JP2010226974A - Gene marker linked to genetic sex of yellowtail, sex determination method for yellowtail, and primer for use in sex determination method - Google Patents

Gene marker linked to genetic sex of yellowtail, sex determination method for yellowtail, and primer for use in sex determination method Download PDF

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JP2010226974A
JP2010226974A JP2009075492A JP2009075492A JP2010226974A JP 2010226974 A JP2010226974 A JP 2010226974A JP 2009075492 A JP2009075492 A JP 2009075492A JP 2009075492 A JP2009075492 A JP 2009075492A JP 2010226974 A JP2010226974 A JP 2010226974A
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yellowtail
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yellowtails
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JP5452047B2 (en
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Takashi Sakamoto
崇 坂本
Kanako Fuji
加菜子 藤
Akiyuki Ozaki
照遵 尾崎
Masanori Okauchi
正典 岡内
Kazuo Araki
和男 荒木
Keita Hattori
圭太 服部
Kazunori Yoshida
一範 吉田
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Tokyo University of Marine Science and Technology NUC
Fisheries Research Agency
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Fisheries Research Agency
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Abstract

<P>PROBLEM TO BE SOLVED: To determine the genetic sex of an immature yellowtail with reliability without killing the yellowtail. <P>SOLUTION: The genetic sex of the immature yellowtail is determined by a nucleic acid amplification reaction method using an yellowtail-derived nucleic acid as a template by using a primer produced from a gene marker linked to genetic sex of the yellowtail, and analyzing the obtained amplification product. The primer comprises an oligonucleotide part comprising at least 10 bases in 5'-TTTCATTGTGGCGCTCAG-3' and an oligonucleotide part comprising at least 10 bases in 5'-GGTTGTAATGTGTCCCAG-3'. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は、ブリ類の遺伝的性に連鎖する遺伝子マーカー、この遺伝子マーカーを利用したブリ類の性判別法、及び、性判別法に用いるプライマーに関する。   The present invention relates to a genetic marker linked to the genetic sex of yellowtails, a sex discrimination method for yellowtails using this genetic marker, and a primer used in the sex discrimination method.

ブリ属は世界に９種であり、日本には４種（ブリ、ヒラマサ、カンパチ、ヒレナガカンパチ）が分布している。それらのほとんどが水産物として重要種であるとともにフィッシング対象種としても重要な位置にある。
食用魚としての価値が高いブリは、出世魚であり、日本では天然魚の漁獲（約7万t/年）とともに、養殖(約15万t/年)が行われている。ブリの養殖は、特に、西日本で行われており、その生産額は約1200億円と、海面魚類養殖の生産額の半分以上を占める。また海外においても、韓国などで養殖されている(約57t・年)。 There are nine species of Brassica in the world, and four species (Buri, Hiramasa, Kampachi, Hirenaga Kampachi) are distributed in Japan. Most of them are important species as fishery products and are also important as fishing species.
The yellowtail, which has high value as an edible fish, is an advanced fish. In Japan, natural fish is caught (approximately 70,000 t / year) and farmed (approximately 150,000 t / year). The culture of yellowtail is carried out especially in western Japan, and its production value is approximately 120 billion yen, accounting for more than half of the production value of sea fish culture. Overseas, it is cultivated in South Korea (approximately 57 tons / year).

ブリ養殖では、モジャコ（1.5〜15cm）と称する天然稚魚を採捕し、養殖種苗とする畜養が主流である。そのため、天然資源であるモジャコの資源管理のため、各県での採捕量は厳しく制限され、また、放流のための種苗生産が行われている。
しかし、今後の養殖業を発展させていくためには、天然資源に頼らず、かつ天然魚に優る病気に強い、味が良いなどの有用(優良経済)形質を持つブリを生産する必要がある。そこで、有用形質を持つブリの家系を保有し、親のもつ有用性質にかかる遺伝情報を利用する育種という観点が重要になる。 In yellowtail culture, livestock farming is the mainstream, where natural juveniles called Mojako (1.5-15cm) are collected and used as cultured seedlings. Therefore, in order to manage the resources of Mojaco, a natural resource, the amount collected in each prefecture is strictly limited, and seedling production for release is carried out.
However, in order to develop the aquaculture industry in the future, it is necessary to produce yellowtail that does not rely on natural resources, has resistance to diseases superior to natural fish, and has good (economic) traits such as good taste. . Therefore, the viewpoint of breeding that possesses a family of yellowtail with useful traits and uses genetic information on the useful properties of parents is important.

魚類の育種においては、効率的に交配を実施していくために、親となる魚の雄と雌の数をバランスよく育てる必要がある。ブリにおいては、成熟期に目的の交配を行うために、ある特定の雌親魚から良質な卵を採取することは、その個体から良質な卵を採取できる期間が短いために、困難とされている。一方、雄親魚からの精子の採取は、どの個体も成熟期は常に排精することが多く、比較的容易である。そのため、ブリにおける育種のために親魚を養成する際には、雌を雄よりも多く養成する必要がある。
また、ブリの雄と雌は、外見上、二次性徴(成熟)を判断できる特徴をもたず、産卵期に生殖口よりチューブを差し込み、体内の卵あるいは精子を確認するしか、雌雄判別の方法はない。産卵期に、卵あるいは精子を作る成熟個体になるまでには、1-2年かかる。そのため、雌雄の数のバランスを調整できないまま、大量の稚魚を育てる必要があり、多くのコストが必要であった。 In fish breeding, in order to carry out mating efficiently, it is necessary to raise the number of males and females of the parent fish in a balanced manner. In yellowtail, it is considered difficult to collect high-quality eggs from a specific female parent fish for the purpose of mating at the mature stage due to the short period during which high-quality eggs can be collected from the individual. . On the other hand, it is relatively easy to collect spermatozoa from male progeny fish, because every individual always semen during maturity. Therefore, when breeding parent fish for breeding in yellowtail, it is necessary to train more females than males.
In addition, males and females of the yellowtail do not have characteristics that can determine secondary sexual characteristics (maturity), and it is only possible to insert a tube from the genital opening during the spawning period and check the eggs or sperm in the body. There is no way. It takes 1-2 years to become a mature individual who makes eggs or sperm during the spawning season. Therefore, it was necessary to raise a large amount of fry without adjusting the balance of the number of males and females, which required a lot of costs.

また、ブリは性決定機構が判明されていないため、魚類の多くで確認されている、ストレス、温度等の環境によって性が変わる（性転換）現象が起こる可能性があり、雌雄のバランス調整は難しかった。
そのため、ブリの人工種苗生産および養殖漁業においては、ブリの性について、稚魚の段階で、親となる雄と雌とを生きたまま判別できることが重要課題であり、さらには、性決定機構の解明が必要である。それゆえ、その解決のために性の管理技術の開発が急がれている。 In addition, the sex determination mechanism of yellowtail has not been clarified, and there is a possibility that the sex changes (sex change) depending on the environment such as stress and temperature, which has been confirmed in many fishes. was difficult.
For this reason, in the artificial seedling production and aquaculture of yellowtail, it is important to be able to discriminate between male and female parents at the juvenile stage, and to elucidate the mechanism of sex determination. is required. Therefore, the development of sexual management technology is urgently needed to solve this problem.

従来、発現に性差のある遺伝子のｍＲＮＡの発現パターンを調べることにより魚の性を判別する方法が知られている（特許文献１参照）。
しかし、この方法は、カレイ目を対象としており、ブリ類の性を判定する方法ではない。また、性分化時期以降の魚に適用し、遺伝的には同一でも発現が異なる遺伝子を利用しているので、性分化期である約６０日を過ぎないと性を判別することができず、発現した性を判別できるのみで、遺伝的な性の判別を行なうことはできない。さらに、一定量以上のＲＮＡを抽出する必要があるため、成魚でない場合は魚を殺してしまう虞がある。 Conventionally, a method for discriminating the sex of a fish by examining the expression pattern of mRNA of a gene having a sex difference in expression is known (see Patent Document 1).
However, this method is aimed at the flounder eye and is not a method for judging the sex of yellowtails. In addition, since it is applied to fish after the sex differentiation period and uses genes that are genetically identical but have different expression, sex cannot be determined until about 60 days, the sex differentiation period, It is only possible to discriminate the expressed sex, not the genetic sex. Furthermore, since it is necessary to extract a certain amount or more of RNA, there is a risk of killing the fish if it is not an adult fish.

特開２０００−６０５６９号公報JP 2000-60569 A

本発明が解決しようとする課題は、成熟前のブリ類の遺伝的性を、ブリ類を殺すことなく確実に判別するのに利用できるブリ類の遺伝的性に連鎖する遺伝子マーカー、この遺伝子マーカーを用いたブリ類の性判別法、及び、性判別法に用いるプライマーを提供することにある。   A problem to be solved by the present invention is to provide a genetic marker linked to the genetic sex of a yellowtail that can be used to reliably determine the genetic sex of a yellowtail before maturing without killing the yellowtail. It is an object to provide a sex discrimination method for yellowtails and a primer used in the sex discrimination method.

本出願人は、五島栽培センターにて飼育されている成熟親魚（天然漁獲魚を飼育しているもの）より、雄と雌とを交配し、Ｆ１集団を作出した。
作出したＦ１集団及びその両親に対してＤＮＡマーカーを用いた連鎖解析を行った結果、発明者らが既に見出していたブリの遺伝子連鎖地図（Ohara et al., 2005）と照らし合わせると、このブリ遺伝子連鎖地図における遺伝子連鎖群（ＬＧ）１２の配列表の配列番号１に示す塩基配列から成る遺伝子マーカーが性決定遺伝子座と連鎖していることがわかった。本出願人は、この遺伝子マーカーをＳｅｑｕ２１と名づけた。
さらに、ブリの性決定要因が、遺伝的要因により決定され、かつ雌由来の遺伝子により雌雄が決定するＺＷ型であることがわかった。 The present applicant mated males and females from mature parent fish (those raising natural catch fish) bred at the Goto Cultivation Center to create an F1 population.
As a result of linkage analysis using DNA markers for the F1 population and its parents, the yellowtail was found against the yellowtail linkage map already found by the inventors (Ohara et al., 2005). It was found that the gene marker consisting of the base sequence shown in SEQ ID NO: 1 in the sequence list of gene linkage group (LG) 12 in the gene linkage map is linked to the sex-determining locus. The applicant named this gene marker Sequ21.
Furthermore, it was found that the sex determining factor of yellowtail is ZW type determined by genetic factors and determined by males and females by female-derived genes.

本発明の遺伝子マーカーは、ブリ類の遺伝的性に連鎖する配列表の配列番号１に示す塩基配列から成る。ブリ類は、ブリ、ヒラマサ、カンパチ、ヒレナガカンパチのいずれか一つから成る。
ブリ類の性判別法は、配列表の配列番号１に示す塩基配列より成る遺伝子マーカーから作成したプライマーを用いて、ブリ由来の核酸を鋳型とした核酸増幅反応法を行い、得られた増幅産物を解析することにより、ブリ類の遺伝的性を判別する。核酸増幅反応法としては、ＰＣＲ法、ＬＡＭＰ法などがある。その中でもＰＣＲ法は最も一般的な方法であり、ＰＣＲ法により得られた産物にゲル電気泳動法を施して解析することにより、ブリ類の遺伝的性を判別する。 The gene marker of the present invention consists of the base sequence shown in SEQ ID NO: 1 in the sequence listing linked to the genetic nature of yellowtail. The yellowtail is composed of any one of yellowtail, Japanese cypress, amberjack, and Japanese amberjack.
The sex discrimination method for yellowtails is carried out by performing a nucleic acid amplification reaction method using a yellowtail-derived nucleic acid as a template, using a primer prepared from a gene marker consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing. By analyzing the above, the genetic sex of yellowtails is determined. Examples of the nucleic acid amplification reaction method include a PCR method and a LAMP method. Among them, the PCR method is the most common method, and the genetic properties of yellowtails are discriminated by subjecting the product obtained by the PCR method to gel electrophoresis analysis.

本発明のブリ類の性判別法に用いるプライマーは、配列表の配列番号１に示す塩基配列より成る遺伝子マーカーから作成した、１０〜５０塩基のオリゴヌクレオチド部分を含有するプライマーが好ましい。例えば、５’−ＴＴＴＣＡＴＴＧＴＧＧＣＧＣＴＣＡＧ−３’（配列表の配列番号２）の内、少なくとも１０個の塩基から成るオリゴヌクレオチド部分、及び、５’−ＧＧＴＴＧＴＡＡＴＧＴＧＴＣＣＣＡＧ−３’（配列表の配列番号３）の内、少なくとも１０個の塩基から成るオリゴヌクレオチド部分より成る。   The primer used for the sex discrimination method for yellowtails of the present invention is preferably a primer containing an oligonucleotide part of 10 to 50 bases prepared from a gene marker consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing. For example, among 5′-TTTCATTGTGGCGCTCAG-3 ′ (SEQ ID NO: 2 in the Sequence Listing), an oligonucleotide part consisting of at least 10 bases, and within 5′-GGTTGTATATGGTCCCCAG-3 ′ (SEQ ID NO: 3 in the Sequence Listing) Consists of an oligonucleotide moiety consisting of at least 10 bases.

本発明によれば、成熟前に生かした状態でブリ類の遺伝的性を判別できるので、親魚として飼育するための稚魚の雌雄を早い時期に調整することが可能になる。   According to the present invention, it is possible to determine the genetic sex of yellowtails in a state where they are alive before maturation, so that it is possible to adjust the sex of fry for breeding as a parent fish at an early stage.

実施例１においてプライマーを用いたＰＣＲ産物のゲル電気泳動像を示す。The gel electrophoresis image of the PCR product which used the primer in Example 1 is shown. 実施例１においてプライマーを用いたＰＣＲ産物のゲル電気泳動の模式図を示す。The schematic diagram of the gel electrophoresis of the PCR product which used the primer in Example 1 is shown. 実施例２においてプライマーを用いたＰＣＲ産物のゲル電気泳動像を示す。The gel electrophoresis image of the PCR product using the primer in Example 2 is shown. 実施例２においてプライマーを用いたＰＣＲ産物のゲル電気泳動の模式図を示す。The schematic diagram of the gel electrophoresis of the PCR product which used the primer in Example 2 is shown.

遺伝子連鎖群ＬＧ１２上の配列表の配列番号１に示す塩基配列から成る遺伝子マーカーを用いて、ブリ類の遺伝的性を判別する。
以下、本発明の実施例について詳細に説明する。
本実施例で用いたのは、標準和名「ブリ」であり、学名はＳｅｒｉｏｌａ ｑｕｉｎｑｕｅｒａｄｉａｔａである。 The genetic sex of yellowtails is discriminated using a genetic marker consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing on the gene linkage group LG12.
Examples of the present invention will be described in detail below.
In this example, the standard Japanese name “Buri” is used, and the scientific name is “Seriola quinqueradiata”.

（解析家系１）
五島栽培センターで飼育している成熟親魚（稚魚サイズを漁獲したものを、養殖業者が２年間畜養したものを五島栽培漁業センターで１年間飼育したもの）より雄と雌とを交配し、得られたＦ１集団の受精卵を１００Ｌ水槽に収容し、１００Ｌの海水で水温２０℃に維持してふ化まで飼育した。ふ化１日後（日齢１）に５００Ｌ水槽２面に移し、２２℃で流水飼育した。日齢３より日齢３０までは、餌料としてワムシを与え、日齢２１から日齢３９までは、アルテミアを加え、日齢３２以降は市販の配合飼料を与えた。 (Analysis family 1)
Obtained by mating males and females from mature parent fish bred at Goto Cultivation Center (fry-sized fish that have been bred for 2 years by a farmer at Goto Cultivation Fishery Center) The fertilized eggs of the F1 population were housed in a 100 L water tank and maintained at 100 ° C. seawater at a water temperature of 20 ° C. until breeding. One day after hatching (age 1), it was transferred to two 500 L water tanks and reared in running water at 22 ° C. From day 3 to day 30, rotifer was given as a feed, from day 21 to day 39, Artemia was added, and from day 32 onwards, commercially available mixed feed was given.

日齢４１以降は、陸上６０トン水槽及び水槽に設営した小割網を使用し日齢１０７まで飼育した。日齢１０８以降は、海上筏において飼育した。
その結果、６４匹のＦ１集団を得た。
飼育開始半年後に、Ｆ１集団６４個体を開腹し、生殖腺を確認することで雌雄の判別を行った。
６４個体の性比は、雄が２８個体、雌が３６個体であった。 After the age of 41, it was reared until the age of 107 using a 60-ton water tank and a small net installed in the tank. After day 108, they were raised in the sea bream.
As a result, 64 F1 populations were obtained.
Six months after the start of breeding, 64 individuals in the F1 population were laparotomized, and the sexes were determined by confirming the gonads.
The sex ratio of 64 individuals was 28 for males and 36 for females.

（解析家系２）
解析家系１で用いた同一の雄と、別の雌とを交配して５５匹のＦ１集団を得た。なお、飼育方法は、解析家系１と同様である。
解析家系１と同様に、飼育開始半年後に、Ｆ１集団５５個体を開腹し、生殖腺を確認することで雌雄の判別を行った。
５５個体の性比は、雄が２４個体、雌が３１個体であった。 (Analysis family 2)
The same male used in Analysis Family 1 and another female were mated to obtain 55 F1 populations. The breeding method is the same as that of the analysis family 1.
Similarly to the analysis family 1, half a year after the breeding, 55 F1 populations were laparotomized, and the sexes were determined by confirming the gonads.
The sex ratio of 55 individuals was 24 males and 31 females.

解析家系１のＦ１集団６４尾全てのブリに対し、次のようにしてＳｅｑｕ２１に関連するＰＣＲプライマーを用いてＰＣＲ法を行い、得られたＰＣＲ産物にゲル電気泳動法を施し、そのマーカー型を確認した。   For all yellowtails in F1 population of Analytical Family 1, PCR was performed using PCR primers related to Sequ21 as follows, gel electrophoresis was applied to the obtained PCR product, and the marker type was determined. confirmed.

まず、全てのブリからのＤＮＡ抽出は、以下の手順で行った。
尾鰭を１ｃｍ角の大きさで採取し、１００ｍＭ ＮａＣｌ、２０ｍＭ Ｔris−ＨＣｌ（pH８．０）、１００ｍＭ ＥＤＴＡ、１．０％ＳＤＳ、１００μｇ／ｍｌ Proteinase Ｋを含む消化溶液を６００μＬ加え、３７℃で一晩静置した。
さらに、フェノール／クロロホルム（１：１）抽出を１回行った後、エタノール沈殿にて染色体ＤＮＡを析出させた。回収したＤＮＡは７０％エタノールで洗浄、乾燥後、ＴＥ溶液（０．０１Ｍ Ｔris−ＨＣｌ pH７．４、２．５ｍＭ ＥＤＴＡ pH８．０）５０μＬに溶解した。 First, DNA extraction from all yellowtails was performed according to the following procedure.
A caudal fin was collected in a 1 cm square size, and 600 μL of a digestion solution containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1.0% SDS, 100 μg / ml Proteinase K was added, and the mixture was added at 37 ° C. I left still overnight.
Further, after extraction with phenol / chloroform (1: 1) once, chromosomal DNA was precipitated by ethanol precipitation. The recovered DNA was washed with 70% ethanol, dried, and then dissolved in 50 μL of a TE solution (0.01 M Tris-HCl pH 7.4, 2.5 mM EDTA pH 8.0).

次に、
Ｆ；５’−ＴＴＴＣＡＴＴＧＴＧＧＣＧＣＴＣＡＧ−３’（配列表の配列番号２）
Ｒ；５’−ＧＧＴＴＧＴＡＡＴＧＴＧＴＣＣＣＡＧ−３’（配列表の配列番号３）
の一組のプライマーを合成し、０．２ｐｍｏｌ Ｆプライマーと［γ-33Ｐ］ＡＴＰ、Ｔ４polynucleotide kinaseによって標識した０．３２ｐｍｏｌ Ｒプライマー、０．２ｍＭ each ｄＮＴＰ、２．０ｍＭ ＭｇＣｌ2、１％ＢＳＡ、０．０２ Ｕ Ex Ｔaq ＤＮＡ polymerase（ＴａｋａｒａＢｉｏ）、５０ｎｇのテンプレートＤＮＡを含む１３μｌの溶液で、Ｇｅｎｅ Ａｍｐ ＰＣＲ ｓｙｓｔｅｍ ９７００ ｔｈｅｒｍａｌ ｃｙｃｌｅｒ（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ）にて、ＰＣＲ法を行った。 next,
F: 5′-TTTTCATTGGGCGCTCAG-3 ′ (SEQ ID NO: 2 in the sequence listing)
R: 5′-GGTTGATAGTGTCCCCAG-3 ′ (SEQ ID NO: 3 in the sequence listing)
A set of primers, 0.2 pmol F primer and [γ- 33 P] ATP, 0.32 pmol R primer labeled with T4polynucleotide kinase, 0.2 mM each dNTP, 2.0 mM MgCl 2 , 1% BSA, A PCR method was performed using a Gene Amp PCR system 9700 thermal cycler (Perkin-Elmer) with 13 μl of a solution containing 0.02 U Ex Taq DNA polymerase (Takara Bio) and 50 ng of template DNA.

なお、このプライマーを用いたＰＣＲ反応組成（１サンプル分）を表１に示し、Ｒプラ
イマーのＲＩ標識用液組成（１００サンプル分）を表２に示す。 The PCR reaction composition (one sample) using this primer is shown in Table 1, and the RI primer solution composition (100 samples) of the R primer is shown in Table 2.

ＰＣＲ条件は、９５℃、２分−（９５℃、３０秒−５２℃、１分−７２℃、１分）×３５サイクル−（７２℃、３分）×１サイクルとした。
反応後、等量のLoading dye（９５％ formamide,１０ｍＭ ＥＤＴＡ，０．０５％ bromophenol blue and xylene cyanol）とよく攪拌し、ＰＣＲ産物を熱変性によって１本鎖にし、６％変性ポリアクリルアミドゲルにて電気泳動を行った。
その後、１時間乾燥させたゲルを、Imaging Plate(ＩＰ)に３〜１２時間感光させ、放射線の感光像が記憶されたＩＰをBio-imaging Analyzer（BAS1000，Fuji Photo Films）で読み取り、コンピュータで映像化した。
その結果を図１及び図２に示す。 PCR conditions were 95 ° C., 2 minutes- (95 ° C., 30 seconds-52 ° C., 1 minute-72 ° C., 1 minute) × 35 cycles− (72 ° C., 3 minutes) × 1 cycle.
After the reaction, stir well with an equal amount of Loading dye (95% formamide, 10 mM EDTA, 0.05% bromophenol blue and xylene cyanol), make the PCR product single-stranded by heat denaturation, and use 6% denaturing polyacrylamide gel. Electrophoresis was performed.
After that, the gel dried for 1 hour is exposed to an Imaging Plate (IP) for 3 to 12 hours, and the IP on which the radiation sensitive image is stored is read with a Bio-imaging Analyzer (BAS1000, Fuji Photo Films), and the image is displayed on a computer. Turned into.
The results are shown in FIGS.

図１及び図２からわかるように、雌親は１６８ｂｐと１５２ｂｐのバンドを保持している。子孫の雌は３６個体中３５個体が雌親由来の１６８ｂｐのバンドを持ち、雄の２８個体中２７個体が雌親由来の１５２ｂｐのバンドを持っていた。
即ち、本家系では、本プライマーにより増幅される雌由来のバンドの種類を調べることで、雌雄を判別することができる。
性決定形質（雌性）とこのバンドとの連鎖関係の確かさはLod scoreで示される。ここで、Lod score３以上というのは、1/103以下の危険率であることを意味している。この危険率が1/103以下である状態のことを、連鎖しているという（Lathropet al., 1984）。
１６８ｂｐ/１５２ｂｐのバンドと性決定形質（雌性）との矛盾は６４個体中２個体であり、Lod score ＝ 2log(2/64)+(64-2)log(1-2/64)-64log0.5となり、この連鎖関係は、1/1015.40の危険率で確実である。よって、配列表の配列番号１のマーカーは、ブリの遺伝的性に連鎖している。
従って、実施例１においては、ＰＣＲ産物に雌親由来の１５２ｂｐのバンドを持つブリは雄のブリ、雌親由来の１６８ｂｐのバンドを持つブリは雌のブリであるといえる。 As can be seen from FIGS. 1 and 2, the female parent holds bands of 168 bp and 152 bp. Of the 36 offspring, 35 out of 36 had a 168 bp band derived from the female parent and 27 out of 28 males had a 152 bp band from the female parent.
That is, in this family, sex can be discriminated by examining the type of female-derived band amplified by this primer.
The certainty of linkage between the sex-determining trait (female) and this band is indicated by the Lod score. Here, a Lod score of 3 or more means a risk rate of 1/10 or less. It is said that this risk rate is 1/10 3 or less (Lathropet al., 1984).
The contradiction between the band of 168 bp / 152 bp and the sex-determining trait (female) is 2 out of 64 individuals, and Lod score = 2log (2/64) + (64-2) log (1-2 / 64) -64log0. 5 and this linkage is certain with a risk factor of 1/10 15.40 . Thus, the marker of SEQ ID NO: 1 in the sequence listing is linked to yellowtail genetics.
Therefore, in Example 1, it can be said that a yellowtail having a 152 bp band derived from a female parent in a PCR product is a male yellowtail, and a yellowtail having a 168 bp band derived from a female parent is a female yellowtail.

解析家系２より得たＦ１集団５５尾についても、実施例１と同様にＰＣＲ法をおこなった。実施例２におけるＰＣＲ産物のゲル電気泳動像を図３及び図４に示す。
図３及び図４からわかるように、雌親は１７４ｂｐと１６２ｂｐのバンドを保持している。子孫の雌は３１個体中３０個体が雌親由来の１６２ｂｐのバンドを持ち、雄の２４個体すべての個体が雌親由来の１７４ｂｐのバンドを持っていた。この場合のLod scoreは、14.39であった。この連鎖関係は、1/1014.39の危険率で確実である。よって、配列表の配列番号１のマーカーは、ブリの遺伝的性に連鎖している。
従って、実施例２においては、ＰＣＲ産物に雌親由来の１７４ｂｐのバンドを持つブリは雄のブリ、雌親由来の１６２ｂｐのバンドを持つブリは雌のブリであるといえる。 The PCR method was performed in the same manner as in Example 1 for 55 F1 populations obtained from analysis family 2. The gel electrophoresis images of the PCR products in Example 2 are shown in FIGS.
As can be seen from FIGS. 3 and 4, the female parent holds bands of 174 bp and 162 bp. Of the offspring females, 30 out of 31 had a 162 bp band derived from the female parent, and all 24 male males had a 174 bp band derived from the female parent. The Lod score in this case was 14.39. This linkage is certain with a risk factor of 1/10 14.39 . Thus, the marker of SEQ ID NO: 1 in the sequence listing is linked to yellowtail genetics.
Therefore, in Example 2, it can be said that a yellowtail having a 174 bp band derived from a female parent in a PCR product is a male yellowtail, and a yellowtail having a 162 bp band derived from a female parent is a female yellowtail.

本発明においては、親から子孫への遺伝情報の伝達についてＤＮＡマーカーを用いて解析している。ブリ類の性判別においては、本発明で提案しているようにＳｅｑｕ２１に関するＰＣＲプライマーを用いて遺伝情報の伝達を解析することによって、全てのブリ家系で性判別を可能にする。親から子孫への遺伝情報の伝達を解析でき、それにより性判別が可能であることがポイントと言える。   In the present invention, transmission of genetic information from a parent to offspring is analyzed using a DNA marker. In the sex discrimination of yellowtails, sex discrimination is enabled in all yellowtail families by analyzing the transmission of genetic information using PCR primers related to Sequ21 as proposed in the present invention. It can be said that it is possible to analyze the transmission of genetic information from parents to offspring, thereby enabling sex discrimination.

なお、上記実施例１、２では、親から子孫への遺伝情報の伝達を解析した際に、性決定形質（雌性）と連鎖するバンドはそれぞれ異なる数値ではあるが、これは本開発に用いたＤＮＡマーカーの特性である。なぜならば、配列表の配列番号２と配列番号３のプライマーは、ＣＡ繰り返し配列（マイクロサテライト）を含む形でＰＣＲプライマーが設計されている。マイクロサテライトを含むＰＣＲプライマーは、遺伝情報の伝達を解析することによって１つの遺伝子座を位置付けることができると共に、同種内のその多型性により各個体間では多くのバンドを検出する（ＤＮＡＭａｋｅｒｓ：Ｐｒｏｔｏｃｏｌｓ，Ａｐｐｌｉｃａｔｉｏｎｓ，ａｎｄ Ｏｖｅｒｖｉｅｗ，ＧＵＳＴＡＶＯ ＣＡＥＴＡＮＯ−ＡＮＯＬＬＥＳ ＰＥＴＥＲ Ｍ．ＧＲＥＳＳＨＯＦＦ）ものであり、解析家系により異なるためである。Ｓｅｑｕ２１で位置づけられるマーカー座が性決定遺伝子の存在する場所であり、それによって性判別が可能になっている。
よって、この実施例で示されるバンドのサイズは、家系によりこれに限定されるものではない。 In Examples 1 and 2 above, when the transmission of genetic information from the parent to the offspring was analyzed, the bands linked to the sex-determining trait (female) were different values, but this was used in this development. It is the characteristic of a DNA marker. This is because the primers of SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing are designed as PCR primers including CA repeat sequences (microsatellite). PCR primers containing microsatellite can locate a single locus by analyzing the transmission of genetic information and detect many bands among individuals due to its polymorphism within the same species (DNAMakers: Protocols) , Applications, and Overview, GUSTAVO CAETANO-ANOLLES PETER M. GRESSHOFF), and this is because it differs depending on the analysis family. The marker locus located in Sequ21 is a place where the sex-determining gene is present, thereby enabling sex discrimination.
Therefore, the size of the band shown in this embodiment is not limited to this depending on the family.

Claims (6)

ブリ類の遺伝的性に連鎖する配列表の配列番号１に示す塩基配列から成る遺伝子マーカー。   A genetic marker comprising the base sequence shown in SEQ ID NO: 1 in the sequence listing linked to the genetics of yellowtail. ブリ類は、ブリ、ヒラマサ、カンパチ、ヒレナガカンパチのいずれか一つから成る請求項１に記載の遺伝子マーカー。   2. The gene marker according to claim 1, wherein the yellowtail is composed of any one of yellowtail, Japanese cypress, amberjack, and Japanese amberjack. 請求項１に記載の遺伝子マーカーから作成したプライマーを用いて、ブリ由来の核酸を鋳型とした核酸増幅反応法を行い、得られた増幅産物を解析することにより、ブリ類の遺伝的性を判別することを特徴とするブリ類の性判別法。   Using a primer prepared from the gene marker according to claim 1, performing a nucleic acid amplification reaction method using a yellowtail-derived nucleic acid as a template, and analyzing the obtained amplification product, thereby determining the genetic nature of the yellowtail A sex discrimination method for yellowtails characterized by 請求項１に記載の遺伝子マーカーから作成したプライマーを用いて、ブリ類のゲノムＤＮＡにポリメラーゼ連鎖反応法(ＰＣＲ法)を行い、得られた産物にゲル電気泳動法を施して解析することにより、ブリ類の遺伝的性を判別することを特徴とするブリ類の性判別法。   By using the primer prepared from the gene marker according to claim 1, performing a polymerase chain reaction method (PCR method) on the genomic DNA of yellowtails, and analyzing the resulting product by gel electrophoresis, A sex discrimination method for yellowtails characterized by determining the genetic sex of yellowtails. 請求項１に記載の遺伝子マーカーから作成した、５’−ＴＴＴＣＡＴＴＧＴＧＧＣＧＣＴＣＡＧ−３’（配列表の配列番号２）の内、少なくとも１０個の塩基から成るオリゴヌクレオチド部分、及び、５’−ＧＧＴＴＧＴＡＡＴＧＴＧＴＣＣＣＡＧ−３’（配列表の配列番号３）の内、少なくとも１０個の塩基から成るオリゴヌクレオチド部分より成ることを特徴としたブリ類の性判別法に用いるプライマー。   An oligonucleotide part composed of at least 10 bases of 5′-TTTCATTGTGGCGCCTCAG-3 ′ (SEQ ID NO: 2 in the sequence listing) prepared from the gene marker according to claim 1, and 5′-GGTTGTATATGTGTCCCAG-3 ′ A primer used in a sex discrimination method for yellowtails, characterized in that it comprises an oligonucleotide portion consisting of at least 10 bases in (SEQ ID NO: 3 in the Sequence Listing). 請求項５に記載のプライマーを用いて、ブリ類のゲノムＤＮＡにポリメラーゼ連鎖反応法(ＰＣＲ法)を行い、得られた産物にゲル電気泳動法を施して解析することにより、ブリ類の遺伝的性を判別することを特徴とするブリ類の性判別法。   Using the primer according to claim 5, the polymerase chain reaction (PCR method) is performed on the genomic DNA of the yellowtail, and the resulting product is subjected to gel electrophoresis to analyze the genetic DNA of the yellowtail A sex discrimination method for yellowtails characterized by sex discrimination.
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JP2014180233A (en) * 2013-03-19 2014-09-29 Tokyo Univ Of Marine Science & Technology Method for discriminating sex of yellowtail group
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JP2019140953A (en) * 2018-02-19 2019-08-29 国立大学法人東京海洋大学 Method for identifying the sex of yellowtail
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