CN114752664B - SNP (Single nucleotide polymorphism) marker for identifying genetic sex of coilia ectenes as well as primer and application of SNP marker - Google Patents
SNP (Single nucleotide polymorphism) marker for identifying genetic sex of coilia ectenes as well as primer and application of SNP marker Download PDFInfo
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- CN114752664B CN114752664B CN202210538110.8A CN202210538110A CN114752664B CN 114752664 B CN114752664 B CN 114752664B CN 202210538110 A CN202210538110 A CN 202210538110A CN 114752664 B CN114752664 B CN 114752664B
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Abstract
The invention relates to the field of SNP markers, and particularly discloses an SNP marker for identifying genetic sex of coilia ectenes, and a primer and application thereof. According to the invention, coilia ectenes sex-specific SNP markers are successfully screened by a 2b-RAD sequencing method, and the markers are positioned at 12186257 th base on the LG18 chromosome of coilia ectenes. Aiming at the SNP marker, the designed primer can accurately and rapidly identify the genetic sex of coilia ectenes by utilizing conventional PCR amplification and Sanger sequencing. Compared with the prior anatomical detection and gonad section observation, the technology has the advantages of accuracy, simplicity, rapidness, less damage to fish bodies and the like, solves the problem of sex identification of coilia ectenes, is beneficial to large-scale cultivation of a total female system of coilia ectenes, and provides a powerful basis for development of breeding industry of coilia ectenes.
Description
Technical Field
The invention relates to the field of SNP markers, in particular to a SNP marker for identifying genetic sex of coilia ectenes, and a primer and application thereof.
Background
Coilia nasus (Coilia nasus) is a kind of middle and small size backtracking fish belonging to the phylum of chordopoda, class of teleostegiaceae, order of herring, family of Engraulis and genus of anchovy, and has high nutritive value and economic value, and is called as "Changjiang Sanxian" with hilus reeves (Tenualosa reevesii) and Fugu obscurus (Takifugu fasciatus). The coilia ectenes have obvious male and female growth bimorphology, the growth speed of the coilia ectenes in the same period is obviously faster than that of the male coilia ectenes, and the female coilia ectenes in the mature period are larger than that of the male coilia ectenes. Therefore, development of coilia ectenes total female breeding and total female breeding technology has important industrial significance. However, before gonad development is mature, female fish and male fish are hardly distinguished from each other in external morphology, and female fish and male fish in embryo and larva stages are hardly distinguished in morphology, so that great difficulty is brought to development of a coilia ectenes total female breeding technology, difficulty is brought to genome breeding value calculation of female and male candidate parent quantitative traits of coilia ectenes genome selective breeding, and development of sex determination molecular mechanism research of genetic sex identification before gonad development and differentiation is hindered. Therefore, a molecular marker capable of accurately and stably identifying the genetic sex of coilia ectenes is developed, and the molecular marker has great promotion effect and production application value for coilia ectenes sex control breeding.
From the genome information of coilia ectenes published at the present stage, only female coilia ectenes genome and annotation files (Xu et al 2020) are provided, no male related information exists, and the coilia ectenes cannot be directly unfolded and compared from the genome level of coilia ectenes with two sexes, so that a certain difficulty is brought to screening of sex related markers of coilia ectenes. With the rapid development of high throughput sequencing technology, simplified genome sequencing based on enzyme digestion (RAD-Seq, restriction-site Associated DNA Sequence) has provided the possibility for developing sex-specific molecular markers for fish as a method for genotyping by sequencing (Gamble et al, 2014; liu et al, 2018; methou et al, 2019; han et al, 2020).
The invention provides a method for simplifying genome sequencing by utilizing RAD, provides a coilia ectenes male and female specificity SNP marker, is successfully applied to rapid identification of coilia ectenes male and female gender, and provides a certain theoretical basis for related researches such as coilia ectenes total female breeding and the like.
Reference to the literature
1.Gamble T,Coryell J,Ezaz T,et al.Restriction site-associated DNA sequencing(RAD-seq)reveals an extraordinary number of transitions among Gecko sex determining systems.Molecular Biology and Evolution,2014,32:1296-1309.
2.Han C,Zhu Q,Lu H,et al.Screening and characterization of sex-specific markers developed by a simple NGS method in mandarin fish(Siniperca chuatsi).Aquaculture,2020,527:735495.
3.Liu H,Pang M,Yu X,et al.Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp(Hypophthalmichthys nobilis)and silver carp(Hypophthalmichthys molitrix).DNA Research,2018,25:257-264.
4.Xu G,Bian C,Nie Z,et al.Genome and population sequencing of a chromosome-level genome assembly of the Chinese tapertail anchovy(Coilia nasus)provides novel insights into migratory adaptation.GigaScience,2020,9:1-13.
5.Zhou Y,Wu J,Wang Z,et al.Identification of sex-specific markers and heterogametic XX/XY sex determination system by 2b-RAD sequencing in redtail catfish(Mystus wyckioides).Aquaculture Research,2019,50:2251-2266.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting 12186257 th base on LG18 chromosome of coilia ectenes in identifying the sex of coilia ectenes.
In order to achieve the above object, the present invention adopts the following technical measures:
the applicant carried out comparison analysis on the sequencing data of the coilia ectenes 2b-RAD, and found a large number of SNP markers between the male and female coilia ectenes. Screening and verifying 10 SNP markers with the most obvious sex difference correlation with coilia ectenes, wherein one SNP marker shows accurate male-female specificity, the SNP locus is positioned at 12186257 th base on the LG18 chromosome of coilia ectenes, and the corresponding genome is NCBI SRA ID, PRJNA422339. Thus, reagents that detect the 12186257 base on the LG18 chromosome of coilia ectenes can be used for sex identification of coilia ectenes.
For the above application, the reagent may be a primer.
Preferably, the primers are:
Forward primer：gcacaaagttgcactgttcc
Reverse primer：atgcacaacacggatcttgc。
and (3) judging the sex by detecting the 178 th base by using the fragments amplified by the primers, wherein the individuals marked with the SNP as T/G genotype are female coilia ectenes, and the individuals marked with the SNP as T/T genotype are male coilia ectenes.
Compared with the prior art, the invention has the following advantages:
(1) The genetic sex identification method provided by the invention can be used for sex identification by taking genomic DNA as a template and simply carrying out PCR and Sanger sequencing. The method is not influenced by the development period and the environment of individuals, can stably identify the genetic sexes of different individuals in each population of coilia ectenes, including embryos, larvae and adults, overcomes the defects of complicated operation, long time consumption and the like of other methods, and is suitable for rapidly identifying large samples.
(2) The invention has simple, quick and accurate operation and is suitable for popularization and application. The SNP marker can be applied to production practice to effectively identify and identify genetic sexes at various development stages, and is helpful for distinguishing coilia ectenes from male and female coilia ectenes, so that preparation is made for cultivating coilia ectenes full female breeding groups; can further improve the cultivation and economic benefits of the coilia ectenes in the pond cultivation.
(3) The SNP marker detection accuracy of the invention reaches 100%.
Drawings
FIG. 1 is a diagram showing SNP sites and peaks thereof of the sequencing result of Sanger sequencing;
wherein A is a sequencing peak diagram of SNP loci corresponding to female fishes; b is a sequencing peak diagram of SNP loci corresponding to the male fish.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
development and verification of coilia ectenes sex-specific SNP markers
Coilia ectenes are diploid organisms and are ZW/ZZ-type sex-determining systems, females ZW and males ZZ. A large number of SNP markers were found between male and female fish by comparing and analyzing 10 groups of 2b-RAD sequencing data of female and male coilia ectenes from the source fishery technology Co., ltd. Screening and verifying 10 SNP markers with the most obvious sex difference correlation with coilia ectenes, wherein one SNP marker shows accurate male-female specificity, and the SNP locus is positioned at 12186257 bases on the coilia ectenes LG18 chromosome.
The results are shown in Table 1, and the results show that the sequence from female fish has two bases of T/G at the SNP site shown in FIG. 1, the sequence from male fish does not have SNP polymorphism at the site, and all the bases of the sequences at the site are T.
TABLE 1 SNP molecular marker information obtained by screening
Primers were designed for the SNP molecular markers of Table 1:
Forward primer：gcacaaagttgcactgttcc
Reverse primer：atgcacaacacggatcttgc。
the sequence amplified in coilia ectenes female fish is shown as SEQ ID NO.1, the 178 th base in the sequence is T/G, the sequence amplified in male fish is shown as SEQ ID NO.2, and the 178 th base in the sequence is T/T.
Example 2:
preliminary verification of SNP locus accuracy by PCR product sequencing
In this example, 44 coilia ectenes samples (22 each) from the breeding group (with determined genetic sex) of fishery technologies limited in Zhenjiang city, jiangsu province were used for male and female identification.
Shearing the tail fin of coilia ectenes with scissors sterilized with alcohol to about 0.5X0.5 cm, and storing in anhydrous ethanol at 4deg.C. And extracting the genome DNA of the fin through an ammonium acetate method. The quality of the genomic DNA was detected by agarose gel electrophoresis at 1.0%, and the concentration was detected by an ultraviolet spectrophotometer (Eppendorf, AG2231 type), and stored at-20℃for further use.
The PCR amplification reaction system comprises:
Forward primer：gcacaaagttgcactgttcc
Reverse primer：atgcacaacacggatcttgc
the PCR amplification reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 30sec,35 cycles; extending at 72℃for 10min.
Results
The PCR products were subjected to 1.0% agarose gel electrophoresis and then sent to Sanger sequencing by Wohan's Biotechnology Co., ltd (Tsingke, wohan, china), and the SNP sites and peak patterns of the sequencing results were counted, and the results are shown in Table 2.
Table 2 statistical results of verification of SNP locus at position 178 in 44 coilia ectenes
Example 3:
PCR product sequencing and amplification sample verification SNP locus accuracy
In this example, 48 coilia ectenes samples (33 females, 15 males) from 3 populations (determined genetic sex) were used for sexing. Group 1 is a breeding group (15 female groups, 1 male group) of a source fishery science and technology limited company in Zhenjiang city, jiangsu province, and group 2 is a breeding group (4 female groups, 10 male groups) of a Wuhan Hengyu Xin science and technology special cooperation company in Wuhan city, hubei province; population 3 is wild population of wild Zhi lake (female 14, male 4) in Hubei province.
Shearing the tail fin of coilia ectenes with scissors sterilized with alcohol to about 0.5X0.5 cm, and storing in anhydrous ethanol at 4deg.C. And extracting the genome DNA of the fin through an ammonium acetate method. The quality of the genomic DNA was detected by agarose gel electrophoresis at 1.0%, and the concentration was detected by an ultraviolet spectrophotometer (Eppendorf, AG2231 type), and stored at-20℃for further use.
The PCR amplification reaction system comprises:
Forward primer：gcacaaagttgcactgttcc
Reverse primer：atgcacaacacggatcttgc
the PCR amplification reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 30sec,35 cycles; extending at 72℃for 10min.
(4) Results
The PCR products were subjected to 1.0% agarose gel electrophoresis and then sent to Sanger sequencing by Wohan's Biotechnology Co., ltd (Tsingke, wohan, china), and the SNP sites and peak patterns of the sequencing results were counted, and the results are shown in Table 3.
Table 3 statistical results of verification of SNP locus at position 178 in 48 coilia ectenes
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Sequence listing
<110> university of agriculture in China
ZHENJIANG JIANGZHIYUAN FISHERY TECHNOLOGY Co.,Ltd.
<120> SNP marker for identifying genetic sex of coilia ectenes, primer and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 390
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gcacaaagtt gcactgttcc tgtggcctac cttcaacaaa ctcaaaatga tgacaccgac 60
agaggtgtca gaggtgcatg tacatgtccg cacactcctg gaagaagaag atgaaggagg 120
agaagccacc actacaactt ttgaaaacga ggaggacgag gcgcacaccc gcaccctgtc 180
tccacctgca gcaaaacgaa aaaggagttc ttttttcgct gagtgggaaa acgtggatga 240
tgacgatgat gctcctgttc gagatgaggt taacctgtac attagtcaaa ggcatgccat 300
ggctgatgac cgcgatttgc ttggctggtg gagaataaac tccagtatat acccgaagct 360
tgccaagtta gcaagatccg tgttgtgcat 390
<210> 2
<211> 390
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gcacaaagtt gcactgttcc tgtggcctac cttcaacaaa ctcaaaatga tgacaccgac 60
agaggtgtca gaggtgcatg tacatgtccg cacactcctg gaagaagaag atgaaggagg 120
agaagccacc actacaactt ttgaaaacga ggaggacgag gcgcacaccc gcaccctttc 180
tccacctgca gcaaaacgaa aaaggagttc ttttttcgct gagtgggaaa acgtggatga 240
tgacgatgat gctcctgttc gagatgaggt taacctgtac attagtcaaa ggcatgccat 300
ggctgatgac cgcgatttgc ttggctggtg gagaataaac tccagtatat acccgaagct 360
tgccaagtta gcaagatccg tgttgtgcat 390
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gcacaaagtt gcactgttcc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atgcacaaca cggatcttgc 20
Claims (4)
1. Use of a reagent for detecting base 178 on the polynucleotide shown in SEQ ID No.1 for identifying the sex of coilia ectenes.
2. The application of the reagent for detecting 178 th base on the polynucleotide shown in SEQ ID NO.1 in coilia ectenes breeding.
3. The use according to claim 1, wherein the agent is a primer.
4. The use according to claim 3, wherein the primers are:
Forward primer：gcacaaagttgcactgttcc
Reverse primer：atgcacaacacggatcttgc。
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| CN102899321A (en) * | 2012-10-11 | 2013-01-30 | 中国水产科学研究院淡水渔业研究中心 | Method for selecting specific SRAP marking tapes for coilia ectenes genders and gender determination method |
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| CN102899321A (en) * | 2012-10-11 | 2013-01-30 | 中国水产科学研究院淡水渔业研究中心 | Method for selecting specific SRAP marking tapes for coilia ectenes genders and gender determination method |
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