JP2014180233A - Method for discriminating sex of yellowtail group - Google Patents
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この発明は、天然のブリ類の性識別方法に関し、より詳細には、一塩基多型を利用してブリ類の性を識別する方法に関する。 The present invention relates to a method for sex identification of natural yellowtails, and more particularly to a method for identifying sex of yellowtail using single nucleotide polymorphism.
我が国の養殖重要種であるブリでは、天然種苗が多く用いられているが、より安定した養殖生産のために、人工種苗による種苗の早期導入と、人工種苗への優良形質付与に関する技術開発が行われている。ブリ人工種苗生産における問題点として、雄は成熟期に常に排精することが多く、精子の採取が比較的容易であり、また凍結保存技術も確立されている。一方で、雌は１個体から良質な卵を採取できる期間が短いため、また抱卵確認の際のハンドリングにより排卵しなくなることが多く、採卵のタイミングが難しいなど、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難しい。また養殖ブリの出荷においては、雌は春先の産卵期になると体重が目減りするため商品として向かないとされている。そのため、雌雄が識別できれば、養成する親魚の雌雄の尾数の調整や出荷時期を雌雄で使い分けることができる等の利点がある。
ブリ類のゲノムは未だ公開されていないが、発明者らは、既に、ブリとヒラマサについて、連鎖群LG12を含むブリ連鎖地図を開示している（非特許文献１）。更に、発明者らは、ブリの性決定遺伝子座が、連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間にあると特定し、このマイクロサテライトDNAマーカー座Sequ21が性判別マーカーとして使える可能性を示唆した（非特許文献２）。更に、発明者らは、マイクロサテライトDNAマーカー座Sequ21の特定の配列の増幅産物のサイズの差により性別を判定できる方法を開発した（特許文献１）。
（特許文献１）
Natural seedlings are commonly used in yellowtail, which is an important aquaculture species in Japan. For the purpose of more stable aquaculture production, technology for early introduction of seedlings using artificial seedlings and the provision of excellent traits to artificial seedlings is being developed. It has been broken. As a problem in yellowtail artificial seedling production, males often excrete at the time of maturity, sperm collection is relatively easy, and cryopreservation techniques have been established. On the other hand, because females have a short period of time to collect good-quality eggs from one individual, and often do not ovulate due to handling when confirming incubation, the timing of egg collection is difficult. It is difficult to collect good-quality eggs from a specific female parent. In the shipment of cultured yellowtail, females are said to be unsuitable as a product because their weight is lost at the spawning season in early spring. Therefore, if the male and female can be identified, there are advantages such as adjustment of the number of males and females of the parent fish to be cultivated and the use of different shipping times.
Although the genome of the yellowtail has not yet been disclosed, the inventors have already disclosed a yellowtail linkage map containing linkage group LG12 for yellowtail and hiramasa (Non-patent Document 1). Furthermore, the inventors have identified that the sex determination locus of yellowtail is between the microsatellite DNA marker loci Sequ17 and Sequ21 on linkage group LG12, and this microsatellite DNA marker loci Sequ21 can be used as a sex discrimination marker. The possibility was suggested (nonpatent literature 2). Furthermore, the inventors have developed a method capable of determining gender based on the difference in size of amplification products of specific sequences of the microsatellite DNA marker locus Sequ21 (Patent Document 1).
(Patent Document 1)
本発明は、ブリ類の、特に天然のブリ類の性を識別するための方法を提供することを目的とする。 The present invention aims to provide a method for identifying the sex of yellowtail, especially natural yellowtail.
本発明者らは、ブリ類の性決定遺伝子座が存在すると考えられる連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間にある塩基配列を検査した結果、複数の一塩基多型(SNP)を見出した（後述の実施例１）。そして、天然のブリのこの一塩基多型と、別途調べた性別とを比較した結果、特定の一塩基多型がブリ類の性別と極めてよく相関していることを見出し（後述の実施例２）、ブリ類の性を識別するための方法を見出した。
即ち、本発明は、ブリ類から採取したＤＮＡの、連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間に存在する配列番号1で示される塩基配列の302番目の塩基及び配列番号２で示される塩基配列の366番目の塩基の少なくとも一方の一塩基多型の遺伝型を調べる段階、及び該遺伝型に基づいてブリ類の遺伝的性を判定する段階から成る、ブリ類の遺伝的性を識別する方法である。
As a result of examining the nucleotide sequence between the microsatellite DNA marker loci Sequ17 and Sequ21 on the linkage group LG12, which is considered to have a sex-determining locus of yellowtails, the present inventors have found that a plurality of single nucleotide polymorphisms ( SNP) was found (Example 1 described later). As a result of comparing this single nucleotide polymorphism of natural yellowtail with the sex examined separately, it was found that a specific single nucleotide polymorphism correlates very well with the sex of yellowtail (Example 2 described later). ) And found a method for identifying the sex of yellowtail.
That is, the present invention relates to the 302nd base of the base sequence represented by SEQ ID NO: 1 and the SEQ ID NO: 2 present between the microsatellite DNA marker loci Sequ17 and Sequ21 on the linkage group LG12 of DNA collected from yellowtails. Genetic analysis of yellowtails comprising the steps of examining the genotype of a single nucleotide polymorphism of at least one of the 366th bases of the nucleotide sequence represented by: and determining the genetic sex of yellowtails based on the genotype It is a method of identifying sex.
また、本発明は、検査の対象であるブリ類の個体からゲノムＤＮＡを抽出し、連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間に存在する配列番号1で示される塩基配列の302番目の塩基及び配列番号２で示される塩基配列の366番目の塩基の少なくとも一方に存在する一塩基多型を含む塩基配列の外側に結合する特異的な配列を有する一対のプライマーを用いてＰＣＲ反応を行い、得られた増幅産物中の該一塩基多型の遺伝型を調べる段階、及び該遺伝型に基づいてブリ類の遺伝的性を判定する段階から成る、ブリ類の遺伝的性を識別する方法である。
更に本発明は、上記のブリ類の遺伝的性を識別する方法に使用するためのプライマーセットであって、連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間に存在する配列番号1で示される塩基配列の302番目の塩基又は配列番号２で示される塩基配列の366番目の塩基を含む領域を検出するためのプライマーであって、該塩基を含む該塩基配列中の５０〜１００塩基の長さの領域の両端に設定された長さが１５塩基以上のフォワードプライマー及びリバースプライマー又はこれらと９０％以上相同のフォワードプライマー及びリバースプライマーから成るプライマーセットである。
Further, the present invention extracts genomic DNA from yellowtail individuals to be examined, and has a nucleotide sequence represented by SEQ ID NO: 1 existing between microsatellite DNA marker loci Sequ17 and Sequ21 on linkage group LG12. PCR using a pair of primers having a specific sequence that binds to the outside of a base sequence containing a single nucleotide polymorphism present in at least one of the 366th base of the 302th base and the base sequence shown in SEQ ID NO: 2 The genetic property of the yellowtail is comprised of a step of performing a reaction and examining the genotype of the single nucleotide polymorphism in the obtained amplification product, and determining the genetic sex of the yellowtail based on the genotype. It is a method of identification.
Furthermore, the present invention provides a primer set for use in the above-described method for identifying the genetic sex of yellowtails, comprising SEQ ID NO: 1 present between microsatellite DNA marker loci Sequ17 and Sequ21 on linkage group LG12. A primer for detecting a region containing the 302 th base of the base sequence shown by or the 366 th base of the base sequence shown by SEQ ID NO: 2 and 50 to 100 bases in the base sequence containing the base Is a primer set consisting of a forward primer and a reverse primer having a length of 15 bases or more, or a forward primer and a reverse primer having a homology of 90% or more.
本発明は、連鎖群ＬＧ１２上のマイクロサテライトDNAマーカー座Sequ17とSequ21との間に存在するSNPマーカー座269k02t7の塩基配列（配列番号１）の302番目の塩基(即ち、遺伝マーカー269k02t7SNP)及びSNPマーカー座163a23t7の塩基配列（配列番号２）の366番目の塩基(即ち、遺伝マーカー163a23t7SNP)の少なくとも一方の一塩基多型を用いて、ブリ類の遺伝的性を識別する方法である。
本発明の方法により性別を調べることのできる魚類は、Seriola 属の魚類であり、和名では、ブリ（学名：Seriola quinqueradiata）、ヒラマサ（学名：Seriola lalandi）、カンパチ（学名：Seriola dumerili）、ヒレナガカンパチ（学名：Seriola rivoliana）、アオブリ、ガンド、ガンドブリ、モジャコ、モジャッコ、イナダ、イナラ、ワラサ、コゾクラ、コズクラ、ツバイソ、フクラギ、フクラゲ、ワカナ、ショウジンゴ、ツバス、ヤズ、ハマチ、メジ、メジロ、ハナジロ、マルゴ、ワカナゴ、オオイナ、スズイナ、オオイオ、ワカシ、マサギ、ヒラサ、ヒラス、ヒラソ、テンコツ、セントク、アガユ、マヤ、外国名では、(Japanese) Amberjack、(Five-ray) Yellowtailと称される魚を含む。本明細書では、これらを総称してブリ類という。
本発明の方法は、特に天然のブリ類の性を識別することに優れる。
The present invention relates to the 302 th base of the SNP marker locus 269k02t7 (SEQ ID NO: 1) existing between the microsatellite DNA marker locus Sequ17 and Sequ21 on linkage group LG12 (ie, genetic marker 269k02t7SNP) and the SNP marker This is a method for identifying the genetic sex of yellowtail using at least one single nucleotide polymorphism of the 366th base (ie, genetic marker 163a23t7SNP) of the base sequence (SEQ ID NO: 2) of locus 163a23t7.
The fish whose sex can be examined by the method of the present invention is a fish of the genus Seriola, and the Japanese names are yellowtail (scientific name: Seriola quinqueradiata), hiramasa (scientific name: Seriola lalandi), amberjack (scientific name: Seriola dumerili), cypress. Amberjack (Seriola rivoliana), Aoburi, Gand, Ganduburi, Mojaco, Mojacco, Inada, Inara, Warasa, Kozokura, Kozukura, Tsubasa, Fukuragi, Fukurage, Wakana, Shojingo, Tsubasa, Yaz, Hamachi, Azalea, Japanese Whiteeye, Hanagi Margo, Wakanago, Ooina, Suzuina, Ooio, Sardine, Masagi, Hirasa, Hiras, Hiraso, Tenkotsu, Sentoku, Agayu, Maya, including the fish called (Japanese) Amberjack, (Five-ray) Yellowtail . In the present specification, these are collectively referred to as yellowtails.
The method of the present invention is particularly excellent in identifying the nature of natural yellowtails.
本発明の一塩基多型は、SNPマーカー座269k02t7（配列番号１）及びSNPマーカー座163a23t7（配列番号２）に存在する。ブリ類の連鎖群ＬＧ１２を図１に示す(非特許文献２)。
図１に示す連鎖地図は、相同染色体間の組換えを利用して作成した、染色体上のDNA配列の並び順を表す地図である。組換えはランダムに起こるため、用いる家系が異なれば特定のDNAマーカー座間で組換えを起こした個体数も異なる。したがって、各DNAマーカー座間の遺伝的距離は可変である。ただし、１.DNAマーカー座の並び順は同じである、ことと、２.DNAマーカー座間の物理的距離（bp）が非常に近ければそれら二つのDNAマーカー座は一つのマーカー座であるかのように振る舞う、ことは種、性別を問わず全ての連鎖地図で不変である。図１に示されているマイクロサテライトDNAマーカー座Sequ17とSequ21の遺伝的距離を見て分かるように、性決定遺伝子座（図１に「Sex」で示す。）とマイクロサテライトDNAマーカー座Sequ17とSequ21との間では複数の組換えが起こっている。SNPマーカー座269k02t7と163a23t7は、性決定遺伝子座と物理的に非常に近く、連鎖地図上では性決定遺伝子座（「Sex」）とほぼ同じ位置に存在する。
The single nucleotide polymorphisms of the present invention exist at the SNP marker locus 269k02t7 (SEQ ID NO: 1) and the SNP marker locus 163a23t7 (SEQ ID NO: 2). A linked group LG12 of yellowtail is shown in FIG. 1 (Non-patent Document 2).
The linkage map shown in FIG. 1 is a map representing the arrangement order of DNA sequences on a chromosome created by using recombination between homologous chromosomes. Since recombination occurs randomly, the number of individuals who have undergone recombination between specific DNA marker loci varies depending on the family used. Thus, the genetic distance between each DNA marker locus is variable. However, 1. The order of DNA marker loci is the same, and 2. If the physical distance (bp) between DNA marker loci is very close, whether these two DNA marker loci are one marker loci. It behaves like all sorts of linkage maps, regardless of species or gender. As can be seen from the genetic distance between the microsatellite DNA marker loci Sequ17 and Sequ21 shown in FIG. 1, the sex-determining loci (shown as “Sex” in FIG. 1) and the microsatellite DNA marker loci Sequ17 and Sequ21. Multiple recombination has occurred between and. SNP marker loci 269k02t7 and 163a23t7 are physically very close to the sex-determining locus and are located on the linkage map at approximately the same position as the sex-determining locus (“Sex”).
このような、ブリ類の性の表現型（雄、雌）と強い相関を示す遺伝マーカーを単離するには、まずマイクロサテライトDNAマーカー座Sequ17とSequ21との間に存在する遺伝マーカーを複数単離し、それらのうち性決定遺伝子座との間でほとんど組換えが観察されないような遺伝マーカーを見付ける。その次に、組換えがほとんど観察されない遺伝マーカー座を起点として、性決定遺伝子座方向に向かって染色体上に新たな遺伝マーカーを複数配置し、各マーカーの遺伝型が性別と一致するかどうかを調べることによって行う。 To isolate such genetic markers that are strongly correlated with the yellowtail sex phenotype (male and female), first, a plurality of genetic markers existing between the microsatellite DNA marker loci Sequ17 and Sequ21 are used. And find a genetic marker in which little recombination is observed between the sex-determining loci. Next, starting from a genetic marker locus where almost no recombination is observed, multiple new genetic markers are placed on the chromosome in the direction of the sex-determining locus, and whether the genotype of each marker matches the gender. Do by examining.
また、本願の性決定遺伝子座周辺のＢＡＣクローンと遺伝マーカーを図２に示す。
図２は、上記で説明したうち、性決定遺伝子座との間でほとんど組換えが観察されない遺伝マーカー（Sequ0485BAC、Sequ776TUF）と、性決定遺伝子座方向に向かって染色体上に新たに配置した遺伝マーカー（237o03sp6SNP、198o21sp6SNP、269k02t7SNP、163a23t7SNP、151d03t7SNP、190h02sp6SNP）を示す。枠内に存在する遺伝マーカーは、調査した家系の個体全てでその遺伝型と性別が完全に一致していたため、性判別遺伝マーカーの有力候補としてまず挙げられた。
後述の実施例に示すように、これらの遺伝マーカーのうち、269k02t7SNPと163a23t7SNPの遺伝型は性決定形質と相関する。
FIG. 2 shows BAC clones and genetic markers around the sex-determining locus of the present application.
Figure 2 shows a genetic marker (Sequ0485BAC, Sequ776TUF) in which recombination is hardly observed with the sex-determining locus, and a genetic marker newly placed on the chromosome in the direction of the sex-determining locus. (237o03sp6SNP, 198o21sp6SNP, 269k02t7SNP, 163a23t7SNP, 151d03t7SNP, 190h02sp6SNP). The genetic markers present in the frame were first listed as potential candidates for sex-discriminating genetic markers because their genotypes and genders were completely consistent in all the individuals in the families studied.
Among these genetic markers, the genotypes of 269k02t7SNP and 163a23t7SNP correlate with sex-determining traits, as shown in the Examples described later.
上記一塩基多型の検出は、検査対象のブリ類の個体の一部、例えば、尾鰭、血液、腎臓筋肉などの核が存在する組織からＤＮＡを採取し、このＤＮＡに含まれる一塩基多型の遺伝型を検査する。一塩基多型の検出は、一塩基多型部分の塩基配列を決定することによって行うことができる。一塩基多型部分の塩基配列の決定には、例えば、TaqMan法、ダイレクトシークエンス法、ARMS-PCR法、飽和型DNA結合色素によるHRM解析、PCR-制限酵素切断断片長多型による方法(PCR-RFLP解析)、MALDI-TOF/MSによるSNPタイピング法、DNAチップを用いた方法などを挙げることができる。
一塩基多型の検出には、例えば、PCR-RFLP解析を用いることができる。具体的には、上記遺伝子における一塩基多型の検出は、例えば、ブリ類からゲノムＤＮＡを抽出し、前記一塩基多型を含む塩基配列の外側に結合する特異的な配列を有する一対のＰＣＲプライマーを用いてＰＣＲ反応を行い、得られたＰＣＲ産物を制限酵素処理し、制限酵素処理により得られたＤＮＡの遺伝型を識別することで行うことができる。
The single nucleotide polymorphism is detected by collecting DNA from a part of an individual of the yellowtail species to be examined, for example, a tissue having nuclei such as caudal fin, blood, kidney muscle, and the like. Test for genotype. Single nucleotide polymorphism can be detected by determining the nucleotide sequence of the single nucleotide polymorphism. For example, TaqMan method, direct sequencing method, ARMS-PCR method, HRM analysis with saturated DNA-binding dye, PCR-restriction fragment length polymorphism method (PCR- RFLP analysis), SNP typing method using MALDI-TOF / MS, and a method using a DNA chip.
For detection of a single nucleotide polymorphism, for example, PCR-RFLP analysis can be used. Specifically, detection of a single nucleotide polymorphism in the above gene is performed by, for example, extracting a pair of PCR from genomic DNA from yellowtails and having a specific sequence that binds outside the nucleotide sequence containing the single nucleotide polymorphism. PCR can be performed using primers, the obtained PCR product is treated with a restriction enzyme, and the genotype of the DNA obtained by the restriction enzyme treatment is identified.
各方法に用いるＰＣＲプライマーは、塩基配列（配列番号１）の302番目の塩基（269k02t7SNP）及び塩基配列（配列番号２）の366番目の塩基（163a23t7SNP）を含む領域を検出するためのプライマーであって、該塩基を含む該塩基配列中の５０〜１００塩基の長さの領域の両端に設定された長さが１５塩基以上、好ましくは１９〜２４塩基のフォワードプライマー及びリバースプライマーから成るプライマーセットである。これらの塩基配列に９０％以上相同であるプライマーを使用してもよい。
遺伝マーカー269k02t7SNPの遺伝型がＡ／Ｃの場合には雌と判定し、又は該遺伝型がＡ／Ａの場合には雄と判定することができる。
また、遺伝マーカー163a23t7SNPの遺伝型がＡ／Ｇの場合には雌と判定し、又は該遺伝型がＡ／Ａの場合には雄と判定することができる。
ブリ類の性を識別するためには、これらいずれか一方を行ってもよいし、両方行ってもよい。
The PCR primer used in each method is a primer for detecting a region containing the 302th base (269k02t7SNP) of the base sequence (SEQ ID NO: 1) and the 366th base (163a23t7SNP) of the base sequence (SEQ ID NO: 2). A primer set consisting of a forward primer and a reverse primer having a length of 15 bases or more, preferably 19 to 24 bases, set at both ends of a region of 50 to 100 bases in the base sequence containing the bases. is there. Primers that are 90% or more homologous to these base sequences may be used.
When the genotype of the genetic marker 269k02t7SNP is A / C, it can be determined as female, or when the genotype is A / A, it can be determined as male.
In addition, when the genetic type of the genetic marker 163a23t7SNP is A / G, it can be determined as a female, or when the genetic type is A / A, it can be determined as a male.
In order to identify the sex of yellowtail, either one of these may be performed, or both may be performed.
以下、実施例にて本発明を例証するが本発明を限定することを意図するものではない。
実施例１
本実施例ではまず、BACベクターにブリゲノムDNAを組み込んだブリBACライブラリーを構築し、性決定遺伝子座周辺のゲノムDNA配列を保持するBACクローンを特定した。
連鎖群LG12上のマイクロサテライトDNAマーカー座Sequ17とSequ21の間にブリ性決定に関与している遺伝子が存在することが既知であるので（非特許文献２）、上記BACライブラリーから、性決定遺伝子座に近いと推定される遺伝マーカーSequ0485BAC（配列番号５）を有するBACクローン056j15を選んだ。この遺伝マーカーSequ0485BACを検出するための下記プライマー対を選んだ。
F: GATTGCATCTGTAAGACCACCA（配列番号６）
R: GGATTTTCCATACTAAGCGTGC（配列番号７）
このプライマーを用いて、上記BACライブラリーについて、フォワードプライマー 2pmolとリバースプライマー 2pmol、0.2mM each dNTPs、2.0mM MgCl2、0.25U Ex taq DNA polymerase（Takara）、50ng BAC DNA poolを含む10μLの反応溶液を調整し、TProfessional 96 Thermocycler（Biometra）にて、PCRを行った。
PCR反応条件は、（95℃、3分）×1サイクル-（95℃、30秒-55℃、30秒、72℃、1分）×30サイクル-（72℃、5分）×1サイクルとした。
得られたPCR産物はアガロースゲル電気泳動によりサイズ分画、可視化し、PCR陽性を示すBACクローンを特定した。以上の実験で陽性BACクローンとして045p01と051i16が特定された。
The following examples illustrate the invention but are not intended to limit the invention.
Example 1
In this example, first, a Buri BAC library in which Buri genome DNA was incorporated into a BAC vector was constructed, and a BAC clone holding the genomic DNA sequence around the sex determination locus was identified.
Since it is known that a gene involved in sex determination is present between the microsatellite DNA marker loci Sequ17 and Sequ21 on linkage group LG12 (Non-patent Document 2), the sex-determining gene is BAC clone 056j15 having the genetic marker Sequ0485BAC (SEQ ID NO: 5) presumed to be close to the locus was selected. The following primer pair was selected to detect this genetic marker Sequ0485BAC.
F: GATTGCATCTGTAAGACCACCA (SEQ ID NO: 6)
R: GGATTTTCCATACTAAGCGTGC (SEQ ID NO: 7)
Using this primer, 10 μL of the reaction solution containing 2 pmol of forward primer and 2 pmol of reverse primer, 0.2 mM each dNTPs, 2.0 mM MgCl2, 0.25 U Ex taq DNA polymerase (Takara), 50 ng BAC DNA pool After adjustment, PCR was performed with a TProfessional 96 Thermocycler (Biometra).
PCR reaction conditions are (95 ° C, 3 minutes) x 1 cycle-(95 ° C, 30 seconds-55 ° C, 30 seconds, 72 ° C, 1 minute) x 30 cycles-(72 ° C, 5 minutes) x 1 cycle did.
The obtained PCR product was size fractionated and visualized by agarose gel electrophoresis, and a BAC clone showing PCR positive was identified. The above experiments identified 045p01 and 051i16 as positive BAC clones.
次に、これらのBACクローンについてDNA抽出し、得られたBACクローンDNAと3.2pmol プライマー（下記のSP6プライマーとT7プライマー）を混合し、BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)と3130xl ジェネティックアナライザ(Applied Biosystems)を用いて塩基配列を決定した。
SP6 5'-ATCTGCCGTTTCGATCCTCC-3'（配列番号８）
T7 5'-TGACATTGTAGGACTATATTGC-3'（配列番号９）
得られた塩基配列(BES)をもとにプライマー対を設計した。設計したプライマー配列と塩基配列(BES)の一部を表１に示す。得られたBACクローン間の位置関係はPCRによるバンドの有無によって決定した。BACクローン間の位置関係を図２に示す。
以上と同様の操作を他のBACクローンについて繰り返し行うことによって、図２に示すように、性決定遺伝子が存在する領域を含む連続したBACクローンの集団とその塩基配列(BES)を取得した。
Next, DNA extraction was performed on these BAC clones, and the obtained BAC clone DNA and 3.2 pmol primer (SP6 primer and T7 primer below) were mixed, and BigDye (R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) The nucleotide sequence was determined using 3130xl Genetic Analyzer (Applied Biosystems).
SP6 5'-ATCTGCCGTTTCGATCCTCC-3 '(SEQ ID NO: 8)
T7 5'-TGACATTGTAGGACTATATTGC-3 '(SEQ ID NO: 9)
Primer pairs were designed based on the obtained base sequence (BES). Table 1 shows a part of the designed primer sequence and base sequence (BES). The positional relationship between the obtained BAC clones was determined by the presence or absence of a band by PCR. The positional relationship between BAC clones is shown in FIG.
By repeating the same operation as described above for other BAC clones, as shown in FIG. 2, a continuous BAC clone group including the region where the sex-determining gene exists and its base sequence (BES) were obtained.
これらの塩基配列から、下記を含む複数の一塩基多型(SNP)が見つかった(図３)。
269k02t7SNP ： 配列番号１の302番目（A/C）
163a23t7SNP ： 配列番号２の366番目（A/G）
151d03t7SNP ： 配列番号３の641番目（T/C）
190h02sp6SNP ： 配列番号４の206番目（A/G）
From these nucleotide sequences, a plurality of single nucleotide polymorphisms (SNPs) including the following were found (FIG. 3).
269k02t7SNP: 302th position of SEQ ID NO: 1 (A / C)
163a23t7SNP: 366th position of SEQ ID NO: 2 (A / G)
151d03t7SNP: 641th position of SEQ ID NO: 3 (T / C)
190h02sp6SNP: 206th position of SEQ ID NO: 4 (A / G)
実施例２
長崎県五島にて採捕した雄47尾、雌47尾の計94尾のブリ野生個体について、開腹して生殖腺を確認することによりその性別を判定した。
これらのブリの尾鰭を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。
次に、表１の各プライマー2pmol、0.2mM each dNTPs、2.0mM MgCl2、0.25U Ex taq DNA polymerase（Takara）、得られたDNA（テンプレート）50ngを含む10μLの反応溶液を調整し、TProfessional 96 Thermocycler（Biometra）を用いてPCR法を行った。
PCR反応条件は、（95℃、3分）×1サイクル-（95℃、30秒-プライマー対固有のアニーリング温度50〜55℃、30秒、72℃、1分）×30サイクル-（72℃、5分）×1サイクルとした。
PCR反応後、PCR反応液7μLとillustra ExoStarキット（GE Healthcare）に含まれるAlkaline Phosphatase 0.1μLとExonuclease1 0.1μLを含む9μLの反応溶液を調整し、TProfessional 96 Thermocycler（Biometra）にて（37℃、1時間）-（80℃、15分）の酵素反応を行った。その後、酵素反応液3.5μLとプライマー6.4pmolを含む14μLのシーケンシング反応液を調整し、サンガー法による塩基配列決定を行った。なお、ここでプライマーとしてPCR反応に用いたフォワードプライマーを用いた。
Example 2
A total of 94 wild yellowtails, 47 males and 47 females, were collected from Goto, Nagasaki Prefecture.
Collect these yellowtail tails in a 1 cm square size, add 600 μL of digestion solution containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1.0% SDS, 100 μg / ml Proteinase K, 37 ° C. And left overnight. Furthermore, after phenol / chloroform (1: 1) extraction was performed once, chromosomal DNA was precipitated by ethanol precipitation.
Next, a 10 μL reaction solution containing 2 pmol of each primer, 0.2 mM each dNTPs, 2.0 mM MgCl 2 , 0.25 U Ex taq DNA polymerase (Takara), and 50 ng of the obtained DNA (template) in Table 1 was prepared, and TProfessional 96 PCR was performed using Thermocycler (Biometra).
PCR reaction conditions are (95 ° C, 3 minutes) x 1 cycle-(95 ° C, 30 seconds-primer pair specific annealing temperature 50-55 ° C, 30 seconds, 72 ° C, 1 minute) x 30 cycles-(72 ° C 5 minutes) × 1 cycle.
After the PCR reaction, prepare 9 μL of the reaction solution containing 7 μL of the PCR reaction solution and 0.1 μL of Alkaline Phosphatase and 0.1 μL of Exonuclease 1 included in the illustra ExoStar kit (GE Healthcare), and use TProfessional 96 Thermocycler (Biometra) at 37 ° C., 1 Time)-(80 ° C, 15 minutes) enzyme reaction was performed. Thereafter, a sequencing reaction solution of 14 μL containing 3.5 μL of the enzyme reaction solution and 6.4 pmol of the primer was prepared, and the base sequence was determined by the Sanger method. Here, the forward primer used in the PCR reaction was used as a primer.
その結果得られた各遺伝マーカー(SNP)について遺伝型とその性別を下表に示す。
表中のスラッシュ(/)を挟む塩基は、各アレルの塩基を表す。
表中、カイ二乗値（χ２）は、観測度数と帰無仮説を「塩基配列パターンの頻度に雌雄間で偏りが無い」として算出した期待度数とを用いて、「（（（観測度数−期待度数）の2乗）÷期待度数）の総和」として算出したもので、その数値が高いほど遺伝型と性決定形質（雌性）との相関が確からしいことを示す。
表から、163a23t7SNP及び/又は269k02t7SNPの遺伝型を調べることで、ブリ野生個体の性別を高確率で判別することができるといえる。
The base between the slashes (/) in the table represents the base of each allele.
In the table, the chi-square value (χ 2 ) is obtained by using the observed frequency and the expected hypothesis calculated by assuming that the null hypothesis is “no frequency difference between male and female base sequence pattern”. The sum of (expected frequency) squared) ÷ expected frequency) ”indicates that the higher the value, the more likely the correlation between genotype and sex-determining trait (female) is.
From the table, by examining the genotype of 163a23t7SNP and / or 269k02t7SNP, it can be said that the sex of yellowtail wild individuals can be discriminated with high probability.
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| WO2019160136A1 (en) * | 2018-02-19 | 2019-08-22 | 国立大学法人東京海洋大学 | Method for determining sex of yellowtails |
| CN113186307A (en) * | 2021-06-21 | 2021-07-30 | 江苏省淡水水产研究所 | Sex-linked SNPs (single nucleotide polymorphisms) marker development method based on channel catfish male specific gene zbtb38-Y and application |
| CN114934121A (en) * | 2022-06-20 | 2022-08-23 | 南方海洋科学与工程广东省实验室(湛江) | MicroRNA (ribonucleic acid) of sex difference indication label of seriolala quinqueradiata as well as kit and application |
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| CN117965711A (en) * | 2024-03-27 | 2024-05-03 | 中国科学院海洋研究所 | Molecular marker, method, primer and kit for sex identification of male and female sex of oplegnathus fasciatus |
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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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