A disease-resistant grass carp sex molecular marker and its development method and application

Abstract

The present invention discloses a molecular marker for the genetic sex of disease-resistant grass carp. Its DNA sequence is shown in Figure 1. The present invention provides two pairs of primers for identifying the genetic sex of males and females: Primer pair 1 comprises: forward primer 1: GCATACGGACAGTACAGGTGAAG; reverse primer 1: GAAACACAAGATGGTGCCAAATA; and primer pair 2 comprises: forward primer 2: TCAGTGACTGGGAGACTTGGATA; reverse primer 2: AATTTTCCTCTGAGCTTCACACC. The present invention has the following advantages over existing technologies: 1. The method for screening male-specific fragments is simple and widely applicable; 2. It provides an efficient technical approach for the parthenogenesis of disease-resistant grass carp; and 3. It can produce an all-female, disease-resistant grass carp population with multiple excellent traits, significantly improving aquaculture efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of disease-resistant grass carp sex molecular markers, and in particular to a disease-resistant grass carp sex molecular marker, a development method and an application thereof. Background Art

[0002] Grass carp, belonging to the genus Ctenophora in the family Cyprinidae, is one of China's four major freshwater aquaculture species. It is primarily found in the Yangtze, Pearl, and Heilongjiang rivers. In 2020, China's annual grass carp production reached 5.5711 million tons. Due to various factors, including genetic degradation and weak disease resistance, grass carp germplasm resources are seriously threatened. Therefore, a team led by Academician Liu Shaojun of the State Key Laboratory of Freshwater Fish Developmental Biology, a joint provincial and ministerial-funded project at Hunan Normal University, is using gynogenesis technology to develop disease-resistant grass carp with rapid growth and strong disease resistance. Disease-resistant grass carp are produced by backcrossing female grass carp with inactivated sperm from male koi. Breeding disease-resistant grass carp can significantly support the sustainable development of the aquaculture industry by improving breeding efficiency and reducing drug dependence.

[0003] Sex molecular markers are effective tools for distinguishing the genetic sex of fish based on the whole-genome identification of sex-determining regions. Sex determination is a relatively conservative trait, but the sex determination mechanism of fish is very diverse compared to mammals and birds, including genetic determination and environmental regulation. Some hermaphroditic fish, such as bluehead croaker and clownfish, can undergo bidirectional sex changes during their life cycle, but the sex of cyprinids is determined by genetics and the influence of environmental factors is limited. Therefore, the development of sex molecular markers for disease-resistant grass carp can stably and efficiently distinguish its genetic sex.

[0004] Controlling the sex ratio of fish is crucial in aquaculture. Certain fish, such as the yellow catfish, exhibit significant sexual dimorphism. Single-sex group rearing can improve aquaculture efficiency and control reproductive rates. Most fish grow rapidly before reaching sexual maturity. Because male grass carp mature earlier than females, males are typically smaller than females. Therefore, using sex markers combined with breeding techniques can create all-female, disease-resistant grass carp populations with a variety of desirable traits, significantly improving aquaculture profitability. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a disease-resistant grass carp sex molecular marker and its development method and application in view of the deficiencies raised in the above background technology.

[0006] To solve the above technical problems, the present invention provides a technical solution: a molecular marker for the genetic sex of disease-resistant grass carp, whose DNA sequence is shown in NO: 1, and two pairs of primers for identifying the genetic sex of males and females:

[0007] Primer pair 1 is:

[0008] Forward primer 1: GCATACGGACAGTACAGGTGAAG;

[0009] Reverse primer 1: GAAACACAAGATGGTGCCAAATA;

[0010] Primer pair 2 is:

[0011] Forward primer 2: TCAGTGACTGGGAGACTTGGATA;

[0012] Reverse primer 2: AATTTTCCTTGAGCTTCACACC.

[0013] A method for developing a molecular marker for the genetic sex of disease-resistant grass carp. The specific steps for developing the molecular marker for the genetic sex of disease-resistant grass carp are as follows:

[0014] Step 1: Determine the sex of 2-year-old disease-resistant grass carp by measuring the pectoral fin length and gonadal biopsy results. Select 5 healthy, normal-looking male and female individuals, cut the tail fin rays, and store them in a 1.5 ml EP tube filled with 95% ethanol.

[0015] Step 2: Genomic DNA was extracted using the Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech NO. B518251, China) according to the manufacturer's instructions;

[0016] Step 3: The concentration of genomic DNA was measured using a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE), and the genomic DNA was stored at −80°C for subsequent Illumina whole-genome resequencing.

[0017] Step 4: After obtaining whole-genome resequencing reads, male-specific fragments were identified through bioinformatics analysis. Specifically, the male and female resequencing reads were aligned to the male grass carp reference genome [GCA_029834665.1] using BWA mem 0.7.18 (r1243) to generate bam files. These bam files were then sorted, merged, filtered, and deduplicated using Samtools 1.18. The deduplicated bam files were then converted into a stacked file using PSASS 3.1.0. The total sequencing depth for males and females was calculated using a 1kb sliding window. Y-linked specific fragments were identified by screening for fragments with a sequencing depth greater than half of the total sequencing depth and for fragments with a sequencing depth less than 5 of the total sequencing depth. Male-specific primers were then designed based on these Y-linked specific fragments.

[0018] Step 5: Two pairs of male-specific primers were screened out, which had 353 and 463 bp bands in male disease-resistant grass carp, but no bands in females. The universality of the sex molecular marker was verified in two populations.

[0019] An application of molecular markers for genetic sex of disease-resistant grass carp, comprising the following specific steps:

[0020] Step 1: Select large, healthy and disease-free individuals from the disease-resistant grass carp pond;

[0021] Step 2: Screening out female disease-resistant grass carp using the constructed sex molecular markers;

[0022] Step 3, then artificial insemination is performed with the sex-reversed pseudo-male disease-resistant grass carp (XX);

[0023] Step 4: Prepare a population of all-female disease-resistant grass carp with strong disease resistance.

[0024] After adopting the above method and application, the present invention has the following advantages: 1. The invention uses bioinformatics methods to statistically analyze the differences in sequencing depth between male and female individuals, and can quickly screen male-specific Y-linked specific fragments. The method of screening male-specific fragments is simple to operate and widely applicable.

[0025] 2. The sex molecular marker of the present invention is dominant in male disease-resistant grass carp. The male-specific primers designed based on the molecular marker can stably and efficiently identify the genetic sex of disease-resistant grass carp. It can also identify the sex of a gynogenetic disease-resistant grass carp population. If it is an all-female population, it further confirms that the disease-resistant grass carp is female isogametic (XX), providing an efficient technical approach for the unisexual breeding of disease-resistant grass carp.

[0026] 3. The sex molecular marker of the present invention can identify the genetic sex of disease-resistant grass carp. Combined with disease resistance screening and sex reversal technology, an all-female disease-resistant grass carp population with multiple excellent traits can be prepared, which can significantly improve breeding efficiency and is of great significance in the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of molecular markers for the genetic sex of disease-resistant grass carp.

[0028] Figure 2 This is a schematic diagram of molecular markers for the genetic sex of disease-resistant grass carp. DETAILED DESCRIPTION

[0029] The present invention will be described in further detail below with reference to the accompanying drawings.

[0030] Combined with attachment Figure 1-2This study provides a molecular sex marker for disease-resistant grass carp, identifying all-female disease-resistant grass carp and improving breeding efficiency. Using whole-genome resequencing technology, the present invention identified DNA fragments specific to male disease-resistant grass carp through genome-wide association analysis (GWAS). Using sex-specific primers designed based on these male-specific fragments, PCR using these primers can effectively distinguish the genetic sex of disease-resistant grass carp. This molecular marker is universal across populations.

[0031] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0032] The present invention provides a molecular marker for the genetic sex of disease-resistant grass carp, the DNA sequence of which is shown in NO:1.

[0033] The present invention provides two pairs of primers for identifying the genetic sex of males and females:

[0034] Primer pair 1 is:

[0035] Forward primer 1: GCATACGGACAGTACAGGTGAAG;

[0036] Reverse primer 1: GAAACACAAGATGGTGCCAAATA;

[0037] Primer pair 2 is:

[0038] Forward primer 2: TCAGTGACTGGGAGACTTGGATA;

[0039] Reverse primer 2: AATTTTCCTTGAGCTTCACACC.

[0040] The above-mentioned sex-specific primers were amplified by PCR and subjected to nucleic acid electrophoresis. The results showed that there were specific bands in males and no bands in females. The specific steps for developing sex-specific molecular markers for disease-resistant grass carp are as follows:

[0041] Step 1: Determine the sex of 2-year-old disease-resistant grass carp by measuring the pectoral fin length and gonadal biopsy results. Select 5 healthy, normal-looking male and female individuals, cut the tail fin rays, and store them in a 1.5 ml EP tube filled with 95% ethanol.

[0042] Step 2: Genomic DNA was extracted using the Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech NO. B518251, China) according to the manufacturer's instructions;

[0043] Step 3: The concentration of genomic DNA was detected using a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE), and the genomic DNA was stored at −80°C for subsequent Illumina whole-genome resequencing.

[0044] Step 4: After obtaining the whole-genome resequencing reads, male-specific fragments were identified through bioinformatics analysis. Specifically, BWA mem 0.7.18 (r1243) was used to align the male and female resequencing reads with the male grass carp reference genome [GCA_029834665.1] to generate bam files. Samtools 1.18 was used to sort, merge, filter, and remove duplicates from the bam files. PSASS 3.1.0 was then used to generate a stacked file from the deduplicated bam files. The total sequencing depth of males and females was calculated using a 1kb sliding window. By screening male fragments with a sequencing depth greater than half of the total sequencing depth and female fragments with a sequencing depth less than 5, Y-linked specific fragments were identified, and male-specific primers were designed based on the Y-linked specific fragments.

[0045] Step 5: Two pairs of male-specific primers were screened out, which had 353 and 463 bp bands in male disease-resistant grass carp, but no bands in females. The universality of the sex molecular marker was verified in two populations.

[0046] The present invention provides an application of a sex molecular marker in preparing a disease-resistant grass carp population with multiple excellent traits. The specific steps are as follows:

[0047] Step 1: Select large, healthy and disease-free individuals from the disease-resistant grass carp pond;

[0048] Step 2: Screening out female disease-resistant grass carp using the constructed sex molecular markers;

[0049] Step 3: then artificially inseminating with sex-reversed pseudo-male disease-resistant grass carp (XX);

[0050] Step 4: Prepare a population of all-female disease-resistant grass carp with strong disease resistance.

[0051] The male-specific DNA sequence of disease-resistant grass carp is as follows:

[0052] DNA sequence length: 7052bp

[0053] AACAATTTTTAACAGCATGGGATTGTAAATAACACAATTTAAGTAAAAGTCAGTGAC

[0054] TGGGAGACTTGGATATTTTATTGAAAATCATGTACATTTGAAAAACAGCCACAGGTAAAT

[0055] TTCCAATCCAACATGGTTCTTACCTGGAGACTGTTGTGCAGAAAATGGCACAAGCATATT

[0056] AACAGAAGTATGTGGTCGTCAAAATTGCCATATCTGTAGCACTGTTTGATATACACTGAA

[0057] ATTCCTGCCAAAAGCAAGTAAAGGGGTTACAGTTTTTCTGTCACTGCTAAACTGTACAA

[0058] CCCTACTAAAATGAGTACATGAAGATATTAATAAGCCTACAAATGGAAAGGAAGACCCT

[0059] GATAATCATGGCCATCAAATGTTGAATTAATGACAAATTTAAAGAAACACAACAAACCGT

[0060] CAGGTTTTTGGCTGTTGAAGTGATGATCAAGGATTCAGTTAGTGGAGTTACCACTGAAC

[0061] TCAAAGGCTTCAGATGGTGTGAAGCTCAGAGGAAAATTATAGTGTTTCACACATCTGAG

[0062] GGGAAGACCGCAAGTAGTTTCTTTTTAACATAGATGTACCTAACCATCCGAGACAGATTC

[0063] TGGCCTTCGTGGTACTAGGCACCTTCCTCTTCCTCAAAACCTTCCACATCAATGGAAAAT

[0064] GTGTAATTCTCTCTGGTTTTCTGGAAGAGTGCCCTGTCCACTTCAAATAAGTGCCACTTT

[0065] GCAACATTTTTATTGTTTAAAGGGGTGATTATGATTTGACTTTTTTAACTTTTAGTTAGTGT

[0066] GTAATGTTGCTGTTTGAACATAAACAACATCTGCAAAGTTACAACGCTCAAAGTTCAATG

[0067] CAAAAAGAGATATTTTCTTTTACAGAATTCTGTTTAAGGACTACAACAAACGGCTGGTAG

[0068] GGACTACAACAAGCTTCTTCCTGGGTTAGTGACATCACTAAAGCTCTGGGTATATTTCGG

[0069] CTGTCCGCGTTTGTGCACTGTCCGCATGACGTAATTTTCCCATCCGCATGCATCCCAACTT

[0070] TCGAGACCGTGCGTACGGATGCGCGGGCGCGATTGCGCGCGAGATGTGTCCGGGCACG

[0071] GAAAGTGACGTATACCCGGGGCTTAACCCTAAAATTTACATAAACCCCATGTTGGGCTGC

[0072] TTTAGAGAAGAGAAAGAGTTGTTGTAGAGTGATGTTGTCATTTTACCACAACCTGTAAGT

[0073] ATGTTTTATTCATTGTTAATGTGTTTTCCATTGATAGTTTCTATAGTTATTTTTTATGTTGTA

[0074] GAAAAGACGTAAACAAAGGGCAATGGCTCCACTATCCAGTTACCTAAATGCTAATTAAA

[0075] ACTTATCTGACAAACACCTACAAACTGTAGTCTGCAGTCTGTAATCAAATAATCCCTTCA

[0076] AGGAATGTAGTATCATTTGTTAGAACAATTATATGAAATGCATGGATATATTTTGCATGTAT

[0077] GAAGCTTTGTTTCCATGGTCTTCTGATTAATATCCAGTAATTCAGTTTTTTTTTTTTTTTTT

[0078] GTAAAAATAGTCCAAGATTAAACTATGTTCCGTTAATAGACAACCACTGTTGTTACTGCT

[0079] ATAAATTGCAATGTTATATATAATGCAATCATGTATTATGCTGTAATAAAGCTTTATTAAGGAT

[0080] AAAACCGTATATTAAAACTTAACATAAACAGCAGCAGTATTTACAAATAACAGTATCTAA

[0081] AAAATATGATACAACTACAAACAAAAAAACATACCAATTTGAGAGATCCTGTAAGAGTC

[0082] GTGCTTGCACAGGTTCTGCTCGATCCTTTTCAATATTCTCTGGGTCTGAATCGAGCTCAA

[0083] ATTGATAAGGCAATATTGACGCCATTGTTTACAATACACCGGAGCACATAACTGTAATGAT

[0084] AAGGGTTGTGAAGTTATGGGAGCTTCACAACACATAGCCAATCAACCAATCTGAGCCC

[0085] TTTGTGTATTTCTGAGGGAGGGGCTTATAGAGGCACACATATCTTCAGGTCAGGACTGTA

[0086] ATAAAACATGTGAAGTTTGAGGCAGATCAGACATTGTATGCCCGAGTTACAACAACTTC

[0087] CTTTTCCTGGTGAAACATCAGACTTGGTTCAGGCTGCCACAGACACGTCCTTCAGTGAA

[0088] AACTCAAGATCTACTCAATTTAATGTCGCAAACCTTTAAATTAGACTGACCAAATATGAC

[0089] ATTGATCTGATTAAAGCTCTAGGAGGAGTTCGTTAAAGTACAACGCATGGAAATGGCAA

[0090] AAACTGCACAAATTTTGCAGAGAAAATTAAAATATCTCATTTCCTGTTGGGTTTTAAAT

[0091] TTTGCACCCAGGGGCTTTTTCGTAGGTATTGGGCTGCTACATGTGTGTACTGAATTTCATA

[0092] CTTGTATGTGAAAGGTAGAGCGAGTGGCGCTTTATTGAAATTTTGTAGGTGGTGCGTGTG

[0093] CAAAGTTTCATGAGTTTGAGTATATTTATGCTTCAGAAATGAGAGAAGAATTGACCGAAC

[0094] AATAACAATAGGGTCCTCACACCATCAGTGCTTGGGCTCTAAGTTTTTACGTTAACAAAC

[0095] ATTTCATGATTACAGGAGTCATATGAATGATACTTTTATGGTGCTTTTGTGTCCTTTTTAAA

[0096] GCATATTCACAATGCTTTGTGTTCCAGAGAGAAAGAAGGTCATACAGGTTTGGAACAACAT

[0097] GAAGCTTATTGAAACATTTGTTTGTTTTTCATTGCCTGCTTCATTCTCTCTCTCTCTCTCTCCT

[0098] CTCTCTCTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTCAGATGTGAACGAGTGTTCAGA

[0099] GGAACTAAATACTAAATGCCTTGTGAAAGGGACTGCAATGGTCTGGAATGCTTGATTCTG

[0100] ATTGTGTGACAGAGGGTGTTTATAAGTTGTTTGCACATTGCTGTGGTGCGAAATGCGGCTG

[0101] GAGGGCAGGGAGTGATTTTTCTGAATCGCTATTCACAAACTCAAAATTCCTATGTTTTGCG

[0102] TTGTTTATGGTTGCTCAGTAGAATTCCGTTTACAATGTTGATCTGGTTACTGTGAGAACA

[0103] GTGTCACGCTCTGTTGCTTCAGCCATCATGTGGTTGAAAATGTCCAAATAAAAAAGTGTT

[0104] CAACAAGTTGACAGAAGTCGATCCATTTTTTTTTTTTTTTTTGTTGGAAGGGTGTAAATG

[0105] TAACTGTAGTTTTAATGTTGTCTTTGTAAATATTCACTTTCCCATATGGCCTCGGAGGGGC

[0106] ATTCAGCAGACAGTCACCGACGCACTGTATTTTTGTATTTCAATAAATGACAACCAAAAT

[0107] CAAACCCATAGAAGAGACTTGAAACTTTGAGGGATGGTAGTACTCATACCGCCTACAAC

[0108] ATCACCAAGGCTTGCTCCGATCGGCCTGACAGGGGCGCTACAGCGGTCAAAAGTACGA

[0109] AATAGCTCATAACTCCTGAGCCATTAGGCCCAGGCTCAAGTGTCTTATATCCTTGGAATC

[0110] CTTGGCTCAAGGCGTAATTTTCGACAGTTTTCACCCGATCAAACCCAAACCAGTACAGA

[0111] AATGTTCTTTGGCATCTGAATATCAATAATTATAAAAAAAAGTTGAAATTTTGATTCACGG

[0112] TCGCTAAGGGGCGCCAAAACGTACGATGAGGGTAGAGCCACTTTTGCTAAAAATGTCTA

[0113] TAACTCGAGAACAATTGAGATATTTGCACCAAACTCGGTACACATGTGTATGGGCTCAA

[0114] TCTTTGGTCACGATAAAATAATCATGTCGATTGGCCACTAGTTGGCGCTATAACTTGAAA

[0115] AAACATGAAAACAGCTCTAACTATGCAACCGTTAGTCCGATCGACTTGAAATTTGGCATG

[0116] CAGTGTCTTGGTCCAAGGGGGCATGATGGTCTATGAGGACATTGGTGTATCTCAAAAAA

[0117] CATGGCCGCCACTGGCCAATGAAATTTGAGCACCTCTTAGACAAGGTCAGTGGAGGCCA

[0118] ATCAGAATGAAACTGGTGGGCATGTTCGACTCATGGTCCTAGAGGTCTGTAAGAATTTTG

[0119] AAAGAAATCGGCCACAAGTTGGTGCTAACGAGTTTTTACACCTCAGTAATCACGTGGTGT

[0120] TTCACAGGATCACAAAATATGCATATCATTTGATAGATCTCCTCATGCTGAACAACTTTGC

[0121] CTCAAGAACTAATGCTGTCAATTAAACTGTTCATTAATTATTTGAGAATATTTGAAAAACA

[0122] TACTTTTGCRAACTAGTCCTAGGTTTTTCGCCCAATCGGAACGAAACCAGTGCAGGACA

[0123] ATTCTCTGGACTCTCTAGATCAATAATTATCAACAAAAGTTGAACTTTATCATTTGAGTCG

[0124] TTATAACTGGGCCATTTAGAAAATGGGCATGGCTAAATATACCCAAAAGCCTATAAAACC

[0125] TAAACAAAAACTCAGAACTTCACGAAAATAGGTGAGCAGATGCAAGATATAAATCTAAA

[0126] GAAGCACACCAATTTTTGTGCAGATCGGACCATAGTTGGCGCTATAACTGTTAAAAAAA

[0127] ACTTTAAAAAACATAAGTTTTTAACAGTTTTGGATGAAAATGTGTGTAATTTTGTGTGTG

[0128] GTTAACTTATTTTCACGGGAGTCTTGCCAGGTTACGGCTTATGATTTGTGATACGTTGTGA

[0129] TTTAGTGCTTTAAAAACTTTTGCGAATGTCATCGAAACCGTTAGTCCGATCGAGACAAAA

[0130] CCACCGTAGGAAACACGGAACCTAAGTTGATTAATGTCAAAATTTTGATAGGAAGTGGC

[0131] AAAAAAATCCAATCAAAGATGTTCTTAAAGTTACAATATGTAATAATTTTGGTGCTAGAG

[0132] GTCAATACAAAGGCGTAGCTTGATGACGCCAAGATTTAGAGCGGAATCTTGGGACATGT

[0133] GGTCTCCATCTCAATGGTAGGTGGATAATAACAGGGATAGGACTCGGGAAGAAATCATG

[0134] CTCATGGATGCGATTATTAATGTTATTGTAGTATGAAGCAGAGCAGGACCGAGTGTTGTG

[0135] GGAGCTGAACGAGGCCGCTGGAGCGATTGCGCAACACACGCCTCAAAAGCAGCGGGA

[0136] CCTTTATTATGACACAGTCGCCGGCGCTGCTTCCGCTTTTCCGGTCATGAGTATGAGGTA

[0137] ACGTAGCTCTGTTTATCATATTAGATACATTTGAGAGTGTTGAAAATGATGTTATAACGTT

[0138] ACTCTGTGCATTCGTTCGGCGGCTGCTGTGAGACACTGTTACACACTGCAGTAAGATCTT

[0139] TAGAATATCATATTAAATGCTGGATGGCTTGAGTTGATAAATGACATGCAATTCATTTTAA

[0140] AACGTATTGTATGATGGAGAAAATGCTGTATTACTGTTACTAAAAATAAAGCTGCATCTGA

[0141] TTATGCTATGTTAACTACTTGACAAAATAGTGTTTTTCTCTGAGGCATGGTAAAGCATAGT

[0142] ACTCGCAAAAAATCAAGAAAATTAGATTTAAACAATAAGACTAAACGTGTTGAGCTATAT

[0143] AACAACAATTAGTTTTTCTGTCTATAAATATATCAAAACAGTTGTTCCCTTGTCTATTAAAC

[0144] ATGTAAATATTAAAGTGTCTTTGGTGTTTCCATGGTTTCTACAAAACAAAACCTGAACCC

[0145] AAGGGTAACGCGGGTATGACGCCATTGACAGGCGACTCCTTGCAATTTTCTCACGATTTA

[0146] CAAATAGTTGGAAACATTTGGGTACTCAAGTGAACAAAATATATAACACTGGCCTAGTGG

[0147] GTTTTTTTTAAATATTTTACTGCAGAAATATTACATATTGCACCTTTAAGTCGTTTTTTTTA

[0148] AATGTACGTCAATGAGCTAGAACTTTAAGTGACATATCAACAAACCTCATAAAACAGTTT

[0149] GGAACAACATTAGGGTGATTAAATGATGACATAATTTGAATATTTTTTAATCTTTTCTCTTA

[0150] CTCCCATTCTGAACCAGCTGCAGTTACACAGGAAAATGTACTTTTAGATATAATTGCAC

[0151] TGATTTGTCAAAGGGCCTTGGTCTCTTGATAATTGAAGCAAAAACACTGAATTATCTCTT

[0152] TATGGGACAATCTAAATGTTTATGTCTGGTTGGCACACATTAAGAGAAGATTCTGGGAAG

[0153] AGAAAAGGGAGTAGAACACCTGCACCCCCCCCCCCCCATCTCTCTCACCCTCCCTCACTT

[0154] CAAATCTGAGCTAGTACAGTGAAACTAACCACGGTCGACCCATCTTCAATGTTTCATCTA

[0155] AAAATCATTTTGCATAAAGTCTGGAGAAGTTTGACAATTAATGAGTTATCAGAGGATCAA

[0156] ATAAACATCTGTGGTGAAGTCAATGATGGACGATAAAGAGCTAAAGAAGAAGAAGAA

[0157] AAGGCAGCAAGTGGAACGTTTCTGTGTTTTTAGATCTTTTTTATTTAATTTGATAATTATAT

[0158] GATCATGAAGGATTCATGTTGTTTTCATTAATATTTTATATCAGGGATTCCCTATCCTGCTC

[0159] CTGGAGATCTATCTTCCTGCAGAGCATATCAACCCTGATATTTCATATACTGCTGGTCATG

[0160] AAACACTGTGCTTTTTCCATGTGAGTTGGTTAACTCCAAATTAACATACAATGTTCAAAA

[0161] AATTCCTCAGTCTTCATATTCTGCCATGTCTCATCAGTTGTATCTGGTCAGCAGTTTGGAT

[0162] GGATGCATACGGACAGTACAGGTGAAGGAATAAAATCAGTCAATTCATAAAATAAACAC

[0163] AATCTAATGTGATTCTTAATTCATTGTCATGTAAAACATCTGGGATCTGAACACAAGTTTC

[0164] TCCATGACTTCACGTTAAGGGTCTTTATATTCCACACTATTTATAAAGTATCAAAGTACAG

[0165] AGGAAATCCATTAGATAATACAGCGTTCAGAGGTCAAATACTTCTGAATAATGACAATAA

[0166] CTGCATCTGTACAATAGACTGCTGCAACTTTCAGCTCTCTTGAATTATTCTGTGGTCTCAC

[0167] TCTCACTTTACACAAACACAAGTTAGGAAAAATATTTGGCACCATCTTGTGTTTCAGACT

[0168] AGCAGTTACAGAGGTTGCAGAGCTAATGATGTGATACTTCAATAAGCAAATTATTACTGT

[0169] AATCAAAAATATGCAATGCTTTATTAAACAGTTTTATATGTTACATGCAGTTACTATAGTAT

[0170] TAATTATACATTATGCATAATTGTA

[0171] The above description of the present invention and its embodiments is non-limiting, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the invention, without creatively designing a structure and embodiment similar to the technical solution, they shall fall within the scope of protection of the present invention.

Claims

1. The use of two pairs of primers for identifying the genetic sex of male and female in identifying the genetic sex of disease-resistant grass carp is characterized by: The disease-resistant grass carp is produced by inducing gynogenetic offspring produced by female grass carp with inactivated sperm of male koi, and then backcrossing with male grass carp. The primers are primer pair 1 and primer pair 2; Primer pair 1 is: Forward primer 1: GCATACGGACAGTACAGGTGAAG; Reverse primer 1: GAAACACAAGATGGTGCCAAATA; Primer pair 2 is: Forward primer 2: TCAGTGACTGGGAGACTTGGATA; Reverse primer 2: AATTTTCCTTGAGCTTCACACC.

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