다 (도 3의 A 참고). 3종류의 프라이머로 이루어진 조합을 가지고 PCR을 실시한 결과, 삽입에 결합하는 프라이머의 효율이 떨어져 이형접합 개체의 경우 항상 열성 유전자형과 관련된 PCR 산물 밴드의 진하기가 우성 유전자형과 연관된 지역을 증폭한 PCR 산물에 비해서 진하기가 떨어지는 현상을 관찰하였다 (도 3의 B 참고). 이러한 문제는 955-bp 삽입이 반복서열이기 때문에 유전체 내에서 여러 지역이 결합하기 때문인 것으로 판단하였다. 따라서 이러한 문제를 극복하고자 RsMs-F1 프라이머 염기서열에 1개의 미스매치된 염기서열을 추가하였다 (RsMs-F1m). 새로운 3종류의 프라이머 (RsMs-F1m은 서열번호 3, RsMs-F2는 서열번호 4, RsMs-R1은 5이다)로 이루어진 조합을 가지고 PCR을 실시한 결과 다시 PCR을 수행한 결과 두 가지 서로 다른 유전자형에서 증폭된 PCR 산물의 진하기가 비로소 비슷하게 되었다. 개발된 분자표지는 신규한 웅성불임을 일으키는 원인유전자에 존재하는 돌연변이인 955-bp 삽입(서열번호 8)에 기초해서 제작되었기 때문에 표현형을 100% 정확도를 가지고 선발할 수 있는 기능 마커라고 볼 수 있다. 개발된 기능 마커는 RsMs1로 명명하였다. 개발된 RsMs1 마커를 활용하여 새로운 웅성불임과 DCGMS 웅성불임이 모두 발견된 최초의 분리집단의 유전자형을 분석하였다. 그 결과를 표 5에 나타내었다. RsMs1 마커 유전자형이 열성 동형접합인 개체는 모두 새로운 웅성불임 형태를 보였으며 DCGMS 웅성불임은 3개의 이형접합 개체에서 관찰되었다. 이러한 결과는 새로운 웅성불임과 DCGMS 웅성불임은 서로 독립된 유전자에 의해서 발생함을 의미한다.In order to easily determine the genotype of the novel RsMs1 locus, which determines male infertility, a molecular marker consisting of three types of primers was designed based on the 955-bp insertion (see FIG. 3A ). As a result of performing PCR with a combination of three types of primers, the efficiency of the primers binding to the insertion is low, and in the case of heterozygous individuals, the PCR product band is always related to the recessive genotype. It was observed that the darkness was lowered compared to that (refer to B of FIG. 3). This problem was determined to be due to the combination of several regions within the genome since the 955-bp insertion is a repeating sequence. Therefore, to overcome this problem, one mismatched sequence was added to the RsMs-F1 primer sequence (RsMs-F1m). As a result of PCR with a combination of three new primers (RsMs-F1m is SEQ ID NO: 3, RsMs-F2 is SEQ ID NO: 4, and RsMs-R1 is 5), PCR was performed again. The thickness of the amplified PCR product finally became similar. Since the developed molecular marker was made based on the 955-bp insertion (SEQ ID NO: 8), which is a mutation present in the gene that causes male infertility, it can be considered as a functional marker that can select the phenotype with 100% accuracy. . The developed functional marker was named RsMs1. Using the developed RsMs1 marker, we analyzed the genotype of the first isolate in which both new male infertility and DCGMS male infertility were found. The results are shown in Table 5. All individuals with a recessive homozygous RsMs1 marker genotype showed a new male infertility morphology, and DCGMS male infertility was observed in three heterozygous individuals. These results suggest that new male infertility and DCGMS male infertility are caused by independent genes.

Figure 112020143805085-pat00005
Figure 112020143805085-pat00005

본 과제에서 새롭게 발견된 웅성불임은 표현형의 특징과 웅성불임을 유발하는 원인유전자의 기능을 바탕으로 종합적으로 판단할 때 세포질·유전자웅성불임성(CGMS)이 아닌 핵내 유전자만 작용하는 유전자웅성불임성(GMS, Genic male sterility)으로 판단할 수 있었다. 이는 무에서 보고된 최초의 GMS로 향후 무 F1 육종에 효과적으로 활용될 수 있을 것이다. 아울러 본 발명을 통해 GMS가 경우에 따라서 임성이 회복되는 복귀돌연변이 개체를 발견하였기 때문에, 향후 GMS의 임성 회복에 대한 온도 또는 일장과 같은 환경 조건에 따라 F1 육종에 매우 효과적으로 활용할 수 있을 것으로 판단되었다.Male infertility newly discovered in this project is genetic male infertility (GMS) in which only genes in the nucleus, not cytoplasmic/genetic male infertility (CGMS), act when comprehensively judged based on the phenotypic characteristics and the function of the causative gene that causes male infertility. , Genic male sterility). This is the first GMS reported in radish, and it can be effectively utilized for future F 1 breeding of radish. In addition, since the present invention found a reversion mutant in which GMS recovers fertility in some cases, it was determined that it could be used very effectively for F 1 breeding depending on environmental conditions such as temperature or sunlight for GMS recovery in the future. .

본 발명에 따른 웅성불임 유발 돌연변이체의 제작Production of male infertility-inducing mutants according to the present invention

야생형 무(Raphanus sativus L.) 식물체 (서열번호 7과 같은 서열을 포함하고 있다)에 서열번호 2에서의 7.5kb 상류 영역에서 955-bp 삽입으로 인한 돌연변이를 유발시켰다. 이를 이용하여 본 발명에 따라 웅성불임 유발시키는 재조합 벡터를 제작하여 형질전환시켰다. 이와 같이 제작된 식물체의 수정률은 현저히 떨어짐을 확인하였다.A wild-type radish (Raphanus sativus L.) plant (which contains the same sequence as SEQ ID NO: 7) was mutated due to a 955-bp insertion in the 7.5 kb upstream region of SEQ ID NO: 2. Using this, a recombinant vector for inducing male infertility was prepared and transformed according to the present invention. It was confirmed that the fertilization rate of the thus produced plants was significantly reduced.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility <130> PDPB204294k01 <150> KR 10/2020/0140344 <151> 2020-10-27 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1276 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 atgggccatc actcgtgctg caacaagcaa aaggtgaaga gagggctttg gtcaccggaa 60 gaagacgaaa agctcatcaa ctacatcaat acatatggcc atggatgctg gagctctgtt 120 cccaaacatg caggcatata tattggcttt gtttgttgaa ttatctactg ataaaaagtc 180 tatgtcgttg gttttaatct atgtgttgtt ttccgtttat ttaattaggt ctgcagagat 240 gtggaaagag ttgtcgatta agatggataa attatctaag acctgatctt aaacgtggaa 300 gcttctcacc tcaagaagct gctcttatca ttgagcttca tagcattctt ggtaacaggt 360 tgttacttaa ttatcattac atgttggttt atatgtgata atataaaact aaattttcat 420 gaagaaactg aactaccttt atactcattt ttttttgtta tggattcttg ataggtgggc 480 acaaattgct aaacatctac ctggaagaac agataacgag gtcaagaatt tctggaactc 540 aagcattaaa aagaagctca tgtctcacca ctatcaccat catcatggtc atcatcatca 600 tcatacctct tcaatggcga gtttgctcac taaccttcct tatcacaacg tgttcaacgc 660 tactgctggc gacgatgaag gttcaagatt catgtccagt atcatcacaa acactaaccc 720 taatttcatc actccaaccc atctccctct tccttctcat catgttatgt ctccattgat 780 gttctcaacc tctagagaag gagatatcaa gtatttaacc gctaccaaca caaaccagtc 840 tcatcaagat aataaccttt acaccaacct cgacattttg tcagccacgc cattcatcaa 900 caaccatcat caccatcatg ataatgaccc ccagtggcca tcgctaccag atctcccagc 960 gagtaccatt tccacttttc acgacaacct tcaagattat gatgatggtg ataaactcaa 1020 cgtgtttgta acaccataca atgataatgg tgatcctcgg agctttcctt tggtggccac 1080 aaagttaata tgtggacaag ttcttgaagg caaagcaatt tcacaagacc acggcagctt 1140 tcttctccca acaacgtaca acttggaaat gcctggtgat catcgacgag tggactcata 1200 cattaatcac atgatcattc cctcatcatc atcctcttca tcgccaatct cttgtggaca 1260 gtacgtcatc acttaa 1276 <210> 2 <211> 7889 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggactt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 acctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatcg aatcaaagcg gaaccaaaaa 360 cttcatttgg aagaaagtca gctttgagtc tggaccgtcc ttttttttca aaattactct 420 tggttatact aaacaaattt aatcttgtta acaaagtttg atttaattag tacgtgttac 480 tgcattatct atgtgtcatt tatgatatat aattctttat aaactcaaca tattttttat 540 aaatgtgcgt gtttgatcaa aaaaaaatat tgaaaaccct ttaacaaaaa aaaaagagta 600 tcctatatat atccatgcat tgagcgtagg cagaaggctt gatggtgtca gctcgaataa 660 atgaatgtaa atggacagat catattacta tttttagaat atggtccgta gaattttcat 720 aaaattgact aatcaattag tttctagttt tggttaaagc caagctcaca cgaagtctaa 780 aacgttcctc taaaaaggaa tcgtaacagt cccgttttta tttcactgtt ttagcaattt 840 ctctaaaaag gaatcgtaac agtccagttt ttatttcact gttttagcaa tttctttaca 900 tcaagtaaaa tatatttata tattcctcat accctttttc ctactttcta caaattcgtt 960 taccaacgta aaaatgaatt tattaggcat ctaccagatt tgaatatcgt ccaagaagta 1020 aatgaataaa gaaaactgtt taaaagtact ccacatttga ctttaataaa gcaaattatt 1080 tacatttgac tcagattaat actttttatt ttacgagtat tattgattat gtgacaaaaa 1140 actgattggc tatgagtgca ttgtactgac acctggagat agctgtgtta atgtcattgt 1200 tatgtgattg gtcattttct acttttatta ttcttttgtt tttgttttga tgtcttggtc 1260 tgggaggaaa tgttacattc tgtgtagctc tctagaaaag ctctctcttt tcactaaaag 1320 cggttcaagc gtggttgcgt tccggtggcc ggtgggtttt cgccgccggt taccacccct 1380 gtctagtttt ttttctagct ttgttttatc ggcggctcta taatcgagcc tttttccgat 1440 atcaatactt gactctgggg ctgcgacgtc tttttcaggc ggtgatcgcc ggcttttgat 1500 ttcggggggt agaggcttcc tcagccaacc tcgccgactt tcgggaacga tttggggttt 1560 gatgatggtc gaggagatca tcgtcgtggt aacttggttt tggtgctatg tgttctggtg 1620 tcaggcttgt tttttactcg gcggaggaga tttcgacatg gtgttcggtg aagctctccg 1680 gcgatggagc acggcgaaga aggaacaaga gtaagggagt gggttattgt cgcaagcgga 1740 gatagcatga accggttgag atctcaagac agacttgatg aatctcttcg ggggaaaatg 1800 gattggcgac atcgatgggt gcggtggttt gtcgacggag ctcagacgaa attagatcgt 1860 ttcgtctttt ccggagaaaa cttcgaccgt cggatcacga agacatgtta gctgacacgt 1920 gttctgagta ttcggtggtt ggcaacacgt gggcgggctt gggttagtct tggacgcgtg 1980 gctttctctt gctgcgtggg tttcggatat gggcttcagg cccattttat tgtgctcttt 2040 atgttgggcc aatgttataa tcgggccctc agggtcttgt gttctcgggt tgggcttggt 2100 ccggttagac caagtttctt taataaaata tcatactaga tagaaaaaaa taataatgtt 2160 acattcttaa taaaataaca taatatcaga acaagatgaa taaattatgg tttaagcata 2220 tgggtcaaca tttactgtct tttattaact atagtctata tataatcata tcatatgaca 2280 tctctcgtgg agtgttgatt atccgagtaa cttcagtcct cgatacttct ttttttggct 2340 aaatttttct ttttggttaa aatttggtgc aacctcaaat acatctttcg accttaatat 2400 agtttttctc tacggatttg taaaactaac atactcaata tattcacatt tttaatgtgg 2460 tgtctcgagg tctcaagtct cagctgattt tgttttttta attatatatt cagatttttg 2520 ttccgttaat ccacaaggtc atacttttag tttttttttc tgacagcgat catacttttc 2580 gttataacga ccggatatac caaaaacttt acaggatttt ttaactgttt atatctcctt 2640 tatgttttaa tctttttcac atatgttagg acccaattcc cactaaatca cgtcaccgca 2700 aatgattttg atttaaagtt cgtctgcaaa aaagctcttg aaatagtgga tcatattgga 2760 tatggatatg aggatcgttt gatattctga agagtcatgc agttgctatc tttatcccca 2820 tgggaactat gaaaatgaac gttgaaccaa attatggtga aaatatgacg agagctttct 2880 tcttgtgatc tctgtaaagc ttttatatta tgtccaagga actttcatct tcttatacaa 2940 tgtattttcc cataatgtat acgtgtgaac atataaatga taaatgaaca tacccattat 3000 tcgagatgaa cggattatat aatccataca taattaaacc gtagctcaca tatgatgata 3060 gttaagtgat gttcaaccta tactaattta ctgatgctca actattcaac taatttaaat 3120 cataatccga gtgtgtaatt tgttgcacaa agtcctataa gaagggctac tgaaacctga 3180 caaacatcac aaaactatat cgactctttt tttcagaggt atattgcaga atcacattaa 3240 ccatcttgct ttatttcaat catgtttctg aattgtcaag aagaagataa atatcaaaac 3300 taaatgtatg gctttttcta atgcaaagag tccatcaatg gaggaaatcg gatgggatca 3360 aagaaattgg ttaagaggtc agtttcagaa ccaccaaaaa gatatatgtc aaattgagaa 3420 aacaagggaa gtttccgaca ggatctcgtg tccgttgagc tttgacttcc ttaaacagca 3480 tataaccaaa ttttacttcg aaagagttga acttattgca attttagggt tttgttatct 3540 tcaagtgtgc ttcacaccaa tgccaaaaca actgggttct cgagtttctt tccttgagaa 3600 ctaggtgttt tacttaccga acatcttgaa ttataactac acaacagaat gatcactgaa 3660 ttgcttggta cagtaaaatg ttggcagatt tatcagtaaa aactttttca caatcaaaat 3720 tttgacaatc tagtaattaa tagtctaaca acttttaaac ttctaacatt taattattta 3780 aatctcttaa caagtgtact agcgttcgtt taattattgt actaacgttc atttaattat 3840 tttactaacg ttcgtttaat aatgaaattt gttagctaat tgctcgctag aaacgagaac 3900 agctaagtaa aagaaaaaaa ggagtgaacc acagagcttt aagctagcag ggtaattttt 3960 tactatactc tgttcacttt tttcctattg ttaccactca cagatatacc agtcttgaaa 4020 atatataacc tattctctta tatattttca aagagacaat acatgaggat cgttagatta 4080 tatatcggtc atccatctgt ttaatcggtt aatttcgggt tttcgtgatt ttcttaaata 4140 tggatatttt taagagatcc taaattgaat tatcagatca ccggattaac cggtaaaatc 4200 agagatttga atcaaattta aaagcactga tttaaatata tatatatata tatatatata 4260 aattctttta aattaacaaa atatttgtta aattattagt aaattttttt tatcgtaagt 4320 gcggtcaagt tctagttatt attattacaa acactacatt ttaatttaac tctatattat 4380 ttggcctctt ctgctgtaaa aagggtcgca taagattcct tttgaaatca aaatggacac 4440 gggaatagtg atactaaata gttaattgaa aaaaaattcg atttattttt ttttaaacat 4500 ccgtttttgc tgttgaataa catcacagta aaatttataa ttactttaca aatcctttct 4560 ggtcattaca attccatgga aatgtcatat atgtactgta tatgaatttg atggcatgga 4620 atgtaaggtg cttttctatt aagcaatgat ctatacagta atacacacat ggtatttcta 4680 tttggaattt atttgtttgc atgtgacagt aattagtaag ccttcatatt tggagaggat 4740 atgctctgaa atttgattta cattcttcaa aaaaatatgt tcttctagaa gcaaaaagcc 4800 aagatgtata gaaagttatt gtcctttgat ggctcatcaa aaatagaaat aaaagtgtga 4860 aagctcttta atagttgcat gtggcatcat cgaaggcaaa atgaaaagaa tcttatgttt 4920 gggcctattg cttcgctcac aagcattaat ttcatccctt agaaataaat taaaagttta 4980 ctaacaaaat ggctttattc ctatgtcctg acaattttaa tgtcagttcg gcaatgttag 5040 gctactgaag taagcataag aagtatgttc tatgtaacaa agtagtgtga ttttggtaga 5100 tttatccata aatttctata tacattacac aagataatta aagagtaatt gagagattct 5160 tataccaatg aataaacata ggactatcgt ggaagcattt taatatattg gttaaaatct 5220 aaagatttct acatccaaat caggttttga acttcagaat atgtaatttt ttgcatatta 5280 taggatgtac atttcatagg attttagaca tagttatttg ttgtgatcat ttgctacgaa 5340 tataacctac ttatcgaaga tatcgtacat gcataactgt ccatcgatcc aagtgatttt 5400 gagagaagtt catcatggaa tttttataca tggaacaatt tatcaatatt ttatatgtta 5460 tatgtagttg atcatcatat aatagaatta tataatgatg taatgtgtgc tacttattgc 5520 tagaaaaaac aacgattatc aaatatgtac ttttaacaat tgttttggtg ttgatttatt 5580 accatagtat ttttgttggt gtgtgttaga cgatttcata ttttatcatc cttttattaa 5640 tatcttttgt gaatagttaa attaactatt tgtaagaaaa attttgtgtt aacaaaacta 5700 gtagtttatg tatcttatct tgtttaaata aatttatata taatattata attaaatata 5760 attatgcaaa tagtttgttt agcatcaaat cttattttta gaataattta cattatttgg 5820 cttagaatat acatacattt agatatatta aatggaatat atggatcatg ctatatatat 5880 gcaggacatg acatgatata gattatgaat ctatcatata tgatatgttg atgcgttttg 5940 ggttttttct atttatagtc tttctattaa atttaattat attgcttttc tcttttgctt 6000 tcccttacat agacatcatt cgtaggacaa ttcaaagttt ttatatattt ttttggcact 6060 acaaaagttc ttaactagtt gatgaataag aaacgttgac aaaaaggtta aggaatagga 6120 aaccatgata aaaaaaaatc gactaaaaac cataataatt gagaactgca gttttcacct 6180 tttttttttt ggtctaaatg ttaagctgca gttttttttt tttgctaaat aaagctgcag 6240 ttttcacttg cgactggcat atgtgttttt tagttcagtt gttccgctaa cgacagacga 6300 tattccttac tcgcgagatt tcacggactc gtaagcatat ctgaggtcct ttgggtctct 6360 tatgtagaaa gctcatggaa aaaaaatacg atatttacac gcacaataaa tattatagat 6420 tacgcgtaag taagtaaagt ggttgtactc taatttatga tagcgatata tcacatatga 6480 tgattaactc tttgattcga aattgtagtt tttctcatgt agcacattga gcttgtttcc 6540 ccctctcaag aaatgacgtt gttattgtta tgagcagcac tataaacttg acataatgat 6600 gatttcactt tttatttatt tatcgtatag agaccggtga atattttctt tgaagtaaat 6660 aaaataagag aaacggtttc gcgtgtttaa aatataccac gttggtaaaa ctattcacat 6720 gtagtgacat caactcctaa gtcaaattaa gaatctaata tataaagcaa tgaagccctt 6780 tatgtcaaac tttaaacttt gcttcccttg atggcaatca tatactgtat aagcattatt 6840 ttagttagag gcaatccgtt tatataaatg tcttttagct ttaacaaaaa aaaagtcttt 6900 cgacttaatt attgattgaa ataacttgca tatttcaccg taggataaag ttagagggac 6960 gacccatagt gagaatcttt taactacaac taggaagatg tattcaattt aacagttgaa 7020 gtgatttgat ttttaatgga gttttagatg atttttataa gttacagaga tttagttgat 7080 ttttattaaa ctattctaga atatcaccta aaacaatgag atttgagttt taattttttt 7140 taactaagaa actccatcca aacattctaa aatcacctca aaattttaag gagggttaat 7200 ttttatcatc aatgattaat atgaatgttt aaaatgatat gaatacccga ttctctacat 7260 taatctaagt ttaattaaga aaataaaata aagttcaaaa gaaaaaaatt acattaacaa 7320 ctaaattaat aacagaggca tatgatctgc actctctgca tgagcagcac gtgtctgtct 7380 agaagaagtc aatgcctcaa tggtcatgat tctgtaataa gccacacagg ctatcactgg 7440 gagaaacttc tgcttttagc cttctaacct tttataaaca caaaacccta aaaagataca 7500 ctggccactc tcgtcttcag tgtgctaaag tgcccaccta aataaaattt ataatattat 7560 tactttgtta tatatttaat gtgccaactc atttcttttt agtcgctaca aaagttccaa 7620 ttattgttaa atttaactta tatatatgat gttttggaat tcagatgggt ccacaaaatt 7680 aggtggtata ttatcatcca ttagtttggt aatcaattaa taggaggagg tttcatttct 7740 ttctcatttt aattgtgtgt aaaatgtgta ttaatctccc cagtttacaa tattatataa 7800 gtttgtacga cccttcaaca ttgttgcaaa cattcttagt gagagaaata gagagaaaag 7860 agagagatta actagtgaga gaaagagag 7889 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 tgggatctgg ttggatcttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 gaatcaaagc ggaaccaaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 acggtccaga ctcaaagctg 20 <210> 6 <211> 10099 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggactt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 acctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatct atcttattaa aagagaagta 360 cccatttaaa aatacccctt agttttcact tttatttaca ataatattaa ttaagcttcc 420 tatttttatg catgtctttt tcactttagt taatgtgttt tccaaaatta aatacacaat 480 taaataatac ttcctatttt aatgcttatc tttttcactt agattaatgt gttttccaaa 540 attaaatttg aattaaatac acttcattaa cttctctatt aaatcaatat caaatttaaa 600 aaaattatat tcttactgga caaacagttc ataaaaatta taatatcaat ttttcattta 660 aacgaatctg aacgaaaaaa atattaccaa atttaaaccg aaactagata atatccaaac 720 ggatttacca ttttggcatc taaagaacca taaccaaacc tgatccgaac gaaatatttc 780 ggatattaca atgtatttaa atcatattta tatacttcaa tatgttagct attacagata 840 aacaattatc aaaacaccaa aaatacttaa tatatatatt tattctccat ccaaatattc 900 aagttaaata tattttaaat tttaaattta ggtactttgg cttacattac tcaaatttac 960 atgttatatt atttttattt ttatatttag ggttatttaa agatataaaa tttttgaaat 1020 ttaaaaatag tttaaacggg ttatcaaacc tgcaaaaatc cgaatcaaag cggaaccaaa 1080 atttataaac acacgaataa agctagaatc tttaaactcg aaaatctcaa atccgaatag 1140 attttaaccg aatttgaatg ggtaaccaaa tattcacccc taacttagtc aatataaaac 1200 gatcaaatat tataaatata ctattttgta taaagaaata aaaactaaaa ttgaaaatta 1260 atatccgtgc ggtcgcacgg gtcaagatct agtacttcat ttggaagaaa gtcagctttg 1320 agtctggacc gtcctttttt ttcaaaatta ctcttggtta tactaaacaa atttaatctt 1380 gttaacaaag tttgatttaa ttagtacgtg ttactgcatt atctatgtgt catttatgat 1440 atataattct ttataaactc aacatatttt ttataaatgt gcgtgtttga tcaaaaaaaa 1500 atattgaaaa ccctttaaca aaaaaaaaag agtatcctat atatatccat gcattgagcg 1560 taggcagaag gcttgatggt gtcagctcga ataaatgaat gtaaatggac agatcatatt 1620 actattttta gaatatggtc cgtagaattt tcataaaatt gactaatcaa ttagtttcta 1680 gttttggtta aagccaagct cacacgaagt ctaaaacgtt cctctaaaaa ggaatcgtaa 1740 cagtcccgtt tttatttcac tgttttagca atttctctaa aaaggaatcg taacagtcca 1800 gtttttattt cactgtttta gcaatttctt tacatcaagt aaaatatatt tatatattcc 1860 tcataccctt tttcctactt tctacaaatt cgtttaccaa cgtaaaaatg aatttattag 1920 gcatctacca gatttgaata tcgtccaaga agtaaatgaa taaagaaaac tgtttaaaag 1980 tactccacat ttgactttaa taaagcaaat tatttacatt tgactcagat taatactttt 2040 tattttacga gtattattga ttatgtgaca aaaaactgat tggctatgag tgcattgtac 2100 tgacacctgg agatagctgt gttaatgtca ttgttatgtg attggtcatt ttctactttt 2160 attattcttt tgtttttgtt ttgatgtctt ggtctgggag gaaatgttac attctgtgta 2220 gctctctaga aaagctctct cttttcacta aaagcggttc aagcgtggtt gcgttccggt 2280 ggccggtggg ttttcgccgc cggttaccac ccctgtctag ttttttttct agctttgttt 2340 tatcggcggc tctataatcg agcctttttc cgatatcaat acttgactct ggggctgcga 2400 cgtctttttc aggcggtgat cgccggcttt tgatttcggg gggtagaggc ttcctcagcc 2460 aacctcgccg actttcggga acgatttggg gtttgatgat ggtcgaggag atcatcgtcg 2520 tggtaacttg gttttggtgc tatgtgttct ggtgtcaggc ttgtttttta ctcggcggag 2580 gagatttcga catggtgttc ggtgaagctc tccggcgatg gagcacggcg aagaaggaac 2640 aagagtaagg gagtgggtta ttgtcgcaag cggagatagc atgaaccggt tgagatctca 2700 agacagactt gatgaatctc ttcgggggaa aatggattgg cgacatcgat gggtgcggtg 2760 gtttgtcgac ggagctcaga cgaaattaga tcgtttcgtc ttttccggag aaaacttcga 2820 ccgtcggatc acgaagacat gttagctgac acgtgttctg agtattcggt ggttggcaac 2880 acgtgggcgg gcttgggtta gtcttggacg cgtggctttc tcttgctgcg tgggtttcgg 2940 atatgggctt caggcccatt ttattgtgct ctttatgttg ggccaatgtt ataatcgggc 3000 cctcagggtc ttgtgttctc gggttgggct tggtccggtt agaccaagtt tctttaataa 3060 aatatcatac tagatagaaa aaaataataa tgttacattc ttaataaaat aacataatat 3120 cagaacaaga tgaataaatt atggtttaag catatgggtc aacatttact gtcttttatt 3180 aactatagtc tatatataat catatcatat gacatctctc gtggagtgtt gattatccga 3240 gtaacttcag tcctcgatac ttcttttttt ggctaaattt ttctttttgg ttaaaatttg 3300 gtgcaacctc aaatacatct ttcgacctta atatagtttt tctctacgga tttgtaaaac 3360 taacatactc aatatattca catttttaat gtggtgtctc gaggtctcaa gtctcagctg 3420 attttgtttt tttaattata tattcagatt tttgttccgt taatccacaa ggtcatactt 3480 ttagtttttt tttctgacag cgatcatact tttcgttata acgaccggat ataccaaaaa 3540 ctttacagga ttttttaact gtttatatct cctttatgtt ttaatctttt tcacatatgt 3600 taggacccaa ttcccactaa atcacgtcac cgcaaatgat tttgatttaa agttcgtctg 3660 caaaaaagct cttgaaatag tggatcatat tggatatgga tatgaggatc gtttgatatt 3720 ctgaagagtc atgcagttgc tatctttatc cccatgggaa ctatgaaaat gaacgttgaa 3780 ccaaattatg gtgaaaatat gacgagagct ttcttcttgt gatctctgta aagcttttat 3840 attatgtcca aggaactttc atcttcttat acaatgtatt ttcccataat gtatacgtgt 3900 gaacatataa atgataaatg aacataccca ttattcgaga tgaacggatt atataatcca 3960 tacataatta aaccgtagct cacatatgat gatagttaag tgatgttcaa cctatactaa 4020 tttactgatg ctcaactatt caactaattt aaatcataat ccgagtgtgt aatttgttgc 4080 acaaagtcct ataagaaggg ctactgaaac ctgacaaaca tcacaaaact atatcgactc 4140 tttttttcag aggtatattg cagaatcaca ttaaccatct tgctttattt caatcatgtt 4200 tctgaattgt caagaagaag ataaatatca aaactaaatg tatggctttt tctaatgcaa 4260 agagtccatc aatggaggaa atcggatggg atcaaagaaa ttggttaaga ggtcagtttc 4320 agaaccacca aaaagatata tgtcaaattg agaaaacaag ggaagtttcc gacaggatct 4380 cgtgtccgtt gagctttgac ttccttaaac agcatataac caaattttac ttcgaaagag 4440 ttgaacttat tgcaatttta gggttttgtt atcttcaagt gtgcttcaca ccaatgccaa 4500 aacaactggg ttctcgagtt tctttccttg agaactaggt gttttactta ccgaacatct 4560 tgaattataa ctacacaaca gaatgatcac tgaattgctt ggtacagtaa aatgttggca 4620 gatttatcag taaaaacttt ttcacaatca aaattttgac aatctagtaa ttaatagtct 4680 aacaactttt aaacttctaa catttaatta tttaaatctc ttaacaagtg tactagcgtt 4740 cgtttaatta ttgtactaac gttcatttaa ttattttact aacgttcgtt taataatgaa 4800 atttgttagc taattgctcg ctagaaacga gaacagctaa gtaaaagaaa aaaaggagtg 4860 aaccacagag ctttaagcta gcagggtaat tttttactat actctgttca cttttttcct 4920 attgttacca ctcacagata taccagtctt gaaaatatat aacctattct cttatatatt 4980 ttcaaagaga caatacatga ggatcgttag attatatatc ggtcatccat ctgtttaatc 5040 ggttaatttc gggttttcgt gattttctta aatatggata tttttaagag atcctaaatt 5100 gaattatcag atcaccggat taaccggtaa aatcagagat ttgaatcaaa tttaaaagca 5160 ctgatttaaa tatatatata tatatatata tataaattct tttaaattaa caaaatattt 5220 gttaaattat tagtaaattt tttttatcgt aagtgcggtc aagttctagt tattattatt 5280 acaaacacta cattttaatt taactctata ttatttggcc tcttctgctg taaaaagggt 5340 cgcataagat tccttttgaa atcaaaatgg acacgggaat agtgatacta aatagttaat 5400 tgaaaaaaaa ttcgatttat ttttttttaa acatccgttt ttgctgttga ataacatcac 5460 agtaaaattt ataattactt tacaaatcct ttctggtcat tacaattcca tggaaatgtc 5520 atatatgtac tgtatatgaa tttgatggca tggaatgtaa ggtgcttttc tattaagcaa 5580 tgatctatac agtaatacac acatggtatt tctatttgga atttatttgt ttgcatgtga 5640 cagtaattag taagccttca tatttggaga ggatatgctc tgaaatttga tttacattct 5700 tcaaaaaaat atgttcttct agaagcaaaa agccaagatg tatagaaagt tattgtcctt 5760 tgatggctca tcaaaaatag aaataaaagt gtgaaagctc tttaatagtt gcatgtggca 5820 tcatcgaagg caaaatgaaa agaatcttat gtttgggcct attgcttcgc tcacaagcat 5880 taatttcatc ccttagaaat aaattaaaag tttactaaca aaatggcttt attcctatgt 5940 cctgacaatt ttaatgtcag ttcggcaatg ttaggctact gaagtaagca taagaagtat 6000 gttctatgta acaaagtagt gtgattttgg tagatttatc cataaatttc tatatacatt 6060 acacaagata attaaagagt aattgagaga ttcttatacc aatgaataaa cataggacta 6120 tcgtggaagc attttaatat attggttaaa atctaaagat ttctacatcc aaatcaggtt 6180 ttgaacttca gaatatgtaa ttttttgcat attataggat gtacatttca taggatttta 6240 gacatagtta tttgttgtga tcatttgcta cgaatataac ctacttatcg aagatatcgt 6300 acatgcataa ctgtccatcg atccaagtga ttttgagaga agttcatcat ggaattttta 6360 tacatggaac aatttatcaa tattttatat gttatatgta gttgatcatc atataataga 6420 attatataat gatgtaatgt gtgctactta ttgctagaaa aaacaacgat tatcaaatat 6480 gtacttttaa caattgtttt ggtgttgatt tattaccata gtatttttgt tggtgtgtgt 6540 tagacgattt catattttat catcctttta ttaatatctt ttgtgaatag ttaaattaac 6600 tatttgtaag aaaaattttg tgttaacaaa actagtagtt tatgtatctt atcttgttta 6660 aataaattta tatataatat tataattaaa tataattatg caaatagttt gtttagcatc 6720 aaatcttatt tttagaataa tttacattat ttggcttaga atatacatac atttagatat 6780 attaaatgga atatatggat catgctatat atatgcagga catgacatga tatagattat 6840 gaatctatca tatatgatat gttgatgcgt tttgggtttt ttctatttat agtctttcta 6900 ttaaatttaa ttatattgct tttctctttt gctttccctt acatagacat cattcgtagg 6960 acaattcaaa gtttttatat atttttttgg cactacaaaa gttcttaact agttgatgaa 7020 taagaaacgt tgacaaaaag gttaaggaat aggaaaccat gataaaaaaa aatcgactaa 7080 aaaccataat aattgagaac tgcagttttc accttttttt ttttggtcta aatgttaagc 7140 tgcagttttt tttttttgct aaataaagct gcagttttca cttgcgactg gcatatgtgt 7200 tttttagttc agttgttccg ctaacgacag acgatattcc ttactcgcga gatttcacgg 7260 actcgtaagc atatctgagg tcctttgggt ctcttatgta gaaagctcat ggaaaaaaaa 7320 tacgatattt acacgcacaa taaatattat agattacgcg taagtaagta aagtggttgt 7380 actctaattt atgatagcga tatatcacat atgatgatta actctttgat tcgaaattgt 7440 agtttttctc atgtagcaca ttgagcttgt ttccccctct caagaaatga cgttgttatt 7500 gttatgagca gcactataaa cttgacataa tgatgatttc actttttatt tatttatcgt 7560 atagagaccg gtgaatattt tctttgaagt aaataaaata agagaaacgg tttcgcgtgt 7620 ttaaaatata ccacgttggt aaaactattc acatgtagtg acatcaactc ctaagtcaaa 7680 ttaagaatct aatatataaa gcaatgaagc cctttatgtc aaactttaaa ctttgcttcc 7740 cttgatggca atcatatact gtataagcat tattttagtt agaggcaatc cgtttatata 7800 aatgtctttt agctttaaca aaaaaaaagt ctttcgactt aattattgat tgaaataact 7860 tgcatatttc accgtaggat aaagttagag ggacgaccca tagtgagaat cttttaacta 7920 caactaggaa gatgtattca atttaacagt tgaagtgatt tgatttttaa tggagtttta 7980 gatgattttt ataagttaca gagatttagt tgatttttat taaactattc tagaatatca 8040 cctaaaacaa tgagatttga gttttaattt tttttaacta agaaactcca tccaaacatt 8100 ctaaaatcac ctcaaaattt taaggagggt taatttttat catcaatgat taatatgaat 8160 gtttaaaatg atatgaatac ccgattctct acattaatct aagtttaatt aagaaaataa 8220 aataaagttc aaaagaaaaa aattacatta acaactaaat taataacaga ggcatatgat 8280 ctgcactctc tgcatgagca gcacgtgtct gtctagaaga agtcaatgcc tcaatggtca 8340 tgattctgta ataagccaca caggctatca ctgggagaaa cttctgcttt tagccttcta 8400 accttttata aacacaaaac cctaaaaaga tacactggcc actctcgtct tcagtgtgct 8460 aaagtgccca cctaaataaa atttataata ttattacttt gttatatatt taatgtgcca 8520 actcatttct ttttagtcgc tacaaaagtt ccaattattg ttaaatttaa cttatatata 8580 tgatgttttg gaattcagat gggtccacaa aattaggtgg tatattatca tccattagtt 8640 tggtaatcaa ttaataggag gaggtttcat ttctttctca ttttaattgt gtgtaaaatg 8700 tgtattaatc tccccagttt acaatattat ataagtttgt acgacccttc aacattgttg 8760 caaacattct tagtgagaga aatagagaga aaagagagag attaactagt gagagaaaga 8820 gagatgggcc atcactcgtg ctgcaacaag caaaaggtga agagagggct ttggtcaccg 8880 gaagaagacg aaaagctcat caactacatc aatacatatg gccatggatg ctggagctct 8940 gttcccaaac atgcaggcat atatattggc tttgtttgtt gaattatcta ctgataaaaa 9000 gtctatgtcg ttggttttaa tctatgtgtt gttttccgtt tatttaatta ggtctgcaga 9060 gatgtggaaa gagttgtcga ttaagatgga taaattatct aagacctgat cttaaacgtg 9120 gaagcttctc acctcaagaa gctgctctta tcattgagct tcatagcatt cttggtaaca 9180 ggttgttact taattatcat tacatgttgg tttatatgtg ataatataaa actaaatttt 9240 catgaagaaa ctgaactacc tttatactca tttttttttg ttatggattc ttgataggtg 9300 ggcacaaatt gctaaacatc tacctggaag aacagataac gaggtcaaga atttctggaa 9360 ctcaagcatt aaaaagaagc tcatgtctca ccactatcac catcatcatg gtcatcatca 9420 tcatcatacc tcttcaatgg cgagtttgct cactaacctt ccttatcaca acgtgttcaa 9480 cgctactgct ggcgacgatg aaggttcaag attcatgtcc agtatcatca caaacactaa 9540 ccctaatttc atcactccaa cccatctccc tcttccttct catcatgtta tgtctccatt 9600 gatgttctca acctctagag aaggagatat caagtattta accgctacca acacaaacca 9660 gtctcatcaa gataataacc tttacaccaa cctcgacatt ttgtcagcca cgccattcat 9720 caacaaccat catcaccatc atgataatga cccccagtgg ccatcgctac cagatctccc 9780 agcgagtacc atttccactt ttcacgacaa ccttcaagat tatgatgatg gtgataaact 9840 caacgtgttt gtaacaccat acaatgataa tggtgatcct cggagctttc ctttggtggc 9900 cacaaagtta atatgtggac aagttcttga aggcaaagca atttcacaag accacggcag 9960 ctttcttctc ccaacaacgt acaacttgga aatgcctggt gatcatcgac gagtggactc 10020 atacattaat cacatgatca ttccctcatc atcatcctct tcatcgccaa tctcttgtgg 10080 acagtacgtc atcacttaa 10099 <210> 7 <211> 9140 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggactt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 acctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatac ttcatttgga agaaagtcag 360 ctttgagtct ggaccgtcct tttttttcaa aattactctt ggttatacta aacaaattta 420 atcttgttaa caaagtttga tttaattagt acgtgttact gcattatcta tgtgtcattt 480 atgatatata attctttata aactcaacat attttttata aatgtgcgtg tttgatcaaa 540 aaaaaatatt gaaaaccctt taacaaaaaa aaaagagtat cctatatata tccatgcatt 600 gagcgtaggc agaaggcttg atggtgtcag ctcgaataaa tgaatgtaaa tggacagatc 660 atattactat ttttagaata tggtccgtag aattttcata aaattgacta atcaattagt 720 ttctagtttt ggttaaagcc aagctcacac gaagtctaaa acgttcctct aaaaaggaat 780 cgtaacagtc ccgtttttat ttcactgttt tagcaatttc tctaaaaagg aatcgtaaca 840 gtccagtttt tatttcactg ttttagcaat ttctttacat caagtaaaat atatttatat 900 attcctcata ccctttttcc tactttctac aaattcgttt accaacgtaa aaatgaattt 960 attaggcatc taccagattt gaatatcgtc caagaagtaa atgaataaag aaaactgttt 1020 aaaagtactc cacatttgac tttaataaag caaattattt acatttgact cagattaata 1080 ctttttattt tacgagtatt attgattatg tgacaaaaaa ctgattggct atgagtgcat 1140 tgtactgaca cctggagata gctgtgttaa tgtcattgtt atgtgattgg tcattttcta 1200 cttttattat tcttttgttt ttgttttgat gtcttggttt gggaggaaat gttacattct 1260 gtgtagctct ctagaaaagc tctctctttt cactaaaagc ggttcaagcg tggttgcgtt 1320 ccggtggccg gtgggttttc gccgccggtt accacccctg tctagttttt tttctagctt 1380 tgttttatcg gcggctctat aatcgagcct ttttccgata tcaatacttg actctggggc 1440 tgcgacgtct ttttcaggcg gtgatcgccg gcttttgatt tcggggggta gaggcttcct 1500 cagccaacct cgccgacttt cgggaacgat ttggggtttg atgatggtcg aggagatcat 1560 cgtcgtggta acttggtttt ggtgctatgt gttctggtgt ccggcttgtt ttttactcgg 1620 cggaggagat ttcgacatgg tgttcggtga agctctccgg cgatggagca cggcgaagaa 1680 ggaacaagag taagggagtg ggttattgtc gcaagcggag atagcatgaa ccggttgaga 1740 tctcaagaca gacttgatga atctcttcgg gggaaaatgg attggcgaca tcgatgggtg 1800 cggtggtttg tcgacggagc tcagacgaaa ttagatcgtt tcgtcttttc cggagaaaac 1860 ttcgaccgtc ggatcacgaa gacatgttag ctgacacgtg ttctgagtat tcggtggttg 1920 gcaacacgtg ggcgggcttg ggttagtctt ggacgcgtgg ctttctcttg ctgcgtgggt 1980 ttcggatatg ggcttcaggc ccattttatt gtgctcttta tgttgggcca atgttataat 2040 cgggccctca gggtcttgtg ttctcgggtt gggcttggtc cggttagacc aagtttcttt 2100 aataaaatat catactagat agaaaaaaat aataatgtta cattcttaat aaaataacat 2160 aatatcagaa caagatgaat aaattatggt ttaagcatat gggtcaacat ttactgtctt 2220 ttattaacta tagtctatat ataatcatat catatgacat ctctcgtgga gtgttgatta 2280 tccgagtaac ttcagtcctc gatacttctt tttttggcta aatttttctt tttggttaaa 2340 atttggtgca acctcaaata catctttcga ccttaatata gtttttctct acggatttgt 2400 aaaactaaca tactcaatat attcacattt ttaatgtggt gtctcgaggt ctcaagtctc 2460 agctgatttt gtttttttaa ttatatattc agatttttgt tccgttaatc cacaaggtca 2520 tacttttagt ttttttttct gacagcgatc atacttttcg ttataacgac cggatatacc 2580 aaaaacttta caggattttt taactgttta tatctccttt atgttttaat ctttttcaca 2640 tatgttagga cccaattccc actaaatcac gtcaccgcaa atgattttga tttaaagttc 2700 gtctgcaaaa aagctcttga aatagtggat catattggat atggatatga ggatcgtttg 2760 atattctgaa gagtcatgca gttgctatct ttatccccat gggaactatg aaaatgaacg 2820 ttgaaccaaa ttatggtgaa aatatgacga gagctttctt cttgtgatct ctgtaaagct 2880 tttatattat gtccaaggaa ctttcatctt cttatacaat gtattttccc ataatgtata 2940 cgtgtgaaca tataaatgat aaatgaacat acccattatt cgagatgaac ggattatata 3000 atccatacat aattaaaccg tagctcacat atgatgatag ttaagtgatg ttcaacctat 3060 actaatttac tgatgctcaa ctattcaact aatttaaatc ataatccgag tgtgtaattt 3120 gttgcacaaa gtcctataag aagggctact gaaacctgac aaacatcaca aaactatatc 3180 gactcttttt ttcagaggta tattgcagaa tcacattaac catcttgctt tatttcaatc 3240 atgtttctga attgtcaaga agaagataaa tatcaaaact aaatgtatgg ctttttctaa 3300 tgcaaagagt ccatcaatgg aggaaatcgg atgggatcaa agaaattggt taagaggtca 3360 gtttcagaac caccaaaaag atatatgtca aattgagaaa acaagggaag tttccgacag 3420 gatctcgtgt ccgttgagct ttgacttcct taaacagcat ataaccaaat tttacttcga 3480 aagagttgaa cttattgcaa ttttagggtt ttgttatctt caagtgtgct tcacaccaat 3540 gccaaaacaa ctgggttctc gagtttcttt ccttgagaac taggtgtttt acttaccgaa 3600 catcttgaat tataactaca caacagaatg atcactgaat tgcttggtac agtaaaatgt 3660 tggcagattt atcagtaaaa actttttcac aatcaaaatt ttgacaatct agtaattaat 3720 agtctaacaa cttttaaact tctaacattt aattatttaa atctcttaac aagtgtacta 3780 gcgttcgttt aattattgta ctaacgttca tttaattatt ttactaacgt tcgtttaata 3840 atgaaatttg ttagctaatt gctcgctaga aacgagaaca gctaagtaaa agaaaaaaag 3900 gagtgaacca cagagcttta agctagcagg gtaatttttt actatactct gttcactttt 3960 ttcctattgt taccactcac agatatacca gtcttgaaaa tatataacct attctcttat 4020 atattttcaa agagacaata catgaggatc gttagattat atatcggtca tccatctgtt 4080 taatcggtta atttcgggtt ttcgtgattt tcttaaatat ggatattttt aagagatcct 4140 aaattgaatt atcagatcac cggattaacc ggtaaaatca gagatttgaa tcaaatttaa 4200 aagcactgat ttaaatatat atatatatat atatataaat tcttttaaat taacaaaata 4260 tttgttaaat tattagtaaa ttttttttat cgtaagtgcg gtcaagttct agttattatt 4320 attacaaaca ctacatttta atttaactct atattatttg gcctcttctg ctgtaaaaag 4380 ggtcgcataa gattcctttt gaaatcaaaa tggacacggg aatagtgata ctaaatagtt 4440 aattgaaaaa aaattcgatt tatttttttt aaacatccgt ttttgctgtt gaataacatc 4500 acagtaaaat ttataattac tttacaaatc ctttctggtc attacaattc catggaaatg 4560 tcatatatgt actgtatatg aatttgatgg catggaatgt aaggtgcttt tctattaagc 4620 aatgatctat acagtaatac acacatggta tttctatttg gaatttattt gtttgcatgt 4680 gacagtaatt agtaagcctt catatttgga gaggatatgc tctgaaattt gatttacatt 4740 cttcaaaaaa atatgttctt ctagaagcaa aaagccaaga tgtatagaaa gttattgtcc 4800 tttgatggct catcaaaaat agaaataaaa gtgtgaaagc tctttaatag ttgcatgtgg 4860 catcatcgaa ggcaaaatga aaagaatctt atgtttgggc ctattgcttc gctcacaagc 4920 attaatttca tcccttagaa ataaattaaa agtttactaa caaaatggct ttattcctat 4980 gtcctgacaa ttttaatgtc agttcggcaa tgttaggcta ctgaagtaag cataagaagt 5040 atgttctatg taacaaagta gtgtgatttt ggtagattta tccataaatt tctatataca 5100 ttacacaaga taattaaaga gtaattgaga gattcttata ccaatgaata aacataggac 5160 tatcgtggaa gcattttaat atattggtta aaatctaaag atttctacat ccaaatcagg 5220 ttttgaactt cagaatatgt aattttttgc atattatagg atgtacattt cataggattt 5280 tagacatagt tatttgttgt gatcatttgc tacgaatata acctacttat cgaagatatc 5340 gtacatgcat aactgtccat cgatccaagt gattttgaga gaagttcatc atggaatttt 5400 tatacatgga acaatttatc aatattttat atgttatatg tagttgatca tcatataata 5460 gaattatata atgatgtaat gtgtgctact tattgctaga aaaaacaacg attatcaaat 5520 atgtactttt aacaattgtt ttggtgttga tttattacca tagtattttt gttggtgtgt 5580 gttagacgat ttcatatttt atcatccttt tattaatatc ttttgtgaat agttaaatta 5640 actatttgta agaaaaattt tgtgttaaca aaactagtag tttatgtatc ttatcttgtt 5700 taaataaatt tatatataat attataatta aatataatta tgcaaatagt ttgtttagca 5760 tcaaatctta tttttagaat aatttacatt atttggctta gaatatacat acatttagat 5820 atattaaatg gaatatatgg atcatgctat atatatgcag gacatgacat gatatagatt 5880 atgaatctat catatatgat atgttgatgc gttttgggtt ttttctattt atagtctttc 5940 tattaaattt aattatattg cttttctctt ttgctttccc ttacatagac atcattcgta 6000 ggacaattca aagtttttat atattttttt ggcactacaa aagttcttaa ctagttgatg 6060 aataagaaac gttgacaaaa aggttaagga ataggaaacc atgataaaaa aaaatcgact 6120 aaaaaccata ataattgaga actgcagttt tcaccttttt tttttggtct aaatgttaag 6180 ctgcagtttt ttttttttgc taaataaagc tgcagttttc acttgcgact ggcatatgtg 6240 ttttttagtt cagttgttcc gctaacgaca gacgatattc cttactcgcg agatttcacg 6300 gactcgtaag catatctgag gtcctttggg tctcttatgt agaaagctca tggaaaaaaa 6360 atacgatatt tacacgcaca ataaatatta tagattacgc gtaagtaagt aaagtggttg 6420 tactctaatt tatgatagcg atatatcaca tatgatgatt aactctttga ttcgaaattg 6480 tagtttttct catgtagcac attgagcttg tttccccctc tcaagaaatg acgttgttat 6540 tgttatgagc agcactataa acttgacata atgatgattt cactttttat ttatttatcg 6600 tatagagacc ggtgaatatt ttctttgaag taaataaaat aagagaaacg gtttcgcgtg 6660 tttaaaatat accacgttgg taaaactatt cacatgtagt gacatcaact cctaagtcaa 6720 attaagaatc taatatataa agcaatgaag ccctttatgt caaactttaa actttgcttc 6780 ccttgatggc aatcatatac tgtataagca ttattttagt tagaggcaat ccgtttatat 6840 aaatgtcttt tagctttaac aaaaaaaaag tctttcgact taattattga ttgaaataac 6900 ttgcatattt caccgtagga taaagttaga gggacgaccc atagtgagaa tcttttaact 6960 acaactagga agatgtattc aatttaacag ttgaagtgat ttgattttta atggagtttt 7020 agatgatttt tataagttac agagatttag ttgattttta ttaaactatt ctagaatatc 7080 acctaaaaca atgagatttg agttttaatt ttttttaact aagaaactcc atccaaacat 7140 tctaaaatca cctcaaaatt ttaaggaggg ttaattttta tcatcaatga ttaatatgaa 7200 tgtttaaaat gatatgaata cccgattctc tacattaatc taagtttaat taagaaaata 7260 aaataaagtt caaaagaaaa aaattacatt aacaactaaa ttaataacag aggcatatga 7320 tctgcactct ctgcatgagc agcacgtgtc tgtctagaag aagtcaatgc ctcaatggtc 7380 atgattctgt aataagccac acaggctatc actgggagaa acttctgctt ttagccttct 7440 aaccttttat aaacacaaaa ccctaaaaag atacactggc cactctcgtc ttcagtgtgc 7500 taaagtgccc acctaaataa aatttataat attattactt tgttatatat ttaatgtgcc 7560 aactcatttc tttttagtcg ctacaaaagt tccaattatt gttaaattta acttatatat 7620 atgatgtttt ggaattcaga tgggtccaca aaattaggtg gtatattatc atccattagt 7680 ttggtaatca attaatagga ggaggtttca tttctttctc attttaattg tgtgtaaaat 7740 gtgtattaat ctccccagtt tacaatatta tataagtttg tacgaccctt caacattgtt 7800 gcaaacattc ttagtgagag aaatagagag aaaagagaga gattaactag tgagagaaag 7860 agagatgggc catcactcgt gctgcaacaa gcaaaaggtg aagagagggc tttggtcacc 7920 ggaagaagac gaaaagctca tcaactacat caatacatat ggccatggat gctggagctc 7980 tgttcccaaa catgcaggca tatatattgg ctttgtttgt tgaattatct actgataaaa 8040 agtctatgtc gttggtttta atctatgtgt tgttttccgt ttatttaatt aggtctgcag 8100 agatgtggaa agagttgtcg attaagatgg ataaattatc taagacctga tcttaaacgt 8160 ggaagcttct cacctcaaga agctgctctt atcattgagc ttcatagcat tcttggtaac 8220 aggttgttac ttaattatca ttacatgttg gtttatatgt gataatataa aactaaattt 8280 tcatgaagaa actgaactac ctttatactc attttttttt gttatggatt cttgataggt 8340 gggcacaaat tgctaaacat ctacctggaa gaacagataa cgaggtcaag aatttctgga 8400 actcaagcat taaaaagaag ctcatgtctc accactatca ccatcatcat ggtcatcatc 8460 atcatcatac ctcttcaatg gcgagtttgc tcactaacct tccttatcac aacgtgttca 8520 acgctactgc tggcgacgat gaaggttcaa gattcatgtc cagtatcatc acaaacacta 8580 accctaattt catcactcca acccatctcc ctcttccttc tcatcatgtt atgtctccat 8640 tgatgttctc aacctctaga gaaggagata tcaagtattt aaccgctacc aacacaaacc 8700 agtctcatca agataataac ctttacacca acctcgacat tttgtcagcc acgccattca 8760 tcaacaacca tcatcaccat catgataatg acccccagtg gccatcgcta ccagatctcc 8820 cagcgagtac catttccact tttcacgata accttcaaga ttatgatgat ggtgataaac 8880 tcaacgtgtt tgtaacacca tacaatgata atggtgatcc tcggagcttt cctttggtgg 8940 ccacaaagtt aatatgtgga caagttcttg aaggcaaagc aatttcacaa gaccacggca 9000 gctttcttct cccaacaacg tacaacttgg aaatgcctgg tgatcatcga cgagtggact 9060 catacattaa tcacatgatc attccctcat catcatcctc ttcatcgcca atctcttgtg 9120 gacagtacgt catcacttaa 9140 <210> 8 <211> 955 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 ctatcttatt aaaagagaag tacccattta aaaatacccc ttagttttca cttttattta 60 caataatatt aattaagctt cctattttta tgcatgtctt tttcacttta gttaatgtgt 120 tttccaaaat taaatacaca attaaataat acttcctatt ttaatgctta tctttttcac 180 ttagattaat gtgttttcca aaattaaatt tgaattaaat acacttcatt aacttctcta 240 ttaaatcaat atcaaattta aaaaaattat attcttactg gacaaacagt tcataaaaat 300 tataatatca atttttcatt taaacgaatc tgaacgaaaa aaatattacc aaatttaaac 360 cgaaactaga taatatccaa acggatttac cattttggca tctaaagaac cataaccaaa 420 cctgatccga acgaaatatt tcggatatta caatgtattt aaatcatatt tatatacttc 480 aatatgttag ctattacaga taaacaatta tcaaaacacc aaaaatactt aatatatata 540 tttattctcc atccaaatat tcaagttaaa tatattttaa attttaaatt taggtacttt 600 ggcttacatt actcaaattt acatgttata ttatttttat ttttatattt agggttattt 660 aaagatataa aatttttgaa atttaaaaat agtttaaacg ggttatcaaa cctgcaaaaa 720 tccgaatcaa agcggaacca aaatttataa acacacgaat aaagctagaa tctttaaact 780 cgaaaatctc aaatccgaat agattttaac cgaatttgaa tgggtaacca aatattcacc 840 cctaacttag tcaatataaa acgatcaaat attataaata tactattttg tataaagaaa 900 taaaaactaa aattgaaaat taatatccgt gcggtcgcac gggtcaagat ctagt 955 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility <130> PDPB204294k01 <150> KR 10/2020/0140344 <151> 2020-10-27 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1276 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 atgggccatc actcgtgctg caacaagcaa aaggtgaaga gagggctttg gtcaccggaa 60 gactagacgaaa agctgcatcaa ctacat gagctctgtt 120 cccaaacatg caggcatata tattggcttt gtttgttgaa ttatctactg ataaaaagtc 180 tatgtcgttg gttttaatct atgtgttgtt ttccgtttat ttaattaggt ctgcagagat 240 gtggaaagag ttgtcgatta agatggataa attatctaag acctgatctt aaacgtggaa 300 gcttctcacc tcaagaagct gctcttatca ttgagcttca tagcattctt ggtaacaggt 360 tgttacttaa ttatcattac atgttggttt atatgtgata atataaaact aaattttcat 420 gaagaaactg aactaccttt atactcattt ttttttgtta tggattcttg ataggtgggc 480 acaaattgct aaacatctac ctggaagaac agataacgag gtcaagaatt tctggaactc 540 aagcattaaa aaga agctca tgtctcacca ctatcaccat catcatggtc atcatcatca 600 tcatacctct tcaatggcga gtttgctcac taaccttcct tatcacaacg tgttcaacgc 660 tactgctggc gacgatgaag gttcaagatt catgtccagt atcatcacaa acactaaccc 720 taatttcatc actccaaccc atctccctct tccttctcat catgttatgt ctccattgat 780 gttctcaacc tctagagaag gagatatcaa gtatttaacc gctaccaaca caaaccagtc 840 tcatcaagat aataaccttt acaccaacct cgacattttg tcagccacgc cattcatcaa 900 caaccatcat caccatcatg ataatgaccc ccagtggcca tcgctaccag atctcccagc 960 gagtaccatt tccacttttc acgacaacct tcaagattat gatgatggtg ataaactcaa 1020 cgtgtttgta acaccataca atgataatgg tgatcctcgg agctttcctt tggtggccac 1080 aaagttaata tgtggacaag ttcttgaagg caaagcaatt tcacaagacc acggcagctt 1140 tcttctccca acaacgtaca acttggaaat gcctggtgat catcgacgag tggactcata 1200 cattaatcac atgatcattc cctcatcatc atcctcttca tcgccaatct cttgtggaca 1260 gtacgtcatc acttaa 1276 <210> 2 <211> 7889 <212> DNA <213 > Artificial Sequence <220> <223> Artificial Sequence <400> 2 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggac tt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 acctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatcg aatcaaagcg gaaccaaaaa 360 cttcatttgg aagaaagtca gctttgagtc tggaccgtcc ttttttttca aaattactct 420 tggttatact aaacaaattt aatcttgtta acaaagtttg atttaattag tacgtgttac 480 tgcattatct atgtgtcatt tatgatatat aattctttat aaactcaaca tattttttat 540 aaatgtgcgt gtttgatcaa aaaaaaatat tgaaaaccct ttaacaaaaa aaaaagagta 600 tcctatatat atccatgcat tgagcgtagg cagaaggctt gatggtgtca gctcgaataa 660 atgaatgtaa atggacagat catattacta tttttagaat atggtccgta gaattttcat 720 aaaattgact aatcaattag tttctagttt tggttaaagc caagctcaca cgaagtctaa 780 aacgttcctc taaaaaggaa tcgtaacagt cccgttttta tttcactgtt ttagcaattt 840 ctctaaaaag gaatcgtaac agtccagttt ttatttcact gttttagcaa tttctttaca 900 tca agtaaaa tatatttata tattcctcat accctttttc ctactttcta caaattcgtt 960 taccaacgta aaaatgaatt tattaggcat ctaccagatt tgaatatcgt ccaagaagta 1020 aatgaataaa gaaaactgtt taaaagtact ccacatttga ctttaataaa gcaaattatt 1080 tacatttgac tcagattaat actttttatt ttacgagtat tattgattat gtgacaaaaa 1140 actgattggc tatgagtgca ttgtactgac acctggagat agctgtgtta atgtcattgt 1200 tatgtgattg gtcattttct acttttatta ttcttttgtt tttgttttga tgtcttggtc 1260 tgggaggaaa tgttacattc tgtgtagctc tctagaaaag ctctctcttt tcactaaaag 1320 cggttcaagc gtggttgcgt tccggtggcc ggtgggtttt cgccgccggt taccacccct 1380 gtctagtttt ttttctagct ttgttttatc ggcggctcta taatcgagcc tttttccgat 1440 atcaatactt gactctgggg ctgcgacgtc tttttcaggc ggtgatcgcc ggcttttgat 1500 ttcggggggt agaggcttcc tcagccaacc tcgccgactt tcgggaacga tttggggttt 1560 gatgatggtc gaggagatca tcgtcgtggt aacttggttt tggtgctatg tgttctggtg 1620 tcaggcttgt tttttactcg gcggaggaga tttcgacatg gtgttcggtg aagctctccg 1680 gcgatggagc acggcgaaga aggaacaaga gtaagggagt gggttattgt cgcaagcgga 1740 gatagcatga accggttgag atctcaagac agacttgatg aatctcttcg ggggaaaatg 1800 gattggcgac atcgatgggt gcggtggttt gtcgacggag ctcagacgaa attagatcgt 1860 ttcgtctttt ccggagaaaa cttcgaccgt cggatcacga agacatgtta gctgacacgt 1920 gttctgagta ttcggtggtt ggcaacacgt gggcgggctt gggttagtct tggacgcgtg 1980 gctttctctt gctgcgtggg tttcggatat gggcttcagg cccattttat tgtgctcttt 2040 atgttgggcc aatgttataa tcgggccctc agggtcttgt gttctcgggt tgggcttggt 2100 ccggttagac caagtttctt taataaaata tcatactaga tagaaaaaaa taataatgtt 2160 acattcttaa taaaataaca taatatcaga acaagatgaa taaattatgg tttaagcata 2220 tgggtcaaca tttactgtct tttattaact atagtctata tataatcata tcatatgaca 2280 tctctcgtgg agtgttgatt atccgagtaa cttcagtcct cgatacttct ttttttggct 2340 aaatttttct ttttggttaa aatttggtgc aacctcaaat acatctttcg accttaatat 2400 agtttttctc tacggatttg taaaactaac atactcaata tattcacatt tttaatgtgg 2460 tgtctcgagg tctcaagtct cagctgattt tgttttttta attatatatt cagatttttg 2520 ttccgttaat ccacaaggtc atacttttag tttttttttc tgacagcgat catacttttc 2580 gttataacga ccgga tatac caaaaacttt acaggatttt ttaactgttt atatctcctt 2640 tatgttttaa tctttttcac atatgttagg acccaattcc cactaaatca cgtcaccgca 2700 aatgattttg atttaaagtt cgtctgcaaa aaagctcttg aaatagtgga tcatattgga 2760 tatggatatg aggatcgttt gatattctga agagtcatgc agttgctatc tttatcccca 2820 tgggaactat gaaaatgaac gttgaaccaa attatggtga aaatatgacg agagctttct 2880 tcttgtgatc tctgtaaagc ttttatatta tgtccaagga actttcatct tcttatacaa 2940 tgtattttcc cataatgtat acgtgtgaac atataaatga taaatgaaca tacccattat 3000 tcgagatgaa cggattatat aatccataca taattaaacc gtagctcaca tatgatgata 3060 gttaagtgat gttcaaccta tactaattta ctgatgctca actattcaac taatttaaat 3120 cataatccga gtgtgtaatt tgttgcacaa agtcctataa gaagggctac tgaaacctga 3180 caaacatcac aaaactatat cgactctttt tttcagaggt atattgcaga atcacattaa 3240 ccatcttgct ttatttcaat catgtttctg aattgtcaag aagaagataa atatcaaaac 3300 taaatgtatg gctttttcta atgcaaagag tccatcaatg gaggaaatcg gatgggatca 3360 aagaaattgg ttaagaggtc agtttcagaa ccaccaaaaa gatatatgtc aaattgagaa 3420 aacaagggaa gtttccgaca ggatctcgtg tccgttgagc tttgacttcc ttaaacagca 3480 tataaccaaa ttttacttcg aaagagttga acttattgca attttagggt tttgttatct 3540 tcaagtgtgc ttcacaccaa tgccaaaaca actgggttct cgagtttctt tccttgagaa 3600 ctaggtgttt tacttaccga acatcttgaa ttataactac acaacagaat gatcactgaa 3660 ttgcttggta cagtaaaatg ttggcagatt tatcagtaaa aactttttca caatcaaaat 3720 tttgacaatc tagtaattaa tagtctaaca acttttaaac ttctaacatt taattattta 3780 aatctcttaa caagtgtact agcgttcgtt taattattgt actaacgttc atttaattat 3840 tttactaacg ttcgtttaat aatgaaattt gttagctaat tgctcgctag aaacgagaac 3900 agctaagtaa aagaaaaaaa ggagtgaacc acagagcttt aagctagcag ggtaattttt 3960 tactatactc tgttcacttt tttcctattg ttaccactca cagatatacc agtcttgaaa 4020 atatataacc tattctctta tatattttca aagagacaat acatgaggat cgttagatta 4080 tatatcggtc atccatctgt ttaatcggtt aatttcgggt tttcgtgatt ttcttaaata 4140 tggatatttt taagagatcc taaattgaat tatcagatca ccggattaac cggtaaaatc 4200 agagatttga atcaaattta aaagcactga tttaaatata tatatatata tatatatata 4260 aattctttta aattaacaaa atattt gtta aattattagt aaattttttt tatcgtaagt 4320 gcggtcaagt tctagttatt attattacaa acactacatt ttaatttaac tctatattat 4380 ttggcctctt ctgctgtaaa aagggtcgca taagattcct tttgaaatca aaatggacac 4440 gggaatagtg atactaaata gttaattgaa aaaaaattcg atttattttt ttttaaacat 4500 ccgtttttgc tgttgaataa catcacagta aaatttataa ttactttaca aatcctttct 4560 ggtcattaca attccatgga aatgtcatat atgtactgta tatgaatttg atggcatgga 4620 atgtaaggtg cttttctatt aagcaatgat ctatacagta atacacacat ggtatttcta 4680 tttggaattt atttgtttgc atgtgacagt aattagtaag ccttcatatt tggagaggat 4740 atgctctgaa atttgattta cattcttcaa aaaaatatgt tcttctagaa gcaaaaagcc 4800 aagatgtata gaaagttatt gtcctttgat ggctcatcaa aaatagaaat aaaagtgtga 4860 aagctcttta atagttgcat gtggcatcat cgaaggcaaa atgaaaagaa tcttatgttt 4920 gggcctattg cttcgctcac aagcattaat ttcatccctt agaaataaat taaaagttta 4980 ctaacaaaat ggctttattc ctatgtcctg acaattttaa tgtcagttcg gcaatgttag 5040 gctactgaag taagcataag aagtatgttc tatgtaacaa agtagtgtga ttttggtaga 5100 tttatccata aatttctata tacattacac a agataatta aagagtaatt gagagattct 5160 tataccaatg aataaacata ggactatcgt ggaagcattt taatatattg gttaaaatct 5220 aaagatttct acatccaaat caggttttga acttcagaat atgtaatttt ttgcatatta 5280 taggatgtac atttcatagg attttagaca tagttatttg ttgtgatcat ttgctacgaa 5340 tataacctac ttatcgaaga tatcgtacat gcataactgt ccatcgatcc aagtgatttt 5400 gagagaagtt catcatggaa tttttataca tggaacaatt tatcaatatt ttatatgtta 5460 tatgtagttg atcatcatat aatagaatta tataatgatg taatgtgtgc tacttattgc 5520 tagaaaaaac aacgattatc aaatatgtac ttttaacaat tgttttggtg ttgatttatt 5580 accatagtat ttttgttggt gtgtgttaga cgatttcata ttttatcatc cttttattaa 5640 tatcttttgt gaatagttaa attaactatt tgtaagaaaa attttgtgtt aacaaaacta 5700 gtagtttatg tatcttatct tgtttaaata aatttatata taatattata attaaatata 5760 attatgcaaa tagtttgttt agcatcaaat cttattttta gaataattta cattatttgg 5820 cttagaatat acatacattt agatatatta aatggaatat atggatcatg ctatatatat 5880 gcaggacatg acatgatata gattatgaat ctatcatata tgatatgttg atgcgttttg 5940 ggttttttct atttatagtc tttctattaa atttaat tat attgcttttc tcttttgctt 6000 tcccttacat agacatcatt cgtaggacaa ttcaaagttt ttatatattt ttttggcact 6060 acaaaagttc ttaactagtt gatgaataag aaacgttgac aaaaaggtta aggaatagga 6120 aaccatgata aaaaaaaatc gactaaaaac cataataatt gagaactgca gttttcacct 6180 tttttttttt ggtctaaatg ttaagctgca gttttttttt tttgctaaat aaagctgcag 6240 ttttcacttg cgactggcat atgtgttttt tagttcagtt gttccgctaa cgacagacga 6300 tattccttac tcgcgagatt tcacggactc gtaagcatat ctgaggtcct ttgggtctct 6360 tatgtagaaa gctcatggaa aaaaaatacg atatttacac gcacaataaa tattatagat 6420 tacgcgtaag taagtaaagt ggttgtactc taatttatga tagcgatata tcacatatga 6480 tgattaactc tttgattcga aattgtagtt tttctcatgt agcacattga gcttgtttcc 6540 ccctctcaag aaatgacgtt gttattgtta tgagcagcac tataaacttg acataatgat 6600 gatttcactt tttatttatt tatcgtatag agaccggtga atattttctt tgaagtaaat 6660 aaaataagag aaacggtttc gcgtgtttaa aatataccac gttggtaaaa ctattcacat 6720 gtagtgacat caactcctaa gtcaaattaa gaatctaata tataaagcaa tgaagccctt 6780 tatgtcaaac tttaaacttt gcttcccttg atggcaatca ta tactgtat aagcattatt 6840 ttagttagag gcaatccgtt tatataaatg tcttttagct ttaacaaaaa aaaagtcttt 6900 cgacttaatt attgattgaa ataacttgca tatttcaccg taggataaag ttagagggac 6960 gacccatagt gagaatcttt taactacaac taggaagatg tattcaattt aacagttgaa 7020 gtgatttgat ttttaatgga gttttagatg atttttataa gttacagaga tttagttgat 7080 ttttattaaa ctattctaga atatcaccta aaacaatgag atttgagttt taattttttt 7140 taactaagaa actccatcca aacattctaa aatcacctca aaattttaag gagggttaat 7200 ttttatcatc aatgattaat atgaatgttt aaaatgatat gaatacccga ttctctacat 7260 taatctaagt ttaattaaga aaataaaata aagttcaaaa gaaaaaaatt acattaacaa 7320 ctaaattaat aacagaggca tatgatctgc actctctgca tgagcagcac gtgtctgtct 7380 agaagaagtc aatgcctcaa tggtcatgat tctgtaataa gccacacagg ctatcactgg 7440 gagaaacttc tgcttttagc cttctaacct tttataaaca caaaacccta aaaagataca 7500 ctggccactc tcgtcttcag tgtgctaaag tgcccaccta aataaaattt ataatattat 7560 tactttgtta tatatttaat gtgccaactc atttcttttt agtcgctaca aaagttccaa 7620 ttattgttaa atttaactta tatatatgat gttttggaat tcagatgg gt ccacaaaatt 7680 aggtggtata ttatcatcca ttagtttggt aatcaattaa taggaggagg tttcatttct 7740 ttctcatttt aattgtgtgt aaaatgtgta ttaatctccc cagtttacaa tattatataa 7800 gtttgtacga cccttcaaca ttgttgcaaa cattcttagt gagagaaata gagagaaaag 7860 agagagatta actagtgaga gaaagagag 7889 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Artificial Sequence <400> 3 tgggatctgg ttggatcttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 gaatcaaagc ggaaccaaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 acggtccaga ctcaaagctg 20 <210> 6 <211> 10099 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence < 400> 6 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggactt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 ac ctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatct atcttattaa aagagaagta 360 cccatttaaa aatacccctt agttttcact tttatttaca ataatattaa ttaagcttcc 420 tatttttatg catgtctttt tcactttagt taatgtgttt tccaaaatta aatacacaat 480 taaataatac ttcctatttt aatgcttatc tttttcactt agattaatgt gttttccaaa 540 attaaatttg aattaaatac acttcattaa cttctctatt aaatcaatat caaatttaaa 600 aaaattatat tcttactgga caaacagttc ataaaaatta taatatcaat ttttcattta 660 aacgaatctg aacgaaaaaa atattaccaa atttaaaccg aaactagata atatccaaac 720 ggatttacca ttttggcatc taaagaacca taaccaaacc tgatccgaac gaaatatttc 780 ggatattaca atgtatttaa atcatattta tatacttcaa tatgttagct attacagata 840 aacaattatc aaaacaccaa aaatacttaa tatatatatt tattctccat ccaaatattc 900 aagttaaata tattttaaat tttaaattta ggtactttgg cttacattac tcaaatttac 960 atgttatatt atttttattt ttatatttag ggttatttaa agatataaaa tttttgaaat 1020 ttaaaaatag tttaaacggg ttatcaaacc tgcaaaaatc cgaatcaaag cggaaccaaa 1080 atttataaac acacgaata a agctagaatc tttaaactcg aaaatctcaa atccgaatag 1140 attttaaccg aatttgaatg ggtaaccaaa tattcacccc taacttagtc aatataaaac 1200 gatcaaatat tataaatata ctattttgta taaagaaata aaaactaaaa ttgaaaatta 1260 atatccgtgc ggtcgcacgg gtcaagatct agtacttcat ttggaagaaa gtcagctttg 1320 agtctggacc gtcctttttt ttcaaaatta ctcttggtta tactaaacaa atttaatctt 1380 gttaacaaag tttgatttaa ttagtacgtg ttactgcatt atctatgtgt catttatgat 1440 atataattct ttataaactc aacatatttt ttataaatgt gcgtgtttga tcaaaaaaaa 1500 atattgaaaa ccctttaaca aaaaaaaaag agtatcctat atatatccat gcattgagcg 1560 taggcagaag gcttgatggt gtcagctcga ataaatgaat gtaaatggac agatcatatt 1620 actattttta gaatatggtc cgtagaattt tcataaaatt gactaatcaa ttagtttcta 1680 gttttggtta aagccaagct cacacgaagt ctaaaacgtt cctctaaaaa ggaatcgtaa 1740 cagtcccgtt tttatttcac tgttttagca atttctctaa aaaggaatcg taacagtcca 1800 gtttttattt cactgtttta gcaatttctt tacatcaagt aaaatatatt tatatattcc 1860 tcataccctt tttcctactt tctacaaatt cgtttaccaa cgtaaaaatg aatttattag 1920 gcatctacca gatttgaata tcgt ccaaga agtaaatgaa taaagaaaac tgtttaaaag 1980 tactccacat ttgactttaa taaagcaaat tatttacatt tgactcagat taatactttt 2040 tattttacga gtattattga ttatgtgaca aaaaactgat tggctatgag tgcattgtac 2100 tgacacctgg agatagctgt gttaatgtca ttgttatgtg attggtcatt ttctactttt 2160 attattcttt tgtttttgtt ttgatgtctt ggtctgggag gaaatgttac attctgtgta 2220 gctctctaga aaagctctct cttttcacta aaagcggttc aagcgtggtt gcgttccggt 2280 ggccggtggg ttttcgccgc cggttaccac ccctgtctag ttttttttct agctttgttt 2340 tatcggcggc tctataatcg agcctttttc cgatatcaat acttgactct ggggctgcga 2400 cgtctttttc aggcggtgat cgccggcttt tgatttcggg gggtagaggc ttcctcagcc 2460 aacctcgccg actttcggga acgatttggg gtttgatgat ggtcgaggag atcatcgtcg 2520 tggtaacttg gttttggtgc tatgtgttct ggtgtcaggc ttgtttttta ctcggcggag 2580 gagatttcga catggtgttc ggtgaagctc tccggcgatg gagcacggcg aagaaggaac 2640 aagagtaagg gagtgggtta ttgtcgcaag cggagatagc atgaaccggt tgagatctca 2700 agacagactt gatgaatctc ttcgggggaa aatggattgg cgacatcgat gggtgcggtg 2760 gtttgtcgac ggagctcaga cgaaattaga tcgtttcgtc ttttccggag aaaacttcga 2820 ccgtcggatc acgaagacat gttagctgac acgtgttctg agtattcggt ggttggcaac 2880 acgtgggcgg gcttgggtta gtcttggacg cgtggctttc tcttgctgcg tgggtttcgg 2940 atatgggctt caggcccatt ttattgtgct ctttatgttg ggccaatgtt ataatcgggc 3000 cctcagggtc ttgtgttctc gggttgggct tggtccggtt agaccaagtt tctttaataa 3060 aatatcatac tagatagaaa aaaataataa tgttacattc ttaataaaat aacataatat 3120 cagaacaaga tgaataaatt atggtttaag catatgggtc aacatttact gtcttttatt 3180 aactatagtc tatatataat catatcatat gacatctctc gtggagtgtt gattatccga 3240 gtaacttcag tcctcgatac ttcttttttt ggctaaattt ttctttttgg ttaaaatttg 3300 gtgcaacctc aaatacatct ttcgacctta atatagtttt tctctacgga tttgtaaaac 3360 taacatactc aatatattca catttttaat gtggtgtctc gaggtctcaa gtctcagctg 3420 attttgtttt tttaattata tattcagatt tttgttccgt taatccacaa ggtcatactt 3480 ttagtttttt tttctgacag cgatcatact tttcgttata acgaccggat ataccaaaaa 3540 ctttacagga ttttttaact gtttatatct cctttatgtt ttaatctttt tcacatatgt 3600 taggacccaa ttcccactaa atcacgtcac cgcaa atgat tttgatttaa agttcgtctg 3660 caaaaaagct cttgaaatag tggatcatat tggatatgga tatgaggatc gtttgatatt 3720 ctgaagagtc atgcagttgc tatctttatc cccatcatgggaa tatactatggtagtgat attatgtcca aggaactttc atcttcttat acaatgtatt ttcccataat gtatacgtgt 3900 gaacatataa atgataaatg aacataccca ttattcgaga tgaacggatt atataatcca 3960 tacataatta aaccgtagct cacatatgat gatagttaag tgatgttcaa cctatactaa 4020 tttactgatg ctcaactatt caactaattt aaatcataat ccgagtgtgt aatttgttgc 4080 acaaagtcct ataagaaggg ctactgaaac ctgacaaaca tcacaaaact atatcgactc 4140 tttttttcag aggtatattg cagaatcaca ttaaccatct tgctttattt caatcatgtt 4200 tctgaattgt caagaagaag ataaatatca aaactaaatg tatggctttt tctaatgcaa 4260 agagtccatc aatggaggaa atcggatggg atcaaagaaa ttggttaaga ggtcagtttc 4320 agaaccacca aaaagatata tgtcaaattg agaaaacaag ggaagtttcc gacaggatct 4380 cgtgtccgtt gagctttgac ttccttaaac agcatataac caaattttac ttcgaaagag 4440 ttgaacttat tgcaatttta gggttttgtt atcttcaagt gtgcttcaca ccaatgccaa 4500 aacaactggg ttctcgagtt tctttccttg agaactaggt gttttactta ccgaacatct 4560 tgaattataa ctacacaaca gaatgatcac tgaattgctt ggtacagtaa aatgttggca 4620 gatttatcag taaaaacttt ttcacaatca aaattttgac aatctagtaa ttaatagtct 4680 aacaac tttt aaacttctaa catttaatta tttaaatctc ttaacaagtg tactagcgtt 4740 cgtttaatta ttgtactaac gttcatttaa ttattttact aacgttcgtt taataatgaa 4800 atttgttagc taattgctcg ctagaaacga gaacagctaa gtaaaagaaa aaaaggagtg 4860 aaccacagag ctttaagcta gcagggtaat tttttactat actctgttca cttttttcct 4920 attgttacca ctcacagata taccagtctt gaaaatatat aacctattct cttatatatt 4980 ttcaaagaga caatacatga ggatcgttag attatatatc ggtcatccat ctgtttaatc 5040 ggttaatttc gggttttcgt gattttctta aatatggata tttttaagag atcctaaatt 5100 gaattatcag atcaccggat taaccggtaa aatcagagat ttgaatcaaa tttaaaagca 5160 ctgatttaaa tatatatata tatatatata tataaattct tttaaattaa caaaatattt 5220 gttaaattat tagtaaattt tttttatcgt aagtgcggtc aagttctagt tattattatt 5280 acaaacacta cattttaatt taactctata ttatttggcc tcttctgctg taaaaagggt 5340 cgcataagat tccttttgaa atcaaaatgg acacgggaat agtgatacta aatagttaat 5400 tgaaaaaaaa ttcgatttat ttttttttaa acatccgttt ttgctgttga ataacatcac 5460 agtaaaattt ataattactt tacaaatcct ttctggtcat tacaattcca tggaaatgtc 5520 atatatgtac t gtatatgaa tttgatggca tggaatgtaa ggtgcttttc tattaagcaa 5580 tgatctatac agtaatacac acatggtatt tctatttgga atttatttgt ttgcatgtga 5640 cagtaattag taagccttca tatttggaga ggatatgctc tgaaatttga tttacattct 5700 tcaaaaaaat atgttcttct agaagcaaaa agccaagatg tatagaaagt tattgtcctt 5760 tgatggctca tcaaaaatag aaataaaagt gtgaaagctc tttaatagtt gcatgtggca 5820 tcatcgaagg caaaatgaaa agaatcttat gtttgggcct attgcttcgc tcacaagcat 5880 taatttcatc ccttagaaat aaattaaaag tttactaaca aaatggcttt attcctatgt 5940 cctgacaatt ttaatgtcag ttcggcaatg ttaggctact gaagtaagca taagaagtat 6000 gttctatgta acaaagtagt gtgattttgg tagatttatc cataaatttc tatatacatt 6060 acacaagata attaaagagt aattgagaga ttcttatacc aatgaataaa cataggacta 6120 tcgtggaagc attttaatat attggttaaa atctaaagat ttctacatcc aaatcaggtt 6180 ttgaacttca gaatatgtaa ttttttgcat attataggat gtacatttca taggatttta 6240 gacatagtta tttgttgtga tcatttgcta cgaatataac ctacttatcg aagatatcgt 6300 acatgcataa ctgtccatcg atccaagtga ttttgagaga agttcatcat ggaattttta 6360 tacatggaac aatttat caa tattttatat gttatatgta gttgatcatc atataataga 6420 attatataat gatgtaatgt gtgctactta ttgctagaaa aaacaacgat tatcaaatat 6480 gtacttttaa caattgtttt ggtgttgatt tattaccata gtatttttgt tggtgtgtgt 6540 tagacgattt catattttat catcctttta ttaatatctt ttgtgaatag ttaaattaac 6600 tatttgtaag aaaaattttg tgttaacaaa actagtagtt tatgtatctt atcttgttta 6660 aataaattta tatataatat tataattaaa tataattatg caaatagttt gtttagcatc 6720 aaatcttatt tttagaataa tttacattat ttggcttaga atatacatac atttagatat 6780 attaaatgga atatatggat catgctatat atatgcagga catgacatga tatagattat 6840 gaatctatca tatatgatat gttgatgcgt tttgggtttt ttctatttat agtctttcta 6900 ttaaatttaa ttatattgct tttctctttt gctttccctt acatagacat cattcgtagg 6960 acaattcaaa gtttttatat atttttttgg cactacaaaa gttcttaact agttgatgaa 7020 taagaaacgt tgacaaaaag gttaaggaat aggaaaccat gataaaaaaa aatcgactaa 7080 aaaccataat aattgagaac tgcagttttc accttttttt ttttggtcta aatgttaagc 7140 tgcagttttt tttttttgct aaataaagct gcagttttca cttgcgactg gcatatgtgt 7200 tttttagttc agttgttccg ct aacgacag acgatattcc ttactcgcga gatttcacgg 7260 actcgtaagc atatctgagg tcctttgggt ctcttatgta gaaagctcat ggaaaaaaaa 7320 tacgatattt acacgcacaa taaatattat agattacgcg taagtaagta aagtggttgt 7380 actctaattt atgatagcga tatatcacat atgatgatta actctttgat tcgaaattgt 7440 agtttttctc atgtagcaca ttgagcttgt ttccccctct caagaaatga cgttgttatt 7500 gttatgagca gcactataaa cttgacataa tgatgatttc actttttatt tatttatcgt 7560 atagagaccg gtgaatattt tctttgaagt aaataaaata agagaaacgg tttcgcgtgt 7620 ttaaaatata ccacgttggt aaaactattc acatgtagtg acatcaactc ctaagtcaaa 7680 ttaagaatct aatatataaa gcaatgaagc cctttatgtc aaactttaaa ctttgcttcc 7740 cttgatggca atcatatact gtataagcat tattttagtt agaggcaatc cgtttatata 7800 aatgtctttt agctttaaca aaaaaaaagt ctttcgactt aattattgat tgaaataact 7860 tgcatatttc accgtaggat aaagttagag ggacgaccca tagtgagaat cttttaacta 7920 caactaggaa gatgtattca atttaacagt tgaagtgatt tgatttttaa tggagtttta 7980 gatgattttt ataagttaca gagatttagt tgatttttat taaactattc tagaatatca 8040 cctaaaacaa tgagatttga gttttaat tt tttttaacta agaaactcca tccaaacatt 8100 ctaaaatcac ctcaaaattt taaggagggt taatttttat catcaatgat taatatgaat 8160 gtttaaaatg atatgaatac ccgattctct acattaatct aagtttaatt aagaaaataa 8220 aataaagttc aaaagaaaaa aattacatta acaactaaat taataacaga ggcatatgat 8280 ctgcactctc tgcatgagca gcacgtgtct gtctagaaga agtcaatgcc tcaatggtca 8340 tgattctgta ataagccaca caggctatca ctgggagaaa cttctgcttt tagccttcta 8400 accttttata aacacaaaac cctaaaaaga tacactggcc actctcgtct tcagtgtgct 8460 aaagtgccca cctaaataaa atttataata ttattacttt gttatatatt taatgtgcca 8520 actcatttct ttttagtcgc tacaaaagtt ccaattattg ttaaatttaa cttatatata 8580 tgatgttttg gaattcagat gggtccacaa aattaggtgg tatattatca tccattagtt 8640 tggtaatcaa ttaataggag gaggtttcat ttctttctca ttttaattgt gtgtaaaatg 8700 tgtattaatc tccccagttt acaatattat ataagtttgt acgacccttc aacattgttg 8760 caaacattct tagtgagaga aatagagaga aaagagagag attaactagt gagagaaaga 8820 gagatgggcc atcactcgtg ctgcaacaag caaaaggtga agagagggct ttggtcaccg 8880 gaagaagacg aaaagctcat caactacatc aat acatatg gccatggatg ctggagctct 8940 gttcccaaac atgcaggcat atatattggc tttgtttgtt gaattatcta ctgataaaaa 9000 gtctatgtcg ttggttttaa tctatgtgtt gttttccgtt tatttaatta ggtctgcaga 9060 gatgtggaaa gagttgtcga ttaagatgga taaattatct aagacctgat cttaaacgtg 9120 gaagcttctc acctcaagaa gctgctctta tcattgagct tcatagcatt cttggtaaca 9180 ggttgttact taattatcat tacatgttgg tttatatgtg ataatataaa actaaatttt 9240 catgaagaaa ctgaactacc tttatactca tttttttttg ttatggattc ttgataggtg 9300 ggcacaaatt gctaaacatc tacctggaag aacagataac gaggtcaaga atttctggaa 9360 ctcaagcatt aaaaagaagc tcatgtctca ccactatcac catcatcatg gtcatcatca 9420 tcatcatacc tcttcaatgg cgagtttgct cactaacctt ccttatcaca acgtgttcaa 9480 cgctactgct ggcgacgatg aaggttcaag attcatgtcc agtatcatca caaacactaa 9540 ccctaatttc atcactccaa cccatctccc tcttccttct catcatgtta tgtctccatt 9600 gatgttctca acctctagag aaggagatat caagtattta accgctacca acacaaacca 9660 gtctcatcaa gataataacc tttacaccaa cctcgacatt ttgtcagcca cgccattcat 9720 caacaaccat catcaccatc atgataatga cccccagtg g ccatcgctac cagatctccc 9780 agcgagtacc atttccactt ttcacgacaa ccttcaagat tatgatgatg gtgataaact 9840 caacgtgttt gtaacaccat acaatgataa tggtgatcct cggagctttc ctttggtggc 9900 cacaaagtta atatgtggac aagttcttga aggcaaagca atttcacaag accacggcag 9960 ctttcttctc ccaacaacgt acaacttgga aatgcctggt gatcatcgac gagtggactc 10020 atacattaat cacatgatca ttccctcatc atcatcctct tcatcgccaa tctcttgtgg 10080 acagtacgtc atcacttaa 10099 <210> 7 <211> 9140 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 tgggatctgg ttggttcttc gatgcggatt ttaggccttg ctttggactt ggttgttttt 60 ggggccgtgc tcggtttgtg ggcagtcggg ctttgatgta ttattggctt attgtaatat 120 tgggctttgg gccttttgaa tatcaatttc tgaaaaaaaa agtaggtctc atgactctca 180 tctatgaaaa aggtctacat ataatttttg tgtgataaat atccacaaat acttcacgtt 240 acctagacgg tagtacatgt acatgcatgt cgtctacatt gagaaatttg attctttttt 300 ggtgatgccg attctttccg tatcgaatca tgatacatac ttcattgga agaaagtcag 360 ctttgagtct ggaccgtcct 420 ttttttttcaa at aattactttttcaa ttgttaa caaagtttga tttaattagt acgtgttact gcattatcta tgtgtcattt 480 atgatatata attctttata aactcaacat attttttata aatgtgcgtg tttgatcaaa 540 aaaaaatatt gaaaaccctt taacaaaaaa aaaagagtat cctatatata tccatgcatt 600 gagcgtaggc agaaggcttg atggtgtcag ctcgaataaa tgaatgtaaa tggacagatc 660 atattactat ttttagaata tggtccgtag aattttcata aaattgacta atcaattagt 720 ttctagtttt ggttaaagcc aagctcacac gaagtctaaa acgttcctct aaaaaggaat 780 cgtaacagtc ccgtttttat ttcactgttt tagcaatttc tctaaaaagg aatcgtaaca 840 gtccagtttt tatttcactg ttttagcaat ttctttacat caagtaaaat atatttatat 900 attcctcata ccctttttcc tactttctac aaattcgttt accaacgtaa aaatgaattt 960 attaggcatc taccagattt gaatatcgtc caagaagtaa atgaataaag aaaactgttt 1020 aaaagtactc cacatttgac tttaataaag caaattattt acatttgact cagattaata 1080 ctttttattt tacgagtatt attgattatg tgacaaaaaa ctgattggct atgagtgcat 1140 tgtactgaca cctggagata gctgtgttaa tgtcattgtt atgtgattgg tcattttcta 1200 cttttattat tcttttgttt ttgttttgat gtcttggttt gggaggaaat gttacattct 1260 gtgtagctct ctagaaa agc tctctctttt cactaaaagc ggttcaagcg tggttgcgtt 1320 ccggtggccg gtgggttttc gccgccggtt accacccctg tctagttttt tttctagctt 1380 tgttttatcg gcggctctat aatcgagcct ttttccgata tcaatacttg actctggggc 1440 tgcgacgtct ttttcaggcg gtgatcgccg gcttttgatt tcggggggta gaggcttcct 1500 cagccaacct cgccgacttt cgggaacgat ttggggtttg atgatggtcg aggagatcat 1560 cgtcgtggta acttggtttt ggtgctatgt gttctggtgt ccggcttgtt ttttactcgg 1620 cggaggagat ttcgacatgg tgttcggtga agctctccgg cgatggagca cggcgaagaa 1680 ggaacaagag taagggagtg ggttattgtc gcaagcggag atagcatgaa ccggttgaga 1740 tctcaagaca gacttgatga atctcttcgg gggaaaatgg attggcgaca tcgatgggtg 1800 cggtggtttg tcgacggagc tcagacgaaa ttagatcgtt tcgtcttttc cggagaaaac 1860 ttcgaccgtc ggatcacgaa gacatgttag ctgacacgtg ttctgagtat tcggtggttg 1920 gcaacacgtg ggcgggcttg ggttagtctt ggacgcgtgg ctttctcttg ctgcgtgggt 1980 ttcggatatg ggcttcaggc ccattttatt gtgctcttta tgttgggcca atgttataat 2040 cgggccctca gggtcttgtg ttctcgggtt gggcttggtc cggttagacc aagtttcttt 2100 aataaaatat catactagat ag aaaaaaat aataatgtta cattcttaat aaaataacat 2160 aatatcagaa caagatgaat aaattatggt ttaagcatat gggtcaacat ttactgtctt 2220 ttattaacta tagtctatat ataatcatat catatgacat ctctcgtgga gtgttgatta 2280 tccgagtaac ttcagtcctc gatacttctt tttttggcta aatttttctt tttggttaaa 2340 atttggtgca acctcaaata catctttcga ccttaatata gtttttctct acggatttgt 2400 aaaactaaca tactcaatat attcacattt ttaatgtggt gtctcgaggt ctcaagtctc 2460 agctgatttt gtttttttaa ttatatattc agatttttgt tccgttaatc cacaaggtca 2520 tacttttagt ttttttttct gacagcgatc atacttttcg ttataacgac cggatatacc 2580 aaaaacttta caggattttt taactgttta tatctccttt atgttttaat ctttttcaca 2640 tatgttagga cccaattccc actaaatcac gtcaccgcaa atgattttga tttaaagttc 2700 gtctgcaaaa aagctcttga aatagtggat catattggat atggatatga ggatcgtttg 2760 atattctgaa gagtcatgca gttgctatct ttatccccat gggaactatg aaaatgaacg 2820 ttgaaccaaa ttatggtgaa aatatgacga gagctttctt cttgtgatct ctgtaaagct 2880 tttatattat gtccaaggaa ctttcatctt cttatacaat gtattttccc ataatgtata 2940 cgtgtgaaca tataaatgat aaatgaac at acccattatt cgagatgaac ggattatata 3000 atccatacat aattaaaccg tagctcacat atgatgatag ttaagtgatg ttcaacctat 3060 actaatttac tgatgctcaa ctattcaact aatttaaatc ataatccgag tgtgtaattt 3120 gttgcacaaa gtcctataag aagggctact gaaacctgac aaacatcaca aaactatatc 3180 gactcttttt ttcagaggta tattgcagaa tcacattaac catcttgctt tatttcaatc 3240 atgtttctga attgtcaaga agaagataaa tatcaaaact aaatgtatgg ctttttctaa 3300 tgcaaagagt ccatcaatgg aggaaatcgg atgggatcaa agaaattggt taagaggtca 3360 gtttcagaac caccaaaaag atatatgtca aattgagaaa acaagggaag tttccgacag 3420 gatctcgtgt ccgttgagct ttgacttcct taaacagcat ataaccaaat tttacttcga 3480 aagagttgaa cttattgcaa ttttagggtt ttgttatctt caagtgtgct tcacaccaat 3540 gccaaaacaa ctgggttctc gagtttcttt ccttgagaac taggtgtttt acttaccgaa 3600 catcttgaat tataactaca caacagaatg atcactgaat tgcttggtac agtaaaatgt 3660 tggcagattt atcagtaaaa actttttcac aatcaaaatt ttgacaatct agtaattaat 3720 agtctaacaa cttttaaact tctaacattt aattatttaa atctcttaac aagtgtacta 3780 gcgttcgttt aattattgta ctaacgttca ttt aattatt ttactaacgt tcgtttaata 3840 atgaaatttg ttagctaatt gctcgctaga aacgagaaca gctaagtaaa agaaaaaaag 3900 gagtgaacca cagagcttta agctagcagg gtaatttttt actatactct gttcactttt 3960 ttcctattgt taccactcac agatatacca gtcttgaaaa tatataacct attctcttat 4020 atattttcaa agagacaata catgaggatc gttagattat atatcggtca tccatctgtt 4080 taatcggtta atttcgggtt ttcgtgattt tcttaaatat ggatattttt aagagatcct 4140 aaattgaatt atcagatcac cggattaacc ggtaaaatca gagatttgaa tcaaatttaa 4200 aagcactgat ttaaatatat atatatatat atatataaat tcttttaaat taacaaaata 4260 tttgttaaat tattagtaaa ttttttttat cgtaagtgcg gtcaagttct agttattatt 4320 attacaaaca ctacatttta atttaactct atattatttg gcctcttctg ctgtaaaaag 4380 ggtcgcataa gattcctttt gaaatcaaaa tggacacggg aatagtgata ctaaatagtt 4440 aattgaaaaa aaattcgatt tatttttttt aaacatccgt ttttgctgtt gaataacatc 4500 acagtaaaat ttataattac tttacaaatc ctttctggtc attacaattc catggaaatg 4560 tcatatatgt actgtatatg aatttgatgg catggaatgt aaggtgcttt tctattaagc 4620 aatgatctat acagtaatac acacatggta tttctattt g gaatttattt gtttgcatgt 4680 gacagtaatt agtaagcctt catatttgga gaggatatgc tctgaaattt gatttacatt 4740 cttcaaaaaa atatgttctt ctagaagcaa aaagccaaga tgtatagaaa gttattgtcc 4800 tttgatggct catcaaaaat agaaataaaa gtgtgaaagc tctttaatag ttgcatgtgg 4860 catcatcgaa ggcaaaatga aaagaatctt atgtttgggc ctattgcttc gctcacaagc 4920 attaatttca tcccttagaa ataaattaaa agtttactaa caaaatggct ttattcctat 4980 gtcctgacaa ttttaatgtc agttcggcaa tgttaggcta ctgaagtaag cataagaagt 5040 atgttctatg taacaaagta gtgtgatttt ggtagattta tccataaatt tctatataca 5100 ttacacaaga taattaaaga gtaattgaga gattcttata ccaatgaata aacataggac 5160 tatcgtggaa gcattttaat atattggtta aaatctaaag atttctacat ccaaatcagg 5220 ttttgaactt cagaatatgt aattttttgc atattatagg atgtacattt cataggattt 5280 tagacatagt tatttgttgt gatcatttgc tacgaatata acctacttat cgaagatatc 5340 gtacatgcat aactgtccat cgatccaagt gattttgaga gaagttcatc atggaatttt 5400 tatacatgga acaatttatc aatattttat atgttatatg tagttgatca tcatataata 5460 gaattatata atgatgtaat gtgtgctact tattgctaga aaaa acaacg attatcaaat 5520 atgtactttt aacaattgtt ttggtgttga tttattacca tagtattttt gttggtgtgt 5580 gttagacgat ttcatatttt atcatccttt tattaatatc ttttgtgaat agttaaatta 5640 actatttgta agaaaaattt tgtgttaaca aaactagtag tttatgtatc ttatcttgtt 5700 taaataaatt tatatataat attataatta aatataatta tgcaaatagt ttgtttagca 5760 tcaaatctta tttttagaat aatttacatt atttggctta gaatatacat acatttagat 5820 atattaaatg gaatatatgg atcatgctat atatatgcag gacatgacat gatatagatt 5880 atgaatctat catatatgat atgttgatgc gttttgggtt ttttctattt atagtctttc 5940 tattaaattt aattatattg cttttctctt ttgctttccc ttacatagac atcattcgta 6000 ggacaattca aagtttttat atattttttt ggcactacaa aagttcttaa ctagttgatg 6060 aataagaaac gttgacaaaa aggttaagga ataggaaacc atgataaaaa aaaatcgact 6120 aaaaaccata ataattgaga actgcagttt tcaccttttt tttttggtct aaatgttaag 6180 ctgcagtttt ttttttttgc taaataaagc tgcagttttc acttgcgact ggcatatgtg 6240 ttttttagtt cagttgttcc gctaacgaca gacgatattc cttactcgcg agatttcacg 6300 gactcgtaag catatctgag gtcctttggg tctcttatgt agaaagctca tggaaaaaaa 6360 atacgatatt tacacgcaca ataaatatta tagattacgc gtaagtaagt aaagtggttg 6420 tactctaatt tatgatagcg atatatcaca tatgatgatt aactctttga ttcgaaattg 6480 tagtttttct catgtagcac attgagcttg tttccccctc tcaagaaatg acgttgttat 6540 tgttatgagc agcactataa acttgacata atgatgattt cactttttat ttatttatcg 6600 tatagagacc ggtgaatatt ttctttgaag taaataaaat aagagaaacg gtttcgcgtg 6660 tttaaaatat accacgttgg taaaactatt cacatgtagt gacatcaact cctaagtcaa 6720 attaagaatc taatatataa agcaatgaag ccctttatgt caaactttaa actttgcttc 6780 ccttgatggc aatcatatac tgtataagca ttattttagt tagaggcaat ccgtttatat 6840 aaatgtcttt tagctttaac aaaaaaaaag tctttcgact taattattga ttgaaataac 6900 ttgcatattt caccgtagga taaagttaga gggacgaccc atagtgagaa tcttttaact 6960 acaactagga agatgtattc aatttaacag ttgaagtgat ttgattttta atggagtttt 7020 agatgatttt tataagttac agagatttag ttgattttta ttaaactatt ctagaatatc 7080 acctaaaaca atgagatttg agttttaatt ttttttaact aagaaactcc atccaaacat 7140 tctaaaatca cctcaaaatt ttaaggaggg ttaattttta tcatcaatga ttaat atgaa 7200 tgtttaaaat gatatgaata cccgattctc tacattaatc taagtttaat taagaaaata 7260 aaataaagtt caaaagaaaa aaattacatt aacaactaaa ttaataacag aggcatatga 7320 tctgcactct ctgcatgagc agcacgtgtc tgtctagaag aagtcaatgc ctcaatggtc 7380 atgattctgt aataagccac acaggctatc actgggagaa acttctgctt ttagccttct 7440 aaccttttat aaacacaaaa ccctaaaaag atacactggc cactctcgtc ttcagtgtgc 7500 taaagtgccc acctaaataa aatttataat attattactt tgttatatat ttaatgtgcc 7560 aactcatttc tttttagtcg ctacaaaagt tccaattatt gttaaattta acttatatat 7620 atgatgtttt ggaattcaga tgggtccaca aaattaggtg gtatattatc atccattagt 7680 ttggtaatca attaatagga ggaggtttca tttctttctc attttaattg tgtgtaaaat 7740 gtgtattaat ctccccagtt tacaatatta tataagtttg tacgaccctt caacattgtt 7800 gcaaacattc ttagtgagag aaatagagag aaaagagaga gattaactag tgagagaaag 7860 agagatgggc catcactcgt gctgcaacaa gcaaaaggtg aagagagggc tttggtcacc 7920 ggaagaagac gaaaagctca tcaactacat caatacatat ggccatggat gctggagctc 7980 tgttcccaaa catgcaggca tatatattgg ctttgtttgt tgaattatct actgataaaa 8040 agtctatgtc gttggtttta atctatgtgt tgttttccgt ttatttaatt aggtctgcag 8100 agatgtggaa agagttgtcg attaagatgg ataaattatc taagacctga tcttaaacgt 8160 ggaagcttct cacctcaaga agctgctctt atcattgagc ttcatagcat tcttggtaac 8220 aggttgttac ttaattatca ttacatgttg gtttatatgt gataatataa aactaaattt 8280 tcatga agaa actgaactac ctttatactc attttttttt gttatggatt cttgataggt 8340 gggcacaaat tgctaaacat ctacctggaa gaacagataa cgaggtcaag aatttctgga 8400 actcaagcat taaaaagaag ctcatgtctc accactatca ccatcatcat ggtcatcatc 8460 atcatcatac ctcttcaatg gcgagtttgc tcactaacct tccttatcac aacgtgttca 8520 acgctactgc tggcgacgat gaaggttcaa gattcatgtc cagtatcatc acaaacacta 8580 accctaattt catcactcca acccatctcc ctcttccttc tcatcatgtt atgtctccat 8640 tgatgttctc aacctctaga gaaggagata tcaagtattt aaccgctacc aacacaaacc 8700 agtctcatca agataataac ctttacacca acctcgacat tttgtcagcc acgccattca 8760 tcaacaacca tcatcaccat catgataatg acccccagtg gccatcgcta ccagatctcc 8820 cagcgagtac catttccact tttcacgata accttcaaga ttatgatgat ggtgataaac 8880 tcaacgtgtt tgtaacacca tacaatgata atggtgatcc tcggagcttt cctttggtgg 8940 ccacaaagtt aatatgtgga caagttcttg aaggcaaagc aatttcacaa gaccacggca 9000 gctttcttct cccaacaacg tacaacttgg aaatgcctgg tgatcatcga cgagtggact 9060 catacattaa tcacatgatc attccctcat catcatcctc ttcatcgcca atctcttgtg 9120 gacagtacgt c atcacttaa 9140 <210> 8 <211> 955 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 ctatcttatt aaaagagaag tacccattta aaaatacccc ttagttttca cttttatttta 60 caataatatt aattaagctt cctattattat tattattattat tattatta tattat tatta tctttttcac 180 ttagattaat gtgttttcca aaattaaatt tgaattaaat acacttcatt aacttctcta 240 ttaaatcaat atcaaattta aaaaaattat attcttactg gacaaacagt tcataaaaat 300 tataatatca atttttcatt taaacgaatc tgaacgaaaa aaatattacc aaatttaaac 360 cgaaactaga taatatccaa acggatttac cattttggca tctaaagaac cataaccaaa 420 cctgatccga acgaaatatt tcggatatta caatgtattt aaatcatatt tatatacttc 480 aatatgttag ctattacaga taaacaatta tcaaaacacc aaaaatactt aatatatata 540 tttattctcc atccaaatat tcaagttaaa tatattttaa attttaaatt taggtacttt 600 ggcttacatt actcaaattt acatgttata ttatttttat ttttatattt agggttattt 660 aaagatataa aatttttgaa atttaaaaat agtttaaacg ggttatcaaa cctgcaaaaaa 720 tccgaatcaa aaatttataa acacacgaat aaatttataa aca aa aa tctttaaact 780 cgaaaatctc aaatccgaat agattttaac cgaatttgaa tgggtaacca aatattcacc 840 cctaacttag tcaatataaa acgatcaaat attataaata tactattttg tataaagaaa 900taaaggatcagt catatagt

Claims (10)

무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역에서 955-bp 삽입을 확인하기 위한 무 웅성불임 판별용 조성물로서,
상기 무의 Rs295350 유전자는 서열번호 1로 표시되는 것이고,
상기 무의 Rs295350 유전자의 상류 7kb 내지 8kb 영역은 서열번호 2로 표시되는 것이며,
상기 955-bp는 서열번호 8로 표시되는 것이며,
상기 조성물은 서열번호 3으로 표시되는 프라이머, 서열번호 4로 표시되는 프라이머, 및 서열번호 5로 표시되는 프라이머를 포함하는 것인, 조성물As a composition for determining non-male infertility for confirming the 955-bp insertion in the upstream 7kb to 8kb region of the radish Rs295350 gene,
The radish Rs295350 gene is represented by SEQ ID NO: 1,
The upstream 7kb to 8kb region of the radish Rs295350 gene is represented by SEQ ID NO: 2,
The 955-bp is represented by SEQ ID NO: 8,
The composition comprises a primer represented by SEQ ID NO: 3, a primer represented by SEQ ID NO: 4, and a primer represented by SEQ ID NO: 5, composition 삭제delete 삭제delete 삭제delete 삭제delete 제 1 항의 조성물을 포함하는 무 웅성불임 판별용 키트.A kit for determining non-male infertility comprising the composition of claim 1. 제 6 항에 있어서,
상기 키트는 서열번호 2와 비교하여 결손, 삽입 또는 치환 돌연변이를 검출하여 무의 웅성불임을 판별하는, 무 웅성불임 판별용 키트.7. The method of claim 6,
The kit is compared to SEQ ID NO: 2 by detecting a deletion, insertion, or substitution mutation to determine the male infertility of the radish, a kit for determining male infertility. (a) 식물체로부터 유래된 시료로부터 게놈 DNA를 분리하는 단계;
(b) 단계 (a)에서 분리한 게놈 DNA에 제 1항의 조성물을 첨가하여 돌연변이 여부를 결정하는 단계; 및
(c) 단계 (b)에서 검출된 돌연변이 부위를 확인하는 단계를 포함하는, 무의 웅성불임 판별 방법.(a) isolating genomic DNA from a sample derived from a plant;
(b) determining whether mutation by adding the composition of claim 1 to the genomic DNA isolated in step (a); and
(c) A method for determining male infertility of radish, comprising the step of identifying the mutation site detected in step (b). 서열번호 8로 표시되는 서열을 포함하는 벡터로 무 식물체를 형질전환시켜서, 웅성불임을 유도하는 방법.A method of inducing male infertility by transforming a radish plant with a vector comprising the sequence represented by SEQ ID NO: 8. 제 9항의 방법으로 형질전환된, 형질전환 식물.A transgenic plant transformed by the method of claim 9 .
KR1020200188644A 2020-10-27 2020-12-31 Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility KR102448347B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20200140344 2020-10-27
KR1020200140344 2020-10-27

Publications (2)

Publication Number Publication Date
KR20220056074A KR20220056074A (en) 2022-05-04
KR102448347B1 true KR102448347B1 (en) 2022-09-28

Family

ID=81584017

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020200188644A KR102448347B1 (en) 2020-10-27 2020-12-31 Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility

Country Status (1)

Country Link
KR (1) KR102448347B1 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679982B (en) * 2007-04-06 2014-07-23 株式会社东部韩农 A method for producing a hybrid seed using plant of novel cytoplasmic-genic male sterility raphanus sativus line and DNA markers for selecting the plant of said raphanus sativus line

Also Published As

Publication number Publication date
KR20220056074A (en) 2022-05-04

Similar Documents

Publication Publication Date Title
AU2021225142B2 (en) Generation of haploid plants
CN106998665B (en) Haploid plant production
JP3782106B2 (en) Genetic control of flowering
CN105602952B (en) A kind of fertile gene and its application
JP6978152B2 (en) Multiphase spore reproductive gene
JP2012514467A (en) Plants producing 2n gametes or apomyotic gametes
CN111153974A (en) Corn disease-resistant gene and molecular marker and application thereof
CN115175556A (en) Novel genetic loci associated with rust resistance in soybean
CN113874388A (en) Parthenogenesis genes
CN112725374A (en) Method for creating plant haploid induction line and application thereof
KR20230088741A (en) Modified promoters of parthenogenetic genes
CN109295071B (en) Rice flower organ development regulation gene PEH1, and encoded protein and application thereof
CN109456396B (en) Rice leaf senescence and panicle type regulation gene HK73, and protein, molecular marker and application encoded by gene HK73
CN105579583B (en) The breeding of Female sterile clone and cross-breeding technology
JP2016507240A (en) Manipulating self-incompatibility in plants
KR102448347B1 (en) Method for determining male-sterility of Raphanus sativus and Raphanus sativus plant having male-sterility
CN108456683B (en) Function and application of gene SID1 for regulating heading stage of rice
KR100527285B1 (en) A cysteine protease gene and a promoter which are expressed specifically in rice anther, a production method of male sterile rice using supression of the gene expression
CN107446031B (en) Plant glutelin transport and storage related protein OsVHA-E1, and coding gene and application thereof
CN109810994A (en) The Gene A/G 1 and its application of one control rice male and female fertility
CN106148297B (en) rice plastid development regulatory gene FLN2 and application thereof
CN113151295A (en) Rice temperature-sensitive male sterile gene OsFMS1 and application thereof
CN107058351B (en) Mutant gene of rice nuclease gene OsGEN-L and application thereof
NL2011980C2 (en) New effects of plant ahl proteins.
KR100455621B1 (en) Method for lowering pollen fertility by using pollen-specific zinc finger transcriptional factor genes

Legal Events

Date Code Title Description
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right