A SNP molecular marker, amplification primer and method for rapid identification of Jian carp varieties

Abstract

The invention discloses a SNP molecular marker, an amplification primer and a method for quickly identifying Jian carp varieties. The SNP molecular marker includes SNP 1 to 5; the physical position of SNP 1 is located at the 27079154th base of Chr17 chromosome, and the base is C or T; the physical position of SNP 2 is located at the 27632650th base of Chr17 chromosome, and the base is A or G; the physical position of SNP 3 is located at the 1441950th base of Chr18 chromosome, and the base is T or C; the physical position of SNP 4 is located at the 5857377th base of Chr18 chromosome, and the base is G or C; The physical position of 5 is located at the 23058224th base of the Chr14 chromosome, and its base is C or G; the SNP molecular marker of the present invention can quickly, accurately and batch identify and screen Jian carp seedlings and parents in the seedling stage and parent stage, and is particularly suitable for variety identification of small-sized fry, solving the problem of difficulty in distinguishing varieties with similar appearance.

Description

Technical Field

[0001] The invention relates to the technical field of Jian carp variety identification, and in particular to a SNP molecular marker, an amplification primer and a method for rapidly identifying Jian carp varieties. Background Art

[0002] Common carp (Cyprius carpio), belonging to the order Cypriniformes and the family Cyprinidae, is an important freshwater farmed fish in China. Its annual output accounts for about 11.3% of my country's freshwater farmed fish. In addition, it has formed a number of distinctive and economically valuable varieties through long-term artificial cultivation, and has accumulated rich carp germplasm resources. Jian carp is a species in the subfamily Cyprinidae and the genus Cyprinidae. It is also the leading variety in the carp farming consumer market. It has the advantages of fast growth, adaptability to a variety of farming methods, strong disease resistance, high meat content, and delicious meat. However, in the practice of introduction and breeding of aquaculture, it is difficult to accurately distinguish the varieties of fry and broodstock of similar-looking carp varieties by appearance, resulting in the phenomenon of gradual introgression and mixing of carp germplasm from different sources, which affects the conservation and breeding of Jian carp, as well as the interests of farmers and consumers.

[0003] At present, there are patents related to the identification of carp germplasm resources. For example, patent No. 202311302944.X discloses the application of a SNP molecular marker combination in the identification of 16 carp varieties, and uses 144 SNP loci to identify 16 carp varieties using a random forest prediction method. Patent No. 201610945256.9 discloses a method for identifying carp germplasm resources, which can be used to identify Songpu mirror carp, Songpu red mirror carp and easy-to-catch carp. Patent No. 201510315761.0 discloses a SNP marker combination, method and use for carp germplasm identification, and uses 48 SNP molecular markers to identify the relationship of Yellow River carp. The existing authorized patents have the following characteristics: (1) The number of molecular markers is large, the detection cost is high and the detection cycle is long, which makes it difficult to conduct mass detection; (2) It is applied to the identification of family relationships within a certain variety; (3) Some carp varieties, such as purse red carp, glass red carp, red and white koi, can be distinguished by their body color and body shape. Therefore, it is urgent to develop a new method with a short development cycle, low cost, simple operation, and the ability to identify Jian carp and similar varieties in batches. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP molecular marker, amplification primer and method for quickly identifying Jian carp varieties. The SNP molecular marker of the present invention can quickly, accurately and batch identify and screen Jian carp seedlings and parents in the seedling stage and parent stage, and is particularly suitable for variety identification of small-sized fry, solving the problem that varieties with similar appearances are difficult to distinguish.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0006] The first aspect of the present invention provides a SNP molecular marker for rapid identification of Jian carp varieties, wherein the SNP molecular marker includes SNP 1 to 5;

[0007] The physical position of SNP 1 is located at base 27079154 of chromosome Chr17, and its base is C or T;

[0008] The physical location of SNP 2 is at base 27632650 of chromosome Chr17, and its base is A or G;

[0009] The physical position of SNP 3 is located at base 1441950 of chromosome Chr18, and its base is T or C;

[0010] The physical position of SNP 4 is located at base 5857377 of chromosome Chr18, and its base is G or C;

[0011] The physical position of SNP 5 is located at base 23058224 of chromosome Chr14, and its base is C or G.

[0012] Preferably, when the genotypes corresponding to the SNP 1 to 5 sites are TT, GG, CC, CC and GG respectively, the carp is identified as Jian carp.

[0013] The second aspect of the present invention provides an amplification primer for rapid identification of SNP molecular markers of Jian carp varieties, the amplification primer comprising:

[0014] The upstream and downstream primer sequences used for amplification of the SNP 1 site are shown in SEQ ID NOs. 6 to 7;

[0015] The upstream and downstream primer sequences used for amplification of the SNP 2 site are shown in SEQ ID NOs. 8 to 9;

[0016] The upstream and downstream primer sequences used for amplification of the SNP 3 site are shown in SEQ ID NOs. 10 to 11;

[0017] The upstream and downstream primer sequences used for amplification of the SNP 4 site are shown in SEQ ID NOs. 12 to 13;

[0018] The upstream and downstream primer sequences used for amplifying the SNP 5 site are shown in SEQ ID NOs. 14-15.

[0019] The third aspect of the present invention provides an application of a SNP molecular marker for rapid identification of Jian carp varieties in the identification and / or screening of Jian carp varieties.

[0020] A fourth aspect of the present invention provides an application of the above-mentioned amplification primers in the identification and / or screening of Jian carp varieties.

[0021] A fifth aspect of the present invention provides a method for rapidly identifying and / or screening Jian carp varieties, comprising the following steps:

[0022] (a) taking samples of carp to be identified;

[0023] (b) extracting sample DNA as a template, performing PCR amplification on SNPs 1 to 5 respectively; sequencing the PCR amplification products to determine the genotype of SNPs 1 to 5 in each individual;

[0024] (c) If the genotypes corresponding to SNPs 1 to 5 are TT, GG, CC, CC and GG respectively, the fish is confirmed to be Jianli.

[0025] Preferably, the product sequence of PCR amplification of the SNP 1 site is as shown in SEQ ID NO.1, and the upstream and downstream primer sequences used are as shown in SEQ ID NO.6-7, and the SNP 1 site is located at the 179th position of the nucleotide sequence shown in SEQ ID NO.1;

[0026] The product sequence of PCR amplification of the SNP 2 site is shown in SEQ ID NO.2, and the upstream and downstream primer sequences used are shown in SEQ ID NO.8-9, and the SNP 2 site is located at the 174th position of the nucleotide sequence shown in SEQ ID NO.2;

[0027] The product sequence of PCR amplification of the SNP 3 site is shown in SEQ ID NO.3, and the upstream and downstream primer sequences used are shown in SEQ ID NO.10-11, and the SNP 3 site is located at the 185th position of the nucleotide sequence shown in SEQ ID NO.3;

[0028] The product sequence of PCR amplification of the SNP 4 site is shown in SEQ ID NO.4, and the upstream and downstream primer sequences used are shown in SEQ ID NO.12-13, and the SNP 4 site is located at the 168th position of the nucleotide sequence shown in SEQ ID NO.4;

[0029] The product sequence of PCR amplification of the SNP 5 site is shown as SEQ ID NO.5, and the upstream and downstream primer sequences used are shown as SEQ ID NOs.14-15. The SNP 5 site is located at the 305th position of the nucleotide sequence shown in SEQ ID NO.5.

[0030] Preferably, the sampling of the carp to be identified comprises:

[0031] For fry with a total length of ≤5cm, whole tissue sampling is carried out; for fry with a total length of >5cm, fin sampling is carried out; for adult fish, fin sampling is carried out.

[0032] Compared with the prior art, the beneficial effects of the present invention include at least:

[0033] The SNP molecular marker combination of the present invention can accurately and efficiently identify Jian carp individuals, and can be used for variety identification and screening in the seed stage of Jian carp, as well as for parent screening in the adult stage of Jian carp. It is especially suitable for variety identification of small-sized fry, and can effectively solve the problem that carp varieties with similar body shape and color, such as Yellow River carp, easy-to-catch carp, and Songpu carp, are difficult to distinguish based on appearance, thereby achieving rapid and accurate variety identification.

[0034] The present invention provides a SNP molecular marker for rapidly identifying Jian carp varieties and its corresponding primers. By detecting the SNP molecular marker, Jian carp fry and parents can be rapidly, accurately and batch-wise identified and screened in the fry and parent stages. The SNP molecular marker is particularly suitable for variety identification of small-sized fry, solving the problem of difficulty in distinguishing varieties with similar appearances, improving the quality of introduced breeding, and ensuring the interests of breeders and consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0036] Figure 1 The genotyping diagram of the SNP1 site Chr17:27079154C>T marker provided in the embodiment of the present invention;

[0037] Figure 2 The genotyping diagram of the SNP2 site Chr17:27632650A>G marker provided in the embodiment of the present invention;

[0038] Figure 3 The genotyping diagram of the SNP3 site Chr18:1441950T>C marker provided in the embodiment of the present invention;

[0039] Figure 4 The genotyping diagram of the SNP4 site Chr18:5857377G>C marker provided in the embodiment of the present invention;

[0040] Figure 5 This is the genotyping diagram of the SNP5 site Chr14:23058224C>G marker provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0041] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0042] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0043] The embodiment of the present invention provides a SNP molecular marker for rapid identification of Jian carp varieties, the SNP molecular marker comprising SNP 1 to 5;

[0044] The physical position of SNP 1 is located at base 27079154 of chromosome Chr17, and its base is C or T;

[0045] The physical location of SNP 2 is at base 27632650 of chromosome Chr17, and its base is A or G;

[0046] The physical position of SNP 3 is located at base 1441950 of chromosome Chr18, and its base is T or C;

[0047] The physical position of SNP 4 is located at base 5857377 of chromosome Chr18, and its base is G or C;

[0048] The physical position of SNP 5 is located at base 23058224 of chromosome Chr14, and its base is C or G.

[0049] In one embodiment, when the genotypes corresponding to the SNP 1 to 5 sites are TT, GG, CC, CC and GG respectively, the fish is identified as Jian carp.

[0050] Another embodiment of the present invention provides an amplification primer for rapid identification of SNP molecular markers of Jian carp varieties, the amplification primer comprising:

[0051] The upstream and downstream primer sequences used for amplifying the SNP 1 site are shown in SEQ ID NOs. 6 to 7, specifically 5'-TTCATGTGGTTTCTGGGCGT-3' and 5'-GTTGATCCGTCAGGAGCACA-3'.

[0052] The upstream and downstream primer sequences used for amplification of the SNP 2 site are shown in SEQ ID NOs. 8 to 9, specifically 5'-TAGTGAGCAGTGTTTGGCCT-3' and 5'-CTTACCAGCTGCTAGAGGGC-3';

[0053] The upstream and downstream primer sequences used for amplification of the SNP 3 site are shown in SEQ ID NOs. 10 to 11, specifically 5'-TCCAATGAAGTGAACACCGCT-3' and 5'-CTGTATGCCATGCGGAATGC-3';

[0054] The upstream and downstream primer sequences used for amplification of the SNP 4 site are shown in SEQ ID NOs. 12 to 13, specifically 5'-CCGTGACGTTAAAACCGAGG-3' and 5'-CAGTGGGCAGCCATTTATGC-3';

[0055] The upstream and downstream primer sequences used for amplifying the SNP 5 site are shown in SEQ ID NOs. 14-15, specifically 5'-TGACTGTGCGTTCCTCTCAG-3' and 5'-ATGCAAAAGTCTTTGTTGCCA-3'.

[0056] Still another embodiment of the present invention provides an application of a SNP molecular marker for rapid identification of Jian carp varieties in the identification and / or screening of Jian carp varieties.

[0057] Another embodiment of the present invention provides an application of the above-mentioned amplification primers in the identification and / or screening of Jian carp varieties.

[0058] Another embodiment of the present invention provides a method for rapidly identifying and / or screening Jian carp varieties, comprising the following steps:

[0059] (a) taking samples of carp to be identified;

[0060] (b) extracting sample DNA as a template, performing PCR amplification on SNPs 1 to 5 respectively; sequencing the PCR amplification products to determine the genotype of SNPs 1 to 5 in each individual;

[0061] (c) If the genotypes corresponding to SNPs 1 to 5 are TT, GG, CC, CC and GG respectively, the fish is confirmed to be Jianli.

[0062] In one embodiment, the product sequence of PCR amplification of the SNP 1 site is shown in SEQ ID NO.1, and the upstream and downstream primer sequences used are shown in SEQ ID NO.6-7, and the SNP 1 site is located at the 179th position of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0063] AAAATGGGTGTTCATGTGGTTTCTGGGCGTCCCAGCGCTGACAGGGGAT

[0064] TCCAGAGGTCGTTTCATTGACTTTGCCCCTGTAATCCTCTCCACGACCCC

[0065] GAAAACAATTAGTGGTGTAGCCGGAGCGAAAACGGCGCTTGGGAGACTCAGCTATAGGATGGAAAGAAAGAAGGCAAGTY(Y=C / T)AAAGAGGAAGA AATTCATGCACTAAAGAGATCTCACTGATTGATCCTTCTTATCGCAGTGTCAATGTAGAGCTGTAACTAGTTGTAAATATGGCTGTATATTGGACAGATGCAGTAATTTGTTGTCCAGTCATCTGTCAATTACAGAAAATAACAGATTTCAGCAACTCCACTTTAGTACCGTTTATATTTTAAAACTCAGCTGTGTGAATGTCACACATGTGCTCCTGACGGATCAAC;

[0066] The product sequence of PCR amplification of the SNP 2 site is shown in SEQ ID NO.2, and the upstream and downstream primer sequences used are shown in SEQ ID NOs.8-9. The SNP 2 site is located at the 174th position of the nucleotide sequence shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.2 is as follows: TAGTGAGCAGTTGTTTGGCCTTAGTAACTGGTTTTGTTGGTCTTCTATAGCTGGTTATATGTTTGGACATGTTTTGGATGAATGGCTTTGGACTTGATTTTTATGCACTTTTTCAGTTTCGAGTATGACATTATTAAAAAGCTTTCTAAATATCACTGTCAATGATTTATTTCCR(R=A / G)ATGCACCCCTAAGAATAAAGGCTCT TCAGTGGTTCTTTAAACCAGACAATTTCCACTATTTGAGTTGTTAATGCTAATGATCACTGATTAATACATATGCATTATCCAAAGAGACTTACATTGTGTTCCCATGTAAATTGTATCAGTTCTCTCTGGGAATCAAACCCATGACCTTGACGTTGCT AGGTCCATGCTGTATTGTTTGAGCAACAGGAAAGCTATTAAATGAATTTAGATCAATATAGTTCATAGTGTATAAGTAAATACGTGTAGATCATTTATTTTAATACAATTTCTTACAAAGTTCTAGCTGGTAAACACTGCCCTCTAGCAGCTGGTAAG;

[0067] The product sequence of PCR amplification of the SNP 3 site is shown in SEQ ID NO.3, and the upstream and downstream primer sequences used are shown in SEQ ID NOs.10-11, and the SNP 3 site is located at the 185th position of the nucleotide sequence shown in SEQ ID NO.3; the nucleotide sequence shown in SEQ ID NO.3 is as follows: TCCAATGAAGTGAACACCGCTATTCCTATCGTACTCATTATGGGAAAAATATTATCGGCACATCCTGACTTTTAAGTATAGTGATAGTGTATTTGGAAAGAGTTTACATGAGATCCGTACCGATGCATGTCGCGGCTTGCCGTCCAAAATGGACGGTGTCCAGACAGACGGTGCAGCGGGCCGCY(Y=T / C)CTCATGTTGA GCCCCTGCGTGAAGCGGTGAGGAGTGTTGTGATGAATCCTGTCCTTCACACGTCTGGTGTACTCTACAGGATCAAACAGACAGACACACTCTCTTAAAGACAGTCTTGCGTCTCTTACACCCGTCTAGATATCTTATGCAAACAGGCCAAAGCA AGCTGAAAATGTCCTCTCCTGAGAACATTTCTCCACCAGAGTGTGGAACCAGCTCGGAGAACACCAGCAGCACTGTACTGTCCAAGCTGTCATTGGTAGCTTGGGTACGAGCATGCAAGGATTTCGGAGTTCTGGCATTCCGCATGGCATACAG;

[0068] The product sequence of PCR amplification of the SNP 4 site is shown in SEQ ID NO.4, and the upstream and downstream primer sequences used are shown in SEQ ID NOs.12-13. The SNP 4 site is located at the 168th position of the nucleotide sequence shown in SEQ ID NO.4; the nucleotide sequence shown in SEQ ID NO.4 is as follows: CCGTGACGTTAAAACCGAGGTATATACCGAACATCATGTTTGTGTACCGTTACACCCCTAGTAAATGATGACAGAATTTTCATTTTTTTGGGTGAACGATCCCTTTAATTATGACTCATGACTTATGTTAACTTGGTTACTGATGGTGAATCAAGACTTAATAAGTAS(S=G / C)TCTGGTTTTAAGGAACATAACCATAATAG GGGGCAGTCGTGGCCTAACGGTTAAAGTGTTGGACTTGCAATCCAAGGGTTGCAAGTTCAAGTCTCGGGCCGGCAGGAATTGTAGGTGGGGGGAGTGAATGTACAGTGCTCTCTCCATCTTCAATACCACGACTGAGGGGCCCTTGAGCAAGGCACCGAACCCCCAACTGCTCCCCGGGCACCGCAGCATAAATGGCTGCCCACTG;

[0069] The product sequence of the PCR amplification of the SNP 5 site is shown in SEQ ID NO.5, and the upstream and downstream primer sequences used are shown in SEQ ID NO.14-15. The SNP 5 site is located at the 305th position of the nucleotide sequence shown in SEQ ID NO.5; The specific nucleotide sequence shown in NO.5 is as follows: TGACTGTGCGTTCCTCTCAGTGCATGTCACCTCTAATCTCTCACTCCTTTTCTGTTCTCCTCTCTTCTACCCTTTTGTTATTTTATACATATGCCAACAGCAGATTGTTCCATTGGTTGAAATTGCTGTTTGACAATTACCAGAATAGTCGAGAGACTTAAGCATTTAT TCAAAGAAGTACTGCACCTCAATTAATTTTAAATTTCAGTCATTTGAGTTTTCAAATAGGAGATTTTGACTTATTGTGTAGTGTTTTTAATATGTTGGAAAACCATTTATAACACAAGTGTGTATTGTAATATGS(S=C / G)TTTTGATGTGAAATATAATTGAAATTTGCGATGTTACAACATGATTA GTTTTTTCATTTGTTAAATTGTAGCATGCATATACTACTGTTCAAAAGTTTGGAGTCAGTATTTT.

[0070] In one embodiment, the sampling of the carp to be identified comprises:

[0071] For fry with a total length of ≤5cm, whole tissue sampling is carried out; for fry with a total length of >5cm, fin sampling is carried out; for adult fish, fin sampling is carried out.

[0072] The technical solution of the present invention is further described in detail below through specific embodiments.

[0073] Example 1

[0074] This embodiment is a method for screening and identifying SNP molecular markers specific to Jian carp species, comprising the following steps:

[0075] (1) Carp individuals were collected from the Fangshan Scientific Experimental Base of the Chinese Academy of Fishery Sciences, including 60 Yellow River carp, 60 Jian carp, 60 Songpu carp, and 60 Yi Chou carp. Fin ray tissues of 240 carp were sampled, temporarily placed in dry ice, and stored at -80°C for future use.

[0076] (2) The whole genome DNA of the fin ray tissues of 240 carp in step (1) was extracted using the phenol-chloroform extraction method, and the extracted whole genome DNA samples were then sent to Beijing Berry Genomics for DNA library construction and whole genome resequencing.

[0077] (3) Trimmomatic-0.39 software was used to perform quality control on the raw resequencing data to obtain clean data for each sample.

[0078] (4) The clean data was aligned to the carp reference genome (the reference genome version was GCA_905221575.1) using the MEM algorithm of the BWA software to obtain an intermediate result file in the sam format; the sam file was converted into a bam file using the samtools software and the results were merged, duplicate sequences were removed, and sorting was performed.

[0079] (5) GATK software was used to detect and type SNP sites in the bam file and generate a vcf file. The sites in the vcf file were quality controlled using vcftools software to obtain high-quality SNP sites that met the screening criteria. The quality control conditions were: minor allele frequency MAF>0.05; P-value in the Hardy-Weinberg test>0.01; site missing rate<0.1; polymorphic information content PIC>0.2; and population site heterozygosity rate<0.2. The core SNP sites were obtained after quality control.

[0080] (6) Based on the genotype differences of core SNP loci in different carp varieties, vcftools software was used to further screen for Jian carp breed-specific polymorphic loci. The screening criteria were: the homozygous mutation rate of polymorphic loci in Jian carp = 1, and the homozygous mutation rate of polymorphic loci in other carp varieties < 0.1. Five SNP loci with significant differences among varieties were screened (as shown in Table 1), namely SNP1 (C / T), SNP 2 (A / G), SNP 3 (T / C), SNP 4 (G / C) and SNP 5 (C / G). The genotyping results of the above five SNP molecular marker loci are shown in Figure 1. Figures 1 to 5 As shown;

[0081] Table 1 The combination information of homozygous SNP loci specific to Jian carp

[0082] SNP loci chromosome Position (bp) Mutation site (ref / alt) SNP1 Chr17 27079154 C / T SNP2 Chr17 27632650 A / G SNP3 Chr18 1441950 T / C SNP4 Chr18 5857377 G / C SNP5 Chr14 23058224 C / G

[0083] The upstream and downstream sequences of the five selected Jian carp-specific homozygous mutation SNP1-5 sites are shown in SEQ ID NOs. 1-5, respectively. Corresponding primers were developed to verify the reliability of the sites in the Jian carp population. The primer sequences are shown in SEQ ID NOs. 6-15.

[0084] (7) 16 carps of Jian carp, Songpu carp, Yellow River carp and Yi Chou carp were selected, and genotyping of the five SNP sites developed was performed using Sanger sequencing; specifically, the following steps were included:

[0085] (a) extracting genomic DNA of carp to be tested;

[0086] (b) using the DNA obtained in step (a) as a template and the nucleotide sequences shown in SEQ ID NOs. 6-15 as primers, PCR amplification was performed on SNP1-5 respectively to obtain PCR amplification products;

[0087] (c) performing Sanger sequencing on the PCR amplification product obtained in step (b) to determine the genotypes of the five SNP sites of the carp to be tested, as shown in Table 2;

[0088] Table 2 Genotypes and species of carp individuals detected at SNP1-5 loci

[0089]

[0090]

[0091] (d) The genotype frequencies of the five SNP loci in Jian carp, Songpu carp, Yellow River carp, and Yi Chou carp were calculated, as shown in Table 3;

[0092] Table 3 Genotype frequencies of SNP1-5 in four carp breeds

[0093]

[0094]

[0095] (e) Combining the sequencing typing results and carp species, it was found that the Jian carp had homozygous mutations at the five SNP sites, that is, the genotyping results of the SNP1 site were all TT, the genotyping results of the SNP 2 site were all GG, the genotyping results of the SNP 3 site were all CC, the genotyping results of the SNP 4 site were all CC, and the genotyping results of the SNP 5 site were all GG; the genotyping results of the Songpu carp at the SNP1-5 sites were 6.25%, 6.25%, 0, 6.25%, 6.25%; the genotyping results of the Yellow River carp at the SNP1-5 sites were 0, 0, 0, 6.25%, 0; the genotyping results of the Easy Carp at the SNP1-5 sites were 12.5%, 6.25%, 0, 6.25%, 0. When the genotyping results of SNP1-5 sites were all homozygous mutations, the proportion of carp identified as Jian carp was 100%; among all carp identified as non-Jian carp, the largest number of homozygous mutation sites detected in a single individual was 2 sites with homozygous mutations. This verification result is consistent with the data analysis results in Example 1, indicating that the developed SNP sites can effectively identify Jian carp in four carp varieties.

[0096] Example 2

[0097] This embodiment is a method for molecular identification, screening and parent selection of Jian carp seedlings using species-specific SNP molecular markers for identifying Jian carp, and the specific steps include:

[0098] (1) Randomly select 50-100 carps to be tested and adopt different sampling strategies for sampling, specifically: for small fry (total length ≤ 5 cm), whole tissue sampling is performed for the identification of the fry of the group; for large fry (total length > 5 cm), fin rays are collected for the species identification of the fry; for adult carps, fin rays are collected for parent selection;

[0099] (2) The sample DNA was extracted by phenol-chloroform extraction method as a template, and the nucleotide sequences shown in SEQ ID NO. 6-15 were used as primers to perform PCR amplification to obtain PCR amplification products; the PCR amplification products were subjected to Sanger sequencing to determine the genotypes of the five SNP sites in each individual;

[0100] (3) When the genotyping results of the sample show that the genotype of the SNP1 site is TT, the genotype of the SNP2 site is GG, the genotype of the SNP3 site is CC, the genotype of the SNP4 site is CC, and the genotype of the SNP5 site is GG, the individual is identified as Jian carp.

[0101] Using the above-mentioned genotype combination and its typing results, the purity of Jian carp seedlings and parents can be identified and screened.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A primer for amplification of SNP molecular markers for rapid identification of Jian carp varieties, characterized in that: The SNP molecular markers include SNPs 1 to 5; the physical position of SNP 1 is located at the 27079154th base of the Chr17 chromosome, and its base is C or T; The physical location of SNP 2 is at base 27632650 of chromosome Chr17, and its base is A or G; The physical position of SNP 3 is located at base 1441950 of chromosome Chr18, and its base is T or C; The physical position of SNP 4 is located at base 5857377 of chromosome Chr18, and its base is G or C; The physical location of SNP 5 is at base 23058224 of chromosome Chr14, and its base is C or G; The amplification primers include: The upstream and downstream primer sequences used for amplification of the SNP 1 site are shown in SEQ ID NOs. 6 to 7; The upstream and downstream primer sequences used for amplification of the SNP 2 site are shown in SEQ ID NOs. 8 to 9; The upstream and downstream primer sequences used for amplification of the SNP 3 site are shown in SEQ ID NOs. 10 to 11; The upstream and downstream primer sequences used for amplification of the SNP 4 site are shown in SEQ ID NOs. 12 to 13; The upstream and downstream primer sequences used for amplifying the SNP 5 site are shown in SEQ ID NOs. 14-15.

2. Application of the amplification primers according to claim 1 in identification and / or screening of carp varieties.

3. A method for rapid identification and / or screening of Jian carp varieties, characterized in that: The steps include: (a) taking samples of carp to be identified; (b) extracting sample DNA as a template, performing PCR amplification on SNPs 1 to 5 in claim 1 respectively; sequencing the PCR amplification products to determine the genotype of SNPs 1 to 5 in each individual; (c) If the genotypes corresponding to SNPs 1 to 5 are TT, GG, CC, CC and GG respectively, the fish is confirmed to be Jianli.

4. The method for rapid identification and / or screening of Jian carp varieties according to claim 3, characterized in that: The product sequence of PCR amplification of the SNP 1 site is shown in SEQ ID NO.1, and the upstream and downstream primer sequences used are shown in SEQ ID NO.6-7, and the SNP 1 site is located at the 179th position of the nucleotide sequence shown in SEQ ID NO.1; The product sequence of PCR amplification of the SNP 2 site is shown in SEQ ID NO.2, and the upstream and downstream primer sequences used are shown in SEQ ID NO.8-9, and the SNP 2 site is located at the 174th position of the nucleotide sequence shown in SEQ ID NO.2; The product sequence of the PCR amplification of the SNP 3 site is shown in SEQ ID NO.3, and the upstream and downstream primer sequences used are shown in SEQ ID NO.10-11, and the SNP 3 site is located at the 185th position of the nucleotide sequence shown in SEQ ID NO.3; The product sequence of PCR amplification of the SNP 4 site is shown in SEQ ID NO.4, and the upstream and downstream primer sequences used are shown in SEQ ID NO.12-13, and the SNP 4 site is located at the 168th position of the nucleotide sequence shown in SEQ ID NO.4; The product sequence of PCR amplification of the SNP 5 site is shown as SEQ ID NO.5, and the upstream and downstream primer sequences used are shown as SEQ ID NOs.14-15. The SNP 5 site is located at the 305th position of the nucleotide sequence shown in SEQ ID NO.

5.

5. The method for rapid identification and / or screening of Jian carp varieties according to claim 3, characterized in that: The sampling of the carp to be identified comprises: For fry with a total length of ≤5cm, whole tissue sampling is carried out; for fry with a total length of >5cm, fin sampling is carried out; for adult fish, fin sampling is carried out.

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