Application of SNP (Single Nucleotide Polymorphism) molecular marker combination in dragon bone length assisted breeding

Abstract

The invention discloses an application of an SNP molecular marker combination in fossil length assisted breeding, the SNP molecular marker combination comprises 10 SNP sites SNP1-SNP10, and in fossil length assisted breeding, TT and CT dominant genotypes of the SNP1 site, GGAA dominant genotypes generated by linkage of SNP2 and SNP3 sites, AA dominant genotypes of the SNP4 site and GG dominant genotypes of the SNP5 site can be selected and reserved, so that the fossil length assisted breeding of the fossil length can be realized, and the fossil length assisted breeding of the fossil length of the fossil length of the fossil length of the fossil length of the fossil length of the fossil length of the fossil length of the fossil length can be realized. According to the method, the breeding of the fossil fragments is improved in an auxiliary manner by selecting individuals of a CC dominant genotype at an SNP6 site, a GG dominant genotype at an SNP7 site, a TT dominant genotype at an SNP8 site, CC and TC dominant genotypes at an SNP9 site and TT and GT dominant genotypes at an SNP10 site, and by eliminating individuals of inferior genotypes, the breeding of the fossil fragments is accelerated, and the progress of generation breeding of the fossil fragments is accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted breeding, and in particular to an application of a SNP molecular marker combination in keel-long assisted breeding. Background Art

[0002] Poultry body size index is an important phenotypic trait for breed selection and breeding. It is significantly correlated with poultry meat performance such as body weight, carcass weight, and eviscerated weight. It is a commonly used index for evaluating poultry weight, body shape, and slaughter performance, and can reflect the growth and development of poultry to a certain extent. Among them, keel length is one of the important body size traits of chickens. Studies have shown that there is a significant strong positive correlation between keel length and breast muscle thickness. The breast muscle is mainly attached to the sternum and keel, and is closely related to the growth of the keel. There is a significant correlation between chicken keel length and body weight. The keel length trait can be selected to improve body weight, breast muscle rate and other indicators. Keel length development is a quantitative trait controlled by multiple genes.

[0003] At present, the keel length is usually directly measured or observed by naked eyes in the breeding process, and the generational inheritance progress is slow. Therefore, finding molecular markers significantly related to keel length is of great significance for improving the progress of generational breeding of keel length. Summary of the invention

[0004] In response to the problem of slow progress in the generational breeding of keel length, the present invention provides an application of a SNP molecular marker combination in assisted breeding of keel length, which assists in improving the breeding of keel length through 10 SNP molecular markers significantly correlated with keel length, thereby accelerating the generational breeding progress of keel length.

[0005] In order to achieve the above object, the present invention provides an application of a SNP molecular marker combination in keel length auxiliary breeding, and the SNP molecular marker combination corresponds to the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI as follows:

[0006] The screening method of the SNP molecular marker is as follows: Using the chicken genome bGalGal.mat.broiler.GRCg7b (GCF_016699485.2) as a reference, GWAS sequencing technology and genome-wide association analysis were used to obtain 10 SNP loci significantly associated with keel length. The association analysis and verification of polymorphic loci genotypes with keel length were performed using univariate analysis of variance in the general linear model of SPSS16.0 software, and the dominant genotypes of the 10 SNP loci were screened.

[0007] GWAS is a powerful tool for analyzing the genetic structure of quantitative traits in livestock and poultry. The GWAS method is used to study the association between SNP sites and phenotypic values, which can identify molecular markers that affect economic traits and is particularly suitable for complex quantitative traits. Based on this, the present invention screened keel length molecular markers through GWAS and obtained 10 SNP molecular markers that were significantly correlated with keel length. Compared with candidate gene and QTL linkage analysis, GWAS has a high marker density, can analyze rare and low-frequency variations, can analyze the genetic structure of complex traits, and can also identify new variations, and the results are more reliable.

[0008] The nucleotide sequences of the primers for the above SNP molecular markers are as follows:

[0009] Specifically, the breeding method for increasing keel length comprises the following steps: (1) Determining the genotype of the chicken to be bred, wherein the genotype is the genotype of the above-mentioned SNP molecular marker combination; (2) Select individuals with the dominant genotypes of TT and CT at SNP1, the dominant genotype of GGAA resulting from the linkage between SNP2 and SNP3, the dominant genotype of AA at SNP4, the dominant genotype of GG at SNP5, the dominant genotype of CC at SNP6, the dominant genotype of GG at SNP7, the dominant genotype of TT at SNP8, the dominant genotypes of CC and TC at SNP9, and the dominant genotypes of TT and GT at SNP10.

[0010] Specifically, in step (1), the method for determining the genotype of the chicken to be bred is: (1.1) Extracting total genomic DNA from the chicken to be tested; preferably, the genomic DNA from the chicken to be tested is obtained by sampling blood from the wing vein of the chicken to be tested; (1.2) According to the SNP molecular marker combination, the target sequence is amplified by PCR method using the corresponding primer pair, and the sequence of the primer pair is: SNP1F: 5' CACCAAGCGTGCATAAGAGA3' SNP1R: 5' ACCTGAAGAGTCGTGGATGG3' SNP2F: 5' CCTGCAGGGAGGAATAACAA3' SNP2R: 5' GGCACCTTTACAGCACCATT3' SNP3F: 5' GAAATGGGCTATGTGGCAGT3' SNP3R: 5' CCAGGACTTGTGCCTTTGTT3' SNP4F: 5' TGAACCCATAGGAGTTTTCCA3' SNP4R: 5'TGCAGCGCATACCATCTTAC3' SNP5F: 5' GAGGGGTTGAAACTGGATGA3' SNP5R: 5'CAAAGCTGGGTGCACAGATA3' SNP6F: 5'GCGTCAGTGGGAATAAGCTC3' SNP6R: 5'AGCAGGTTACAGGGCAGAAA3' SNP7F: 5' CTGAAAGGGAGTCAGCCAAG3' SNP7R: 5'TCCAAAAGCAGCACTAAGCA3' SNP8F: 5' TGCTGAAAATGTGTTGCTGA3' SNP8R: 5'CTGCAGGTGTTTCTGTTCCA3' SNP9F: 5' CTGCAGGGTTTGGAGCAGGC3' SNP9R: 5'CTGATGGGCTCAGAGCAAG3' SNP10F: 5'GAGCAGGAGGGGGAAGTG3' SNP10R: 5'CTTCCATTGGCCGAGAAC3'; (1.3) After sequencing the PCR amplification product, determine the genotype.

[0011] The PCR product was sent to a biological company for sequencing, and the resulting sequence was compared with the reference genome of chicken to find the polymorphic site. The nucleotide sequence of the PCR product of the SNP site is as follows: SNP1—C / T mutation at 199bp, PCR product length is 223bp: CACCAAGCGTGCATAAGAGATTGAATGAGTTCAGCATTAATAACAGGGGCTGTCCTTCTCATTACACCGACTGCCATTTCTAAAAGGCGTGTTTTAATTAATAAGGAAGCTGAAGAAAAGCATTCAGAGGAAGCACTGTCATTAGTGCAGACGGGTAAGCACAGTGTGTCATGCACGCACTTGGGCAGAACTTTTAATK(C / T)TGCTCCATCCACGACTCTTCAGGT SNP2 - G / A mutation at 143 bp, PCR product length is 224 bp: CCTGCAGGGAGGAATAACAACTTCAGGTTTTTGAGGTCTCACTCATGTCACAGAGGTAATAGTGTACCTTGAACTCAGCATCCCTGTTACAGCTTCCTCTAGTGGAGCTATGCAGCAAAGCGTTCTCTCTTCGTCCTTAGAGK(G / A)GAAATGGGCTATGTGGCAGTATGGTTCATCTGCCCAGTTTCAGCTCCTCGTGGCTAACATTAATGGTGCTGTAAAGGTGCC SNP3 - A / G mutation at 112 bp, PCR product length is 219 bp: GAAATGGGCTATGTGGCAGTATGGTTCATCTGCCCAGTTTCAGCTCCTCGTGGCTAACATTAATGGTGCTGTAAAGGTGCCCAGCCAACAATGCCTGTGCAGAGCGGTCACK(A / G)TTTCAGGTATAGTTACCAGTGGCATTGGTTGCCCCAGGATGCCTCTTGGAGTTGAGGACACTGGGTAGCACTGTTTCCACAGAGAAAAACAAAGGCACAAGTCCTGG SNP4 - G / A mutation at 157 bp, PCR product length is 248 bp: TGAACCCATAGGAGTTTTCCAGCTGAGCAATTACTGAAATGTCAGGATGCCTAGTCTGAATTTGCACATAAAAATGGTATACATGCATTAAAACTTAGCCTTCAAAATTATCTTGTTAAATAGTTAGAAATTAGATAGTGTAAAACAACTGACAATK(G / A)AGAAGGGAAAAGAAAAAAAAGATATTTTTAGTATGAAATACATGTATAATTGCAACTGGTGACTTTTACCTGTAAGATGGTATGCGCTGCA SNP5 - A / G mutation at 136 bp, PCR product length is 219 bp: GAGGGGTTGAAACTGGATGATCATTGTGGTCCTTTTCAACTCAGGCCATTCTATGATTCTATGATTCTGTATCCTGAAGGAAGTCATAAAATCAGATCATATGATAAAATTACTAACGTAAGAGGATTTGATAGGK(A / G)CTAACAACATCAAATATTTGATACGATCAGATTTGAATTTTCACATAGAATAGTCCTGCAAAATATCTGTGCACCCAGCTTTG SNP6 - A / G mutation at 180 bp, PCR product length is 237 bp: GCGTCAGTGGGAATAAGCTCAACAGACCAAGCTTATTAGAAATTCATGAACAACTGGCAAAGGTGAGTGAAAAGGAAACATTTGATTTTTCTCTGTTCCATTTCCAGGCTGTTAATTCTGTTTATTAATATGAAAAAGTCTTTAAATTCACGGATCTTGCCCAAGACAAAAGTACAACTK(C / A)ATCACTTACCATATTTCCTGTGGTAGCTGCAGTATGTTTTCTGCCCTGTAACCTGCT SNP7 - A / G mutation at 63 bp, PCR product length is 227 bp: CTGAAAGGGAGTCAGCCAAGAATCCAAGTAGGCCAATGATAAGCTTAGCACATTAGAAAGTTK(A / G)AAATATAAACATGACTCTTATAATTCCAGAAAATAACTCAAATTGTAGGCAAGTATTTTAAAATGCTGTACTTGCAGGCAAAGTAATTGCAGTAAATAATCAATGTAAAAGAGATCAAATATATAAAAACAACCTTCTGAAAACTGCTTAGTGCTGCTTTTGGA SNP8 - G / T mutation at position 102bp, PCR product length is 202bp: TGCTGAAAATGTGTTGCTGAAGGAAACCAGTTTATAGGAATACACCATTATACTGGTGCTACCCATAAGGTAGGGATCAGAAACTGACAAAAACTGGTGATK(G / T)TTCTTATCTCAGAGATTTTCTCTCTTGATTTTAAGCAGGAAATCTGCAGACTACAACAGGCATGAGACAAGTCTTCTGCATGGAACAGAAACACCTGCAG SNP9 - T / C mutation at position 60bp, PCR product length is 233bp: CTGCAGGGTTTGGAGCAGGCTGGCGTCCCTGCTTGGCGCCCTAGAGTGGAACCTGGCAGK(T / C)CAAACGGAAAACGTAAAGCTGGGAGAAGATACGTGTGTGCCTGAACAACGTCGCGAGCGTCCTGGCAGGATGGAAGCGGGGATTGCTGGGCTGTGGCTGTGGGGCAGACGCGGGCCTGAGCCCTACATCCCGCTCTGCTGCCGCCGCTGCGCTCCTTGCTCTGAGCCCATCAG SNP10 - G / T mutation at position 171bp, PCR product length is 218bp: GAGCAGGAGGGGGAAGTGCCGTGCGGTTCCCATCGCTGCCACCACCGCCCGCCGCCCAAGCGCTACTGCGGCCCCCGCGCCCCCTGCGCGGCGCGATGGGAGCCGCCTCACCAAGCCCCGCCCCGCAGCCAATGGGAGCGCGGCGGAGGTGGGACTCCGGGGAACGAGGGGK(G / T)ATTGGGCGAGAGCCACACCCGCCTCGCCGTTCTCGGCCAATGGGAAG Note: The K marked in the above sequence is the mutation site, and the mutated bases in brackets are allele mutations.

[0012] The specific method for determining the dominant genotype individuals is as follows: Firstly, the correlation between single SNP markers and keel length was analyzed. The keel lengths of different genotypes of the 10 SNPs loci were significantly different. SNP1 (rs315701680) had three genotypes: CC, CT and TT. The keel lengths of individuals with TT and CT genotypes were significantly longer than those with CC genotypes (P < 0.05). SNP2 (rs738740137) had three genotypes: AA, GA and GG. The keel lengths of individuals with GG genotypes were significantly longer than those with AA and GA genotypes (P < 0.05). SNP3 (rs317223723) has three genotypes: AA, AG, and GG. The keel length of individuals with the AA genotype was significantly longer than those with the GG and AG genotypes (P < 0.05). SNP4 (rs732443622) has three genotypes: AA, GA, and GG. The keel length of individuals with the AA genotype was significantly longer than those with the GG and GA genotypes (P < 0.05). SNP5 (rs315667756) has three genotypes: AA, AG, and GG. The keel length of individuals with the GG genotype was significantly longer than those with the GG and GA genotypes (P < 0.05). The keel length of individuals with CC genotype was significantly longer than that of AA and GA genotypes (P < 0.05); SNP6 (rs314381113) had three genotypes: AA, CA, and CC, among which the keel length of individuals with CC genotype was significantly longer than that of individuals with AA and CA genotypes (P < 0.05); SNP7 (rs732811384) had three genotypes: AA, AG, and GG, among which the keel length of individuals with GG genotype was significantly longer than that of individuals with AA and AG genotypes (P < 0.05); SNP8 (rs314197610) had three genotypes: The keel length of individuals with TT genotype was significantly longer than that of individuals with GG and GT genotypes (P < 0.05); SNP9 (rs13782000) had three genotypes, CC, TC and TT, and the keel length of individuals with CC and TC genotypes was significantly longer than that of individuals with TT genotype (P < 0.05); SNP10 (rs737401141) had three genotypes, GG, GT and TT, and the keel length of individuals with TT and GT genotypes was significantly longer than that of individuals with GG genotype (P < 0.05).

[0013] Haploview software was used to analyze the linkage disequilibrium (LD) of the 10 SNPs loci. The results of the linkage disequilibrium analysis showed that SNP2 and SNP3 were in a strong linkage state (D' value = 1, R 2 =100), the other 8 SNPs were not in a strong linkage state (D' value <1, R 2<100), the linkage between SNP2 and SNP3 produced two haplotypes: H1 (AG) and H2 (GA), with haplotype frequencies of 0.893 and 0.098, respectively. Therefore, only the combined genotype of SNP2 and SNP3 was analyzed for correlation with keel length, and the combined genotype of 8 molecular markers was no longer analyzed for correlation with keel length. The combination of the two haplotypes produced by the linkage of SNP2 and SNP3 produced five genotypes. Association analysis found that the keel length of the H1H1 haplotype (GGAA genotype) was significantly higher than that of other haplotype combinations.

[0014] The TT and CT genotypes of SNP1, the GGAA genotype resulting from the linkage between SNP2 and SNP3, the AA genotype of SNP4, the GG genotype of SNP5, the CC genotype of SNP6, the GG genotype of SNP7, the TT genotype of SNP8, the CC and TC genotypes of SNP9 and the TT and GT genotypes of SNP10 are the dominant genotypes for keel length and can serve as important molecular markers for molecular-assisted breeding of keel length.

[0015] Through the above technical solution, the present invention achieves the following beneficial effects: In molecular marker-assisted breeding of keel length, the method of retaining individuals with superior genotypes at 10 SNP loci (SNP1-SNP10) and eliminating individuals with inferior genotypes at SNP1-SNP10 can assist in improving the selection and breeding of keel length and accelerate the progress of generational selection and breeding of keel length. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Manhattan plots for labeling molecules related to keel length; Figure 2 QQ plot of molecular markers related to keel length; Figure 3 Linkage disequilibrium analysis diagram of 10 SNP molecular markers. DETAILED DESCRIPTION

[0017] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0018] Example 1 Screening of molecular markers significantly associated with keel length 1. Experimental Materials The test subjects selected were the terminal paternal line A of Lihua yellow-footed chickens. The main selected traits of this line are body weight, feed conversion rate and slaughter rate, etc. It has undergone six generations of closed breeding. The experimental chickens were raised at the Jintan breeding base of Jiangsu Lihua Animal Husbandry Co., Ltd. in Changzhou City, Jiangsu Province. All experimental chickens were hatched from the same batch and raised in the same chicken house. The experimental chickens were raised in two stages. They were raised in cages from 1 to 4 weeks of age and transferred to individual cages after 5 weeks of age. The company's standard breeder feed was used for feeding, and the chickens were free to drink water and eat during the test period. Immunization followed the standard procedures established by the company.

[0019] 2. Keel length measurement 400 roosters from the specialized strain A of Lihua yellow-footed chicken were randomly selected and weighed after fasting for 12 hours. The leg numbers were recorded and the keel length of the 60-day-old roosters was measured. The specific measurement method was as follows: a vernier caliper was used to measure the distance from the front end to the end of the keel of the chicken. All keel length measurements were made by the same person and the measured parts were basically the same.

[0020] 3. Discovery of molecular markers of keel length At 60 days of age, 1.5 mL of blood was collected from the subwing vein of all groups whose keel lengths were measured. EDTA was used for anticoagulation. After blood collection, the anticoagulation tube was slowly shaken up and down to allow the EDTA in the tube to fully contact and mix the blood. The anticoagulation tube was stored in an incubator containing ice packs. After blood collection, it was quickly transported to the laboratory and stored at -20°C for future use. DNA was extracted using the TIANGEN Blood Genomic DNA Extraction Kit (spin column type) (YDP348). DNA integrity and purity were determined by gel migration (1% agarose gel electrophoresis), and the DNA concentration was determined using the Qubit 4 fluorescence quantifier (Thermo Fisher, Shanghai) to ensure that the concentration of the extracted DNA sample was greater than 15 ng / μL.

[0021] Whole genome resequencing: DNA samples that have passed quality inspection are uniformly delivered to Beijing Boya Geya Biotechnology Co., Ltd. for whole genome resequencing, including preparation of genomic libraries and sequencing on the MGISEQ-2000 platform.

[0022] Quality control: The main purpose is to filter the original genome reads of the MGISEQ-2000 platform double-end sequencing, and use the fastp (v.0.20.0) preprocessor (set to default parameters) to remove low-quality reads, adapters, and reads containing poly-N. Finally, the reads obtained after quality control were aligned to the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI using BWA (v0.7.17) software; the reads with repeated alignments were removed using Picard software; and high-quality SNP sites were obtained through GATK 4.0 software analysis.

[0023] Genome-wide association analysis: PLINK (v1.90p) software was used to perform principal component analysis (PCA) on the SNP loci after quality control to prevent false positive results caused by population stratification. The linear mixed model (LMM) of GEMMA (v0.98.5) software was used to perform association analysis on weight traits.

[0024] y=Wα+xβ+u+ , u~MVNn(0,λτ -1 K), ~MVNn(0,λτ -1 I n ) Where y is the phenotype vector; W is the fixed effect matrix (the first three principal components including PCA); α is the fixed effect vector; x is the genotype vector, β is the SNP effect vector; u is the random effect vector; represents the residual vector. MVNn is an n-dimensional multivariate normal distribution, λ is the genetic variance and the residual variance (random effect vector u and residual vector The ratio of -1 is the residual variance, K is the kinship matrix calculated based on SNP using GEMMA, and I n is the identity matrix.

[0025] The (-indep-pairwise 50 10 0.2) parameter in PLINK software was used to infer the number of valid SNPs for independent testing, and the final inference was that the number of valid independent testing SNPs was 432,5210. After multiple testing, the Bonferroni correction method was used to set the significant threshold. The genome-wide significant level p in this study was 1.16×10 -7(0.05 / number of effective independent test SNPs), and then the negative logarithm of log10 is taken for the p value, which is the threshold of the genome-wide significant level; the genome-wide potential significant level p is 2.31×10 -6 (1 / 432529), the negative logarithm of log10 is taken, which is the potential significant level threshold of 5.63. The Cmplot package in R was used to visualize the GWAS results. The adjacent genes within 100 Kb upstream and downstream of the significant site were annotated using Bedtools (v2.30.0) software.

[0026] After quality inspection, genome-wide association analysis was performed on the keel length phenotype values ​​of 366 chickens. Figure 1 , Figure 2 As shown in the Manhattan plot, a total of 10 SNP loci with potential significant correlation were located on chromosomes 1, 3, 4, 10, 14, and 24 of chicken (threshold value above 5.63). The QQ plot further verifies the reliability of the GWAS results. The significantly correlated keel length molecular markers screened are summarized as shown in Table 1: Table 1 Molecular markers significantly associated with keel length

[0027] The physical position of the marker chromosome is referenced to the chicken whole genome (bGalGal1.mat broiler .GRCg7b).

[0028] 4. Genetic polymorphism analysis of 10 SNPs significantly associated with keel length The 10 SNP molecular markers obtained by PopGene (version 1.31) were used to analyze the genotype frequency, gene frequency, and heterozygosity (He), and the chi-square test was used to detect whether the SNP loci were in Hardy-Weinberg (HW) equilibrium. The analysis results are shown in Table 2.

[0029] As shown in Table 2, all 10 SNP sites have 3 genotypes. After HW equilibrium detection, among the 10 SNPs sites, except for SNP5, SNP6, and SNP10, which deviate from the HW equilibrium state (P<0.05), the other 7 SNP sites are in the HW equilibrium state (P>0.05). The genetic diversity of 4 SNP sites (SNP1, SNP2, SNP3, and SNP10) is low (He<0.25), and the other 6 SNP sites (SNP4, SNP5, SNP6, SNP7, SNP8, and SNP9) are moderately genetically diverse (0.25 <He<0.50)。

[0030] Table 2 Genetic polymorphism of 10 SNP loci and Hardy-Weinberg equilibrium test

[0031] 5. Analysis of the dominant genotype of keel length SNP molecular markers The association analysis between polymorphic locus genotype and keel length was performed using univariate analysis of variance in the general linear model of SPSS 16.0 software. Fixed factors: different genotypes of SNP markers, dependent variables: keel length, Tukey HSD method was used to compare the significance of keel length between different marker genotypes, P < 0.05 indicated significant difference.

[0032] Firstly, the correlation between single SNP markers and keel length was analyzed. The keel lengths of different genotypes of the 10 SNPs loci were significantly different. SNP1 (rs315701680) had three genotypes: CC, CT and TT. The keel lengths of individuals with TT and CT genotypes were significantly longer than those with CC genotypes (P < 0.05). SNP2 (rs738740137) had three genotypes: AA, GA and GG. The keel lengths of individuals with GG genotypes were significantly longer than those with AA and GA genotypes (P < 0.05). SNP3 (rs317223723) has three genotypes: AA, AG, and GG. The keel length of individuals with the AA genotype was significantly longer than those with the GG and AG genotypes (P < 0.05). SNP4 (rs732443622) has three genotypes: AA, GA, and GG. The keel length of individuals with the AA genotype was significantly longer than those with the GG and GA genotypes (P < 0.05). SNP5 (rs315667756) has three genotypes: AA, AG, and GG. The keel length of individuals with the GG genotype was significantly longer than those with the GG and GA genotypes (P < 0.05). The keel length of individuals with CC genotype was significantly longer than that of AA and GA genotypes (P < 0.05); SNP6 (rs314381113) had three genotypes: AA, CA, and CC, among which the keel length of individuals with CC genotype was significantly longer than that of individuals with AA and CA genotypes (P < 0.05); SNP7 (rs732811384) had three genotypes: AA, AG, and GG, among which the keel length of individuals with GG genotype was significantly longer than that of individuals with AA and AG genotypes (P < 0.05); SNP8 (rs314197610) had three genotypes: The keel length of individuals with TT genotype was significantly longer than that of individuals with GG and GT genotypes (P < 0.05); SNP9 (rs13782000) had three genotypes, CC, TC and TT, and the keel length of individuals with CC and TC genotypes was significantly longer than that of individuals with TT genotype (P < 0.05); SNP10 (rs737401141) had three genotypes, GG, GT and TT, and the keel length of individuals with TT and GT genotypes was significantly longer than that of individuals with GG genotype (P < 0.05).

[0033] Haploview software was used to analyze the linkage disequilibrium (LD) of the 10 SNPs loci. The results of linkage disequilibrium analysis are shown in Figure 3 . Figure 3 The value R in the box 2 It is obtained by multiplying the D' value by 100. Figure 3 It can be seen that SNP2 and SNP3 are in a strong linkage state (D' value = 1, R 2 =100), the other 8 SNPs were not in a strong linkage state (D' value <1, R 2 <100), the linkage between SNP2 and SNP3 produced two haplotypes: H1 (AG) and H2 (GA), with haplotype frequencies of 0.893 and 0.098, respectively. Therefore, only the combined genotype of SNP2 and SNP3 was analyzed for correlation with keel length, and the combined genotype of 8 molecular markers was no longer analyzed for correlation with keel length. The combination of the two haplotypes produced by the linkage of SNP2 and SNP3 produced five genotypes (Table 4). As can be seen from Table 4, the association analysis found that the keel length of the H1H1 haplotype (GGAA genotype) was significantly higher than that of other haplotype combinations.

[0034] The TT and CT genotypes of SNP1, the GGAA genotype resulting from the linkage between SNP2 and SNP3, the AA genotype of SNP4, the GG genotype of SNP5, the CC genotype of SNP6, the GG genotype of SNP7, the TT genotype of SNP8, the CC and TC genotypes of SNP9 and the TT and GT genotypes of SNP10 are the dominant genotypes for keel length and can serve as important molecular markers for molecular-assisted breeding of keel length.

[0035] In the breeding of keel long, the method of retaining individuals with superior genotypes at 10 SNP loci (SNP1-SNP10) and eliminating individuals with inferior genotypes at SNP1-SNP10 can be used to assist in improving the selection of keel long and accelerate the progress of keel long generation breeding.

[0036] Table 3 Association analysis between gene loci and keel length traits (mean ± SD)

[0037] Table 4 Association analysis between haplotype combinations of SNP2 and SNP3 and keel length traits (mean ± SD)

[0038] Example 2 Verification of molecular marker-assisted breeding for keel length At 60 days of age, the chickens in the terminal paternal line A of Lihua Huangjiao Ma chicken were genotyped, and the dominant genotype individuals with larger keel length were retained. The specific plan is as follows: (1) At 60 days of age, blood was collected from the wing vein of 800 A-line roosters using a disposable syringe, and DNA was extracted using the phenol-chloroform method to extract the total genomic DNA of the chickens to be tested; PCR amplification primers: DNA template sequence information was downloaded from the NCBI website, and primers for 10 SNP sites of related genes (rs315701680, rs738740137, rs317223723, rs732443622, rs315667756, rs314381113, rs732811384, rs314197610, rs13782000, rs737401141) were designed using primer premier software. The relevant information of primer sequences is shown in Table 5.

[0039] Table 5 Primer sequence related information

[0040] (2) PCR amplification, electrophoresis and sequencing genotyping: The PCR amplification products were analyzed by 1.5% agarose gel electrophoresis and sequenced for genotyping. Genotyping was performed on the A-line male roosters of Lihua Mahuang Chicken, and the dominant genotype individuals with longer keel length were retained.

[0041] PCR total reaction system 50 μL: DNA template 4 μL, dNTP (2 mmol / L) 2 μL, Mg 2+ (3 mmol·L -1 ) 0.6 μL, 1× PCR reaction buffer 5 μL, upstream and downstream primers (10 μmol·L -1 ) 1 μL each, Taq polymerase (1U·μL -1 ) 2.5 μL, add ultrapure water to 50 μL.

[0042] PCR reaction program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles; 72℃ extension for 5 min. The PCR amplified target fragment was detected by 1.5% agarose gel electrophoresis.

[0043] The PCR amplification products were sent to a sequencing company for sequence polymorphism detection. The nucleotide sequences of the PCR products of the 10 SNPs sites are as follows: SNP1—C / T mutation at 199bp, PCR product length is 223bp: CACCAAGCGTGCATAAGAGATTGAATGAGTTCAGCATTAATAACAGGGGCTGTCCTTCTCATTACACCGACTGCCATTTCTAAAAGGCGTGTTTTAATTAATAAGGAAGCTGAAGAAAAGCATTCAGAGGAAGCACTGTCATTAGTGCAGACGGGTAAGCACAGTGTGTCATGCACGCACTTGGGCAGAACTTTTAATK(C / T)TGCTCCATCCACGACTCTTCAGGT SNP2 - G / A mutation at 143 bp, PCR product length is 224 bp: CCTGCAGGGAGGAATAACAACTTCAGGTTTTTGAGGTCTCACTCATGTCACAGAGGTAATAGTGTACCTTGAACTCAGCATCCCTGTTACAGCTTCCTCTAGTGGAGCTATGCAGCAAAGCGTTCTCTCTTCGTCCTTAGAGK(G / A)GAAATGGGCTATGTGGCAGTATGGTTCATCTGCCCAGTTTCAGCTCCTCGTGGCTAACATTAATGGTGCTGTAAAGGTGCC SNP3 - A / G mutation at 112 bp, PCR product length is 219 bp: GAAATGGGCTATGTGGCAGTATGGTTCATCTGCCCAGTTTCAGCTCCTCGTGGCTAACATTAATGGTGCTGTAAAGGTGCCCAGCCAACAATGCCTGTGCAGAGCGGTCACK(A / G)TTTCAGGTATAGTTACCAGTGGCATTGGTTGCCCCAGGATGCCTCTTGGAGTTGAGGACACTGGGTAGCACTGTTTCCACAGAGAAAAACAAAGGCACAAGTCCTGG SNP4 - G / A mutation at 157 bp, PCR product length is 248 bp: TGAACCCATAGGAGTTTTCCAGCTGAGCAATTACTGAAATGTCAGGATGCCTAGTCTGAATTTGCACATAAAAATGGTATACATGCATTAAAACTTAGCCTTCAAAATTATCTTGTTAAATAGTTAGAAATTAGATAGTGTAAAACAACTGACAATK(G / A)AGAAGGGAAAAGAAAAAAAAGATATTTTTAGTATGAAATACATGTATAATTGCAACTGGTGACTTTTACCTGTAAGATGGTATGCGCTGCA SNP5 - A / G mutation at 136 bp, PCR product length is 219 bp: GAGGGGTTGAAACTGGATGATCATTGTGGTCCTTTTCAACTCAGGCCATTCTATGATTCTATGATTCTGTATCCTGAAGGAAGTCATAAAATCAGATCATATGATAAAATTACTAACGTAAGAGGATTTGATAGGK(A / G)CTAACAACATCAAATATTTGATACGATCAGATTTGAATTTTCACATAGAATAGTCCTGCAAAATATCTGTGCACCCAGCTTTG SNP6 - A / G mutation at 180 bp, PCR product length is 237 bp: GCGTCAGTGGGAATAAGCTCAACAGACCAAGCTTATTAGAAATTCATGAACAACTGGCAAAGGTGAGTGAAAAGGAAACATTTGATTTTTCTCTGTTCCATTTCCAGGCTGTTAATTCTGTTTATTAATATGAAAAAGTCTTTAAATTCACGGATCTTGCCCAAGACAAAAGTACAACTK(C / A)ATCACTTACCATATTTCCTGTGGTAGCTGCAGTATGTTTTCTGCCCTGTAACCTGCT SNP7 - A / G mutation at 63 bp, PCR product length is 227 bp: CTGAAAGGGAGTCAGCCAAGAATCCAAGTAGGCCAATGATAAGCTTAGCACATTAGAAAGTTK(A / G)AAATATAAACATGACTCTTATAATTCCAGAAAATAACTCAAATTGTAGGCAAGTATTTTAAAATGCTGTACTTGCAGGCAAAGTAATTGCAGTAAATAATCAATGTAAAAGAGATCAAATATATAAAAACAACCTTCTGAAAACTGCTTAGTGCTGCTTTTGGA SNP8 — G / T mutation at 102 bp, PCR product length is 202 bp: TGCTGAAAATGTGTTGCTGAAGGAAACCAGTTTATAGGAATACACCATTATACTGGTGCTACCCATAAGGTAGGGATCAGAAACTGACAAAAACTGGTGATK(G / T)TTCTTATCTCAGAGATTTTCTCTCTTGATTTTAAGCAGGAAATCTGCAGACTACAACAGGCATGAGACAAGTCTTCTGCATGGAACAGAAACACCTGCAG SNP9 — T / C mutation at 60 bp, PCR product length is 233 bp: CTGCAGGGTTTGGAGCAGGCTGGCGTCCCTGCTTGGCGCCCTAGAGTGGAACCTGGCAGK(T / C)CAAACGGAAAACGTAAAGCTGGGAGAAGATACGTGTGTGCCTGAACAACGTCGCGAGCGTCCTGGCAGGATGGAAGCGGGGATTGCTGGGCTGTGGCTGTGGGGCAGACGCGGGCCTGAGCCCTACATCCCGCTCTGCTGCCGCCGCTGCGCTCCTTGCTCTGAGCCCATCAG SNP10 — G / T mutation at 171 bp, PCR product length is 218 bp: GAGCAGGAGGGGGAAGTGCCGTGCGGTTCCCATCGCTGCCACCACCGCCCGCCGCCCAAGCGCTACTGCGGCCCCCGCGCCCCCTGCGCGGCGCGATGGGAGCCGCCTCACCAAGCCCCGCCCCGCAGCCAATGGGAGCGCGGCGGAGGTGGGACTCCGGGGAACGAGGGGK(G / T)ATTGGGCGAGAGCCACACCCGCCTCGCCGTTCTCGGCCAATGGGAAG Note: The K marked in the above sequence is the mutation site, and the mutated bases in brackets are allele mutations.

[0044] (3) Method for measuring keel length at 60 days of age: Use a vernier caliper to measure the distance from the front end to the end of the keel. All keel length measurements are made by the same person, and the measured parts are basically the same.

[0045] (4) Molecular marker-assisted breeding for keel length The TT and CT genotypes of SNP1, the GGAA genotype resulting from the linkage between SNP2 and SNP3, the AA genotype of SNP4, the GG genotype of SNP5, the CC genotype of SNP6, the GG genotype of SNP7, the TT genotype of SNP8, the CC and TC genotypes of SNP9 and the TT and GT genotypes of SNP10 were the dominant genotypes for keel length.

[0046] In the molecular marker-assisted breeding of keel length, the method of retaining individuals with superior genotypes at SNP1-SNP10 loci and eliminating individuals with inferior genotypes is used to assist in improving the selection of keel length and accelerate the progress of generational selection of keel length.

[0047] Through whole genome association analysis, 10 SNP molecular markers significantly associated with keel length were screened. The molecular marker-assisted breeding verification experiment on the keel length of the 5th and 6th generations of Lihua Huangjiao Ma Chicken's terminal paternal line A showed that the operation is simple and can improve the keel length more quickly. As shown in Table 6, after 2 generations of breeding, the keel length of roosters in the 5th generation increased by 1.18mm compared with the 4th generation, and the 6th generation increased by 1.02mm compared with the 5th generation, with an average increase of 1.1mm per generation. Compared with the 2nd to 4th generations, the keel length of each generation increased by about 0.5mm, the keel length of the 5th and 6th generations increased significantly, the coefficient of variation also decreased significantly, and the uniformity was significantly improved, which accelerated the progress of keel length breeding.

[0048] Table 6 Results of keel length measurement in different generations of Lihua Mahuang chicken strain A

[0049] Measurement age: 60 days The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0050] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0051] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Application of a SNP molecular marker combination in keel length assisted breeding, characterized in that: The SNP molecular marker combination includes the following 10 SNP sites SNP1 to SNP10: SNP1 corresponds to position 170194700 of the positive strand of chromosome 1 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a C or T polymorphism and is numbered rs315701680; SNP2 corresponds to position 15305633 of the positive strand of chromosome 3 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a G or A polymorphism and is numbered rs738740137; SNP3 corresponds to position 15305745 of the positive strand of chromosome 3 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is an A or G polymorphism and is numbered rs317223723; SNP4 corresponds to position 78036233 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a G or A polymorphism and is numbered rs732443622; SNP5 corresponds to position 78750083 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is an A or G polymorphism and is numbered rs315667756; SNP6 corresponds to position 10111788 of the positive strand of chromosome 10 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a C or A polymorphism and is numbered as rs314381113; SNP7 corresponds to position 10468445 of the positive strand of chromosome 10 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is an A or G polymorphism and is numbered rs732811384; SNP8 corresponds to position 10534417 of the positive strand of chromosome 10 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a G or T polymorphism and is numbered as rs314197610; SNP9 corresponds to position 13797770 of the positive strand of chromosome 14 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, which is a T or C polymorphism and is numbered as rs13782000; SNP10 corresponds to position 1536829 of the positive strand of chromosome 24 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI. It is a G or T polymorphism and is numbered rs737401141.

2. A breeding method for increasing keel length, characterized in that: The steps include: (1) Determining the genotype of the chicken to be bred, wherein the genotype is the genotype of the SNP molecular marker combination according to claim 1; (2) Select individuals with the dominant genotypes of TT and CT at SNP1, the dominant genotype of GGAA resulting from the linkage between SNP2 and SNP3, the dominant genotype of AA at SNP4, the dominant genotype of GG at SNP5, the dominant genotype of CC at SNP6, the dominant genotype of GG at SNP7, the dominant genotype of TT at SNP8, the dominant genotypes of CC and TC at SNP9, and the dominant genotypes of TT and GT at SNP10.

3. The breeding method according to claim 2, characterized in that: In step (1), the method for determining the genotype of the chicken to be bred is: (1.1) Extracting the total genomic DNA of the chicken to be tested; (1.2) According to the SNP molecular marker combination, the target sequence is amplified by PCR method using the corresponding primer pair, and the sequence of the primer pair is: SNP1F: 5' CACCAAGCGTGCATAAGAGA3' SNP1R: 5' ACCTGAAGAGTCGTGGATGG3' SNP2F: 5' CCTGCAGGGAGGAATAACAA3' SNP2R: 5' GGCACCTTTACAGCACCATT3' SNP3F: 5' GAAATGGGCTATGTGGCAGT3' SNP3R: 5' CCAGGACTTGTGCCTTTGTT3' SNP4F: 5' TGAACCCATAGGAGTTTTCCA3' SNP4R: 5'TGCAGCGCATACCATCTTAC3' SNP5F: 5' GAGGGGTTGAAACTGGATGA3' SNP5R: 5'CAAAGCTGGGTGCACAGATA3' SNP6F: 5'GCGTCAGTGGGAATAAGCTC3' SNP6R: 5'AGCAGGTTACAGGGCAGAAA3' SNP7F: 5' CTGAAAGGGAGTCAGCCAAG3' SNP7R: 5'TCCAAAAGCAGCACTAAGCA3' SNP8F: 5' TGCTGAAAATGTGTTGCTGA3' SNP8R: 5'CTGCAGGTGTTTCTGTTCCA3' SNP9F: 5' CTGCAGGGTTTGGAGCAGGC3' SNP9R: 5'CTGATGGGCTCAGAGCAAG3' SNP10F: 5'GAGCAGGAGGGGGAAGTG3' SNP10R: 5'CTTCCATTGGCCGAGAAC3'; (1.3) After sequencing the PCR amplification product, determine the genotype.

4. The breeding method according to claim 3, characterized in that: The nucleotide sequences of the PCR amplification products are shown in SEQ ID NO.1 to SEQ ID NO.10, and the lengths of the PCR products are 223 bp, 224 bp, 219 bp, 248 bp, 219 bp, 237 bp, 227 bp, 202 bp, 233 bp, and 218 bp.

Images