Method for screening SNP of fast-growing individuals in naozhou stock of larimichthys crocea

Abstract

The present application relates to the technical field of genome selection breeding, and provides a method for screening SNP of fast-growing individuals in Naozhou stock of Larimichthys crocea. The method for screening SNP of fast-growing individuals in Naozhou stock of Larimichthys crocea provided in the present application comprises the following steps: step 1: sample collection; step 2: DNA extraction, PCR amplification and sequence determination; and step 3: data processing and analysis, including association analysis of SNP loci and growth traits of wild Naozhou stock of Larimichthys crocea and status analysis of germplasm genetic resources.

Description

SNP method for screening fast-growing individuals of Pseudosciaena kowalevskii

[0001] The present application claims priority to the Chinese patent application No. 202411073038.1 filed on August 6, 2024, and entitled "SNP method for screening fast-growing individuals of Pseudosciaena kowalevskii", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of genomic selection breeding technology, in particular to a SNP method for screening fast-growing individuals of Pseudosciaena kowalevskii. BACKGROUND

[0003] Pseudosciaena kowalevskii is an important fishery resource in the southeast coastal areas of China. The fishery science and technology community believes that the Pseudosciaena kowalevskii population is divided into the Daiqu tribe, the Min-Yue East tribe, and the Naozhou tribe. The Daiqu tribe is the Jiangsu and Zhejiang sea area population, the Min-Yue East tribe is the Fujian and northern Guangdong sea area population, and the Naozhou tribe is mainly the population near Naozhou Island in Guangdong Province. However, due to overfishing, global climate change, marine environmental pollution, and habitat destruction, the wild Pseudosciaena kowalevskii resource has rapidly declined since the 1970s. In order to protect the wild germplasm resource and achieve aquaculture, Pseudosciaena kowalevskii has successfully transformed from marine capture to artificial breeding. From 2014 to 2022, the average annual output of Pseudosciaena kowalevskii aquaculture reached 200,400 tons, ranking first in China's marine fish aquaculture industry for nine consecutive years. The existing Pseudosciaena kowalevskii aquaculture industry is highly dependent on the breeding population developed from the Guanjingyang wild population (belonging to the Min-Yue East tribe), and faces problems such as slow growth, early sexual maturity, small body size, low disease resistance, and significant reduction in genetic diversity. It is urgent to find high-quality wild Pseudosciaena kowalevskii germplasm to supplement the breeding population.

[0004] Deep-sea aquaculture is an effective way to increase and protect fishery resources in China. The high-quality development of deep-sea aquaculture in Guangdong requires the exploration of high-quality candidate breeding objects suitable for Guangdong waters. The Naozhou tribe of Pseudosciaena kowalevskii, which is unique to Guangdong, has good high-temperature tolerance and large-scale fry breeding technology (which has been broken through), and is expected to become an advantage candidate fish for deep-sea aquaculture in Guangdong and other coastal provinces and cities in the South China Sea. Germplasm resource evaluation is an indispensable key link. In addition, compared with the Pseudosciaena kowalevskii in the East China Sea, the Naozhou tribe is relatively small, and it is necessary to screen fast-growing parents and families to breed fast-growing individuals. Therefore, it is urgent and necessary to screen convenient, fast, and effective fast-growing individual identification markers as soon as possible. SUMMARY

[0005] 1. Technical problems to be solved

[0006] The purpose of the present application is to solve the problem of the relatively small Pseudosciaena kowarum in the prior art, which just breaks through the seedling cultivation technology, and the obvious genetic degradation phenomenon of the East China Sea breeding population, and propose a SNP method for screening fast-growing individuals of Pseudosciaena kowarum.

[0007] 2. Technical solution

[0008] In order to achieve the above purpose, the present application adopts the following technical solution:

[0009] The present application provides a SNP method for screening fast-growing individuals of Pseudosciaena kowarum, comprising the following steps:

[0010] Step 1: sample collection, collecting a certain number of Pseudosciaena kowarum samples in the relevant sea area, and purchasing a certain number of cultured Pseudosciaena kowarum, taking the dorsal muscle tissue and immersing it in 95% alcohol solution, and storing it in a-20℃ refrigerator for subsequent experiments;

[0011] Step 2: DNA extraction, PCR amplification and sequence determination, extracting Pseudosciaena kowarum genomic DNA; based on the Pseudosciaena kowarum GH gene sequence, designing primers GH_2F (5'-CAGAGGTAAGACATTCAGACT-3') and GH_2R (5'-CAGACAGCAGAGCATCAA-3') using Primer Premier 6, and performing PCR amplification, the PCR product contains intron 1, intron 2 and exon 2; using 1% TBE agarose gel electrophoresis to detect the PCR product, and sequencing the qualified sample, the sequence is shown as SEQ ID NO: 1;

[0012] Step 3: data processing and analysis, including correlation analysis of SNP sites of Pseudosciaena kowarum wild and growth traits, and analysis of the current situation of genetic resources.

[0013] The present application also provides a SNP method for screening fast-growing individuals of Pseudosciaena kowarum, comprising the following steps:

[0014] Step 1: sample collection, collecting Pseudosciaena kowarum samples, and collecting cultured Pseudosciaena kowarum, taking muscle tissue for subsequent experiments;

[0015] Step 2: DNA extraction, PCR amplification and sequence determination, extracting Pseudosciaena kowarum genomic DNA; using the primer pair with the nucleotide sequence shown as SEQ ID NO: 2 and SEQ ID NO: 3 for PCR amplification; electrophoretic detection of PCR products, and sequencing the qualified sample, the sequence is shown as SEQ ID NO: 1;

[0016] Step 3: data processing and analysis, including correlation analysis of SNP sites of wild Pseudosciaena crocea in Naozhou population and growth traits and / or analysis of current status of genetic resources of germplasm.

[0017] The application also provides the SNP method for screening fast-growing individuals of Pseudosciaena crocea in Naozhou population or application of the method in management of germplasm resources of Pseudosciaena crocea in Naozhou population or breeding of fine varieties. BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 is a schematic diagram of sample collection and experimental process of the SNP method for screening fast-growing individuals of Pseudosciaena crocea in Naozhou population according to the application. DETAILED DESCRIPTION

[0019] The application provides a SNP method for screening fast-growing individuals of Pseudosciaena crocea in Naozhou population, which comprises the following steps:

[0020] Step 1: sample collection, a certain number of samples of Pseudosciaena crocea in Naozhou population are collected in a relevant sea area, and a certain number of farmed Pseudosciaena crocea are purchased, and muscle tissues of the back of the farmed Pseudosciaena crocea are taken and immersed in a 95% alcohol solution and stored in a -20℃ refrigerator for later experiments;

[0021] Step 2: DNA extraction, PCR amplification and sequence determination, genomic DNA of Pseudosciaena crocea is extracted; based on the sequence of Pseudosciaena crocea GH gene, primers GH_2F 5'-CAGAGGTAAGACATTCAGACT-3'(SEQ ID NO: 2) and GH_2R 5'-CAGACAGCAGAGCATCAA-3'(SEQ ID NO: 3) are designed by using Primer Premier 6, and PCR amplification is performed, and the PCR product contains intron 1, intron 2 and exon 2; the PCR product is detected by 1% TBE agarose gel electrophoresis, and qualified samples are sequenced, and the sequence is shown as SEQ ID NO: 1;

[0022] Step 3: data processing and analysis, including correlation analysis of SNP sites of wild Pseudosciaena crocea in Naozhou population and growth traits and / or analysis of current status of genetic resources of germplasm.

[0023] The application uses growth-related GH gene, screens growth-related polymorphic SNP sites of Pseudosciaena crocea in Naozhou population by correlation analysis, and evaluates the genetic resources of germplasm of Pseudosciaena crocea in Naozhou population. The research results can provide necessary scientific and technological support for management of germplasm resources of Pseudosciaena crocea in Naozhou population, breeding of fine varieties and replenishment of Pseudosciaena crocea breeding population in the East China Sea.

[0024] In some embodiments of the application, in step 1, morphological identification is performed on the samples of Pseudosciaena crocea in Naozhou population and farmed Pseudosciaena crocea, and biological indexes are measured, and in some embodiments of the application, the biological indexes are body weight and body length.

[0025] In some embodiments of the present application, the Ezup column animal genomic DNA extraction kit (Sangon) is used to extract the genomic DNA of P. olivaceus in step 2. The PCR reaction system in step 2 is 50 μL, including 5 μL of 10x reaction buffer, 1 μL of each of the forward and reverse primers, 1 μL of dNTPs, 0.5 μL of Taq DNA polymerase, 2 μL of DNA template, and 39.5 μL of ddH2O. In some embodiments of the present application, the concentration of the Taq DNA polymerase is 2.5 U / μL. The amplification reaction program in step 2 is: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 52°C annealing for 45 s, 72°C extension for 90 s, 35 cycles; and 72°C final extension for 10 min.

[0026] In some embodiments of the present application, the specific steps of the association analysis of the SNP site and the growth trait in step 3 are as follows: the SNP site is analyzed by using Chromas and the SeqMan program in DNAStar, the site with a proportion of different bases greater than 30% at the same site is identified as a SNP site, the genotype of the SNP site in each individual is recorded, and IBM SPSS Statistics for Windows 27.0 is used for association analysis of the screened SNP site and the body length and weight.

[0027] In some embodiments of the present application, the specific steps of the present status analysis of the wild P. olivaceus germplasm genetic resources of the Naozhou population in step 3 are as follows: the haplotype is analyzed by using DnaSP 6, the haplotype diversity (h), nucleotide diversity (π), average nucleotide difference (K), and the number of polymorphic sites are detected by using Arlequin 3.5, the genetic difference between two populations is evaluated by using the population genetic differentiation coefficient Fst, and the distribution of genetic variation is estimated by using the analysis of molecular variance (AMOVA).

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments.

[0029] Embodiment 1:

[0030] Referring to FIG. 1, a SNP method for screening fast-growing individuals of P. olivaceus of the Naozhou population includes the following steps:

[0031] Step 1: sample collection. A total of 53 samples of P. olivaceus of the Naozhou population are collected in Wuchuan (23 tails) and Leizhou Bay (30 tails) in Guangdong, after morphological identification, biological indicators such as body weight and body length are measured, and the dorsal muscle tissue is immersed in 95% alcohol solution and stored in a -20°C refrigerator for subsequent experiments (FIG. 1);

[0032] Step 2: DNA extraction, PCR amplification and sequence determination. The Ezup column animal genomic DNA extraction kit (Sangon) was used to extract the genomic DNA of P. major. Based on the sequence of P. major GH gene (AY090592), the primers GH_2F (5'-CAGAGGTAAGACATTCAGACT-3', SEQ ID NO: 2) and GH_2R (5'-CAGACAGCAGAGCATCAA-3', SEQ ID NO: 3) were designed by Primer Premier 6 and PCR amplification was performed. The PCR reaction system was 50 μL, including 10x reaction buffer 5 μL, forward and reverse primers 1 μL each, dNTPs 1 μL, Taq DNA polymerase (2.5 U / μL) 0.5 μL, DNA template 2 μL and ddH2O 39.5 μL. The amplification reaction program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 52°C annealing for 45 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 10 min. The PCR products were detected by 1% TBE agarose gel electrophoresis, and the qualified samples were sent to Guangzhou Shengong Biological Engineering Technology Service Co., Ltd. for sequencing, and the sequence is shown as SEQ ID: 1;

[0033] Step 3: Data processing and analysis. The SNP site was analyzed by Chromas and SeqMan program in DNAStar. The site with more than 30% of different bases at the same site was identified as a SNP site, and the genotype of the SNP site in each individual was recorded. The screened SNP sites were associated with body length and weight by using IBM SPSS Statistics for Windows 27.0.

[0034] The results are shown in Table 1. In this embodiment, the association of GH gene SNP site of P. major in Naozhou population with growth traits: one polymorphic SNP site was detected in intron 2 of the non-coding region of GH gene of Naozhou wild population; among 53 wild P. major in Naozhou population, 37 individuals were of GG genotype, 14 individuals were of GA genotype, and 2 individuals were of AA genotype; the frequencies of alleles G and A were 83.02% and 16.98%, respectively; the body length and weight of AA genotype individuals were significantly higher than those of GG genotype individuals (P<0.01) and GA genotype individuals (P<0.05), and there was no difference in body length and weight between GA and GG genotype individuals (P>0.05).

[0035] Table 1 Association analysis of SNP site of GH gene of P. major in Naozhou population with growth traits

[0036] In this embodiment, three genotypes (GG, GA and AA) of SNP sites in P. major GH gene were detected, among which AA genotype was only detected in Leizhou Bay population, and the body weight and body length of AA genotype individuals were significantly greater than those of GG and GA genotypes. It can be seen that the allele A is positively correlated with body weight and body length, and may play an important role in the selection of growth and development traits of Naozhou population of P. major.

[0037] In this embodiment, Naozhou population of P. major is relatively small compared to East China Sea population, and body length and body weight are important economic traits affecting the yield and quality of Naozhou population of P. major in deep sea culture. In the breeding process of Naozhou population of P. major, it is recommended to screen the parents and families based on the genotyping results of GH gene, that is, to select AA genotype parents for fast-growing elite breeding and high-quality seed production, thereby providing a convenient, fast and effective screening marker for the selection of fast-growing large individual elite of Naozhou population of P. major. It is worth noting that due to insufficient sample size and fewer sampling sites, AA genotype has not been detected in Wuchuan population at present, and further verification is needed in the future.

[0038] Example 2:

[0039] Evaluation of the current status of Naozhou population of P. major wild germplasm genetic resources.

[0040] Sample collection, DNA extraction, PCR amplification and sequence determination were performed according to steps 1 and 2 of Reference Example 1. A total of 100 samples were detected, including 53 wild Naozhou population of P. major samples (23 from Wuchuan, Guangdong, and 30 from Leizhou Bay) and 47 cultured Min-Yue East population of P. major (purchased from Ningde Yeyue Aquaculture Technology Co., Ltd.).

[0041] In this embodiment, Chromas and SeqMan program in DNAStar were used to analyze SNP sites, and the genotypes of SNP sites in each individual were recorded. IBM SPSS Statistics for Windows 27.0 was used for association analysis of the screened SNP sites and body length and body weight. DnaSP 6 was used to analyze haplotypes, and Arlequin 3.5 was used to detect haplotype diversity (h), nucleotide diversity (π), average nucleotide difference (K) and polymorphic site number, and population genetic differentiation coefficient F st The genetic differences between each pair of populations were evaluated, and the distribution of genetic variation was estimated by analysis of molecular variance (AMOVA).

[0042] The results are shown in Table 2. In this embodiment, the polymorphism analysis of the GH gene SNP site in large yellow croaker showed that among 53 wild large yellow croaker individuals from the Naozhou group, 37 individuals had the GG genotype, 14 individuals had the GA genotype, and 2 individuals had the AA genotype, with allele frequencies of 83.02% and 16.98%, respectively. Among 47 farmed large yellow croaker individuals from the Fujian-Eastern Guangdong group, 37 individuals had the GG genotype, and 10 individuals had the GA genotype, with allele frequencies of 89.36% and 10.64%, respectively. The wild large yellow croaker population from the Naozhou group possesses three genotypes: GG, GA, and AA, while the farmed population from the Fujian-Eastern Guangdong group only possesses two genotypes: GG and GA, demonstrating that the wild Naozhou group exhibits richer genetic polymorphism.

[0043] Table 2. Genotype analysis of different SNP loci in the GH gene of large yellow croaker.

[0044] In this embodiment, the genetic diversity and genetic structure of large yellow croaker were analyzed: compared with the East China Sea farmed population, the wild large yellow croaker from Naozhou Island exhibited relatively high genetic diversity (Table 3), while the Leizhou Bay population showed the highest haplotype diversity, nucleotide diversity, average number of nucleotide differences, and number of polymorphic sites. The F1 generation among the three populations was also analyzed. st The values ​​were all relatively small (Table 4), and the statistical tests were not significant (p > 0.05). AMOVA results (Table 5) showed that 2.83% of the genetic variation originated between populations, and 97.17% originated between individuals within a population, with no statistical significance. In summary, the wild populations in Leizhou Bay exhibit relatively high genetic diversity, the two wild populations of the Naozhou tribe show high genetic homogeneity, and there is no significant genetic differentiation between the wild Naozhou tribe population and the farmed population of the Min-Yue East ethnic group.

[0045] Table 3. Sampling information and genetic diversity parameters of three populations of large yellow croaker.

[0046] Table 4. Genetic differentiation index Fst (bottom left diagonal) and P-value (top right diagonal) among the three populations of large yellow croaker.

[0047] Table 5. AMOVA analysis among the three populations of large yellow croaker.

[0048] In this embodiment, the current status of large yellow croaker germplasm genetic resources and the screening of high-quality germplasm resource library: the genetic diversity level and gene polymorphism (3 genotypes) of the Leizhou Bay population of the Chauzhou tribe wild large yellow croaker are significantly higher than those of the Wuchuan population and the Min-Yue East tribe farming population, and the AA genotype of large individuals is only detected in the Leizhou Bay population; at the same time, there is no obvious genetic differentiation between the Leizhou Bay population and the Wuchuan population and the Min-Yue East tribe farming population. Therefore, it is believed that the Leizhou Bay wild population of the Chauzhou tribe is a relatively good high-quality germplasm resource library, which can provide high-quality wild germplasm resources for the deep sea culture of the Chauzhou tribe large yellow croaker in the northern South China Sea and the breeding population of the East China Sea large yellow croaker.

[0049] The above is only the preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes within the technical scope disclosed in the present application according to the technical scheme and inventive concept of the present application, which should be covered within the protection scope of the present application.

Claims

1. A SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena crocea group, characterized in that, The method comprises the following steps: Step 1: sample collection, collecting a certain number of samples of Pseudosciaena crocea in the relevant sea area, and purchasing a certain number of cultured Pseudosciaena crocea, taking the back muscle tissue and immersing it in 95% alcohol solution, and storing it in a-20 DEG C refrigerator for subsequent experiments; Step 2: DNA extraction, PCR amplification and sequence determination, extracting Pseudosciaena crocea genomic DNA; based on the Pseudosciaena crocea GH gene sequence, the primer GH_2F 5'-CAGAGGTAAGACATTCAGACT-3' and GH_2R 5'-CAGACAGCAGAGCATCAA-3' are designed by using Primer Premier 6, and PCR amplification is carried out, and the PCR product contains intron 1, intron 2 and exon 2; the PCR product is detected by 1% TBE agarose gel electrophoresis, and the qualified sample is sequenced, and the sequence is shown as SEQ ID NO: 1; Step 3: data processing and analysis, including SNP site and growth trait correlation analysis of wild Pseudosciaena crocea in the Diaoyu Islands population, and analysis of the current status of germplasm genetic resources.

2. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 1, characterized in that, In step 1, the Pseudosciaena crocea samples and the cultured Pseudosciaena crocea are morphologically identified, and biological indicators are measured.

3. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 2, characterized in that, The biological indicators include body weight and body length.

4. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena coreensis group according to claim 1, characterized in that, In step 2, the Ezup column animal genomic DNA extraction kit is used to extract the Pseudosciaena crocea genomic DNA.

5. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 1, characterized in that, In step 2, the PCR reaction system is 50 μL, including 5 μL of 10x reaction buffer, 1 μL of each of the forward and reverse primers, 1 μL of dNTPs, 0.5 μL of Taq DNA polymerase, 2 μL of DNA template and 39.5 μL of ddH2O.

6. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 5, characterized in that, The concentration of the Taq DNA polymerase is 2.5 U / μL.

7. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena coreensis group according to claim 1, characterized in that, In step 2, the amplification reaction program is: 94 DEG C pre-denaturation for 5 min; 94 DEG C denaturation for 45 s, 52 DEG C annealing for 45 s, 72 DEG C extension for 90 s, 35 cycles; 72 DEG C final extension for 10 min.

8. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena coreensis group according to claim 1, characterized in that, In step 3, the SNP site and growth trait correlation analysis includes: using Chromas and SeqMan program in DNAStar to analyze the SNP site, and the same site with more than 30% of different bases is identified as a SNP site.

9. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 8, characterized in that, In step 3, the SNP site and growth trait correlation analysis further includes: recording the genotype of the SNP site in each individual, and using IBM SPSS Statistics for Windows 27.0 to perform correlation analysis on the screened SNP site and body length and body weight.

10. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena coreensis group according to claim 1, characterized in that, In step 3, the current status of germplasm genetic resources of wild Pseudosciaena crocea in the Diaoyu Islands population includes: using DnaSP 6 to analyze haplotypes.

11. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaenidae according to claim 1, characterized in that, In step 3, the current status of germplasm genetic resources of wild Pseudosciaena crocea in the Diaoyu Islands population includes: using Arlequin 3.5 to detect haplotype diversity h, nucleotide diversity π, average nucleotide difference K and polymorphic site number.

12. The SNP method for screening fast-growing individuals of Pseudosciaena crocea of the Pseudosciaena coreensis group according to claim 1, characterized in that, The step 3 includes: the population genetic differentiation coefficient F st The genetic difference between two groups is evaluated, and the distribution of genetic variation is estimated by AMOVA.

13. A method for screening a SNP of Pseudosciaena crocea of Pseudosciaenidae for fast-growing individuals, characterized in that, The method comprises the following steps: Step 1: sample collection, collecting Pseudosciaena crocea samples in the Diaoyu Islands population, and collecting cultured Pseudosciaena crocea, taking muscle tissue for subsequent experiments; Step 2: DNA extraction, PCR amplification and sequence determination, extracting the genomic DNA of P. major; performing PCR amplification by using the primer pair with nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3; detecting the PCR product by electrophoresis, and sequencing the qualified sample, and the sequence is shown in SEQ ID NO: 1; Step 3: data processing and analysis, including the correlation analysis of SNP sites of wild P. major in Naozhou population and growth traits and / or the analysis of the current situation of germplasm genetic resources.

14. The method of claim 13, wherein, The muscle tissue is dorsal muscle.

15. The method of claim 13, characterized in that, The step 1 further comprises the following steps: morphological identification of the P. major samples in Naozhou population and the cultured P. major, and measuring the body weight and body length.

16. The method of claim 13, wherein, In the step 2, the genomic DNA of P. major is extracted by using Ezup column animal genomic DNA extraction kit.

17. The method of claim 13, wherein, In the step 2, the PCR reaction system according to 50 μL includes 10x reaction buffer 5 μL, forward and reverse primers 1 μL each, dNTPs 1 μL, Taq DNA polymerase 0.5 μL, DNA template 2 μL and ddH2O 39.5 μL.

18. The method of claim 17, wherein, The concentration of the Taq DNA polymerase is 2.5 U / μL.

19. The method of claim 13, wherein, In the step 2, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 52℃ annealing for 45 s, 72℃ extension for 90 s, 35 cycles; and 72℃ final extension for 10 min.

20. The method of claim 13, wherein, In the step 3, the correlation analysis of SNP sites and growth traits includes: analyzing the SNP sites by using Chromas and SeqMan program in DNAStar, the sites with the proportion of different bases at the same site greater than 30% are identified as SNP sites, the genotypes of the SNP sites in each individual are recorded, and the IBM SPSS Statistics for Windows 27.0 is used for the correlation analysis of the screened SNP sites and body length and body weight.

21. The method of claim 13, wherein, The step 3 includes: analyzing haplotype by DnaSP 6, detecting haplotype diversity (h), nucleotide diversity (π), average nucleotide difference (K) and the number of polymorphic sites by Arlequin 3.5, and using population genetic differentiation coefficient F st The genetic differences between two groups are evaluated, and the distribution of genetic variation is estimated by molecular variance analysis (AMOVA).

22. The application of the SNP method for screening fast-growing individuals of P. major in Naozhou population according to any one of claims 1-12 or the method according to any one of claims 13-21 in the management of P. major germplasm resources in Naozhou population or the breeding of fine varieties.

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