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

Abstract

The invention discloses an application of an SNP molecular marker combination in oblique body length assisted breeding, the SNP molecular marker combination comprises 10 SNP sites SNP1-SNP10, and in oblique body length assisted breeding, the SNP molecular marker combination can be obtained by selecting and remaining an AA dominant genotype of an SNP1 site, AA and GA dominant genotypes of an SNP2 site, GG dominant genotypes of an SNP3 site and AA and GA dominant genotypes of an SNP4 site. According to the method, a CCCC dominant genotype individual, an SNP7 CC dominant genotype individual, an SNP8 CC dominant genotype individual, an SNP9 AA dominant genotype individual and an SNP10 CC dominant genotype individual are generated after SNP5 and SNP6 loci are linked, and a method for eliminating SNP1-SNP10 locus disadvantageous genotype individuals is used for assisting in improving the breeding of the oblique length of the body and accelerating the breeding progress of the generation of the oblique length of the body.

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 body elongation-assisted breeding. Background Art

[0002] Poultry body size index is an important phenotypic trait for breed selection. It is significantly correlated with poultry meat performance such as body weight, carcass weight, and eviscerated weight. It is a commonly used indicator for evaluating poultry weight, body shape, and slaughter performance, and can reflect the growth and development of poultry to a certain extent. As an important body size trait, the growth and development of body slant length has a direct or indirect relationship with many economic traits such as body weight, body shape, and slaughter performance. It is also one of the candidate traits for early selection of chicken growth traits. The development of body slant length is a quantitative trait controlled by multiple genes.

[0003] At present, the process of body skew length breeding is usually carried out by directly measuring body skew length or observing body skew length with the naked eye, and the generational genetic progress is slow. Therefore, finding molecular markers significantly related to body skew length is of great significance for accelerating the progress of generational breeding of body skew length. Summary of the invention

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

[0005] In order to achieve the above object, the present invention provides an application of a SNP molecular marker combination in body length assisted 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 body skew length. The univariate analysis of variance in the general linear model of SPSS16.0 software was used to perform association analysis and verification of the genotype of the polymorphic loci and body skew length, 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 body oblique length molecular markers through GWAS and obtained 10 SNP molecular markers that were significantly correlated with body oblique 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 the body oblique 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 AA dominant genotype at SNP1, the AA and GA dominant genotypes at SNP2, the GG dominant genotype at SNP3, the AA and GA dominant genotypes at SNP4, the CCCC dominant genotype resulting from the linkage between SNP5 and SNP6, the CC dominant genotype at SNP7, the CC dominant genotype at SNP8, the AA dominant genotype at SNP9, and the CC dominant genotype 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' AGGGAGTTCTGGATTCTTGCT3' SNP1R: 5' CAACACCCAAAGATTGAGACC3' SNP2F: 5'CATTCCCCCTTGTCCTATCA3' SNP2R: 5'TTCTGTGGTGGAATTGCTCA3' SNP3F: 5' GATGCTGGGGATGCTACTGT3' SNP3R: 5' CATCCCACCATGTTTCTGTG3' SNP4F: 5' GCCTGTTGCTCTCCATTGAT3' SNP4R: 5'AGTGGCACACAGCAGCATAC3' SNP5F: 5' TGAGGCAAGATCAAGTGCAG3' SNP5R: 5'TCATCCAGTCCAACCATTCA3' SNP6F: 5' AAATGGGCCAGTGTTTTCAG3' SNP6R: 5'GAAGGCAAGTCTGAGCAAGG3' SNP7F: 5' TTCTGTAAGCCTGCATACTG3' SNP7R: 5'CGGATGCTGCACTGTAGAAA3' SNP8F: 5' AGGACGGTTGGAATCTGATG3' SNP8R: 5'GCATGGAAGTTTGGTTGACTG3' SNP9F: 5' TGCAGAAGGATGCAGAGAGA3' SNP9R: 5'GGAAGATTAGTCTTGGGCAAA3' SNP10F: 5'TGGGGGAGATGAAAACAG3' SNP10R: 5'TGATATTGGTGGCAGGTGAA3'; (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—G / A mutation at 59bp, PCR product length 220bp AGGGAGTTCTGGATTCTTGCTAGAAATCATTATTCTCTTTTGAAACTTCTACATCTATK(G / A)TCTCCTAAGAAGTGTATGGAATGTTTTGTTTAAGAAATGTGCCACCTGTTGTGATCAGACATGCTGCCTGAAAAATGGAAATCGATTGATAAATTCAATATATCTAAATTCCTTCTTCCTTTGACTTTAAAATAAAATTAGGTCTCAATCTTTGGGTGTTG.

[0012] SNP2—A / G mutation at 161 bp, PCR product length is 210 bp CATTCCCCCTTGTCCTATCACTATCCACCCTTGTAAACAGACATTCCCTCTCCTGTTTATATGCTCCCTTCAAGTATTGGAAGGCCACAATAAGGTCTCCCCGGAGCCTTCTTTTCCAAGCTAAAAGACGAATCATACATCTTTTAAAAAAAGAGAACK(A / G)CAAACATCATGAGAAGGTGACCAATTTGGTGAGCAATTCCACCACAGAA.

[0013] SNP3—G / A mutation at 114 bp, PCR product length is 184 bp GATGCTGGGGATGCTACTGTTCTAGGGGATCCTAGATCCTCTATAAAGGCGGTGAGACGGAAAGCCCAGCTGAAGTGCCTTTTATACCGGTGCACACAGCCTGAGTAATAAACAK(G / A)GACAAGTTGGAAACTGTGATGCACTTGGAAAGTTATGACCTTATTGCTATCACAGAAACATGGTGGGATG.

[0014] SNP4-34bp G / A mutation, PCR product length is 240 bp GCCTGTTGCTCTCCATTGATTGCAGAAAGCACTK(G / A)TGTGAAACTACCTGAATTGTACTGATGGCAAAGAGAACTCAGCGTGGTTTTAACCCATATCTACCCTGACCATGCTTTAACCCAGACAATTCATGAGCACAGCATTAATTACAGGCCAAAGAGCAGTGCCAGCAGGCAGACAATCTGGCCGTGTGCTTTCATGGATAAGACTATGTTATAATATGTGTATGCTGCTGTGTGCCACT。

[0015] SNP5 - T / C mutation at 74 bp, PCR product length is 230 bp TGAGGCAAGATCAAGTGCAGCATAAAAACTAGTCTGAAAGCTCTCAACCTCCAGTTGGATGTTGGGAAAAAATK(T / C)CTTCTCTGAAAGAGTGGTCACACGCTGGAATGGGCTTCCCAGGGAGGTGGTGGGGTCACCATCTCTGGAGGTTTTCAAGAAATGTGTAAATGTGGTAGTAAGGGACATGGTTTAGCAGGGAAATATTGGTGGTAGGTGAATGGTTGGACTGGATGA。

[0016] SNP6 - G / C mutation at 110 bp, PCR product length is 192 bp AAATGGGCCAGTGTTTTCAGCTTATTTTGAGTTTCCCGTATTGGATTACCAGTTGCTGTTGGTTACTTACTTTGAAGAGGTCTTTCTTGCTGAAATTGCCCATGAAACAK(G / C)CTAAGTCTCTGATTTTGTGAATTATATCTTAGTGCAACCTTTCTCGATGATTGAGTTGCTGGCCTTGCTCAGACTTGCCTTC。

[0017] SNP7 - T / C mutation at 92 bp, PCR product length is 238 bp TTCTGTAAGCCTGCATACTGTTTATTCCTTACAGTGTAAAAAAAACCTTTTCTTTCCGACATTGCAGTTTCAGAGCTATTTCGGTCTTTCAK(T / C)CCTCATCCCCTGTGAGAAAGAAAGGTGGGAACAGAAGAAAACTTTTTTCCCTCTTCCGTACTTTAGAGAATTCTCCTGCTGAAGGGTTCTTCCTTTTTCTTACCAGCTCCATAGGAATCTGGCTAATTTCTACAGTGCAGCATCCG。

[0018] SNP8 - C / T mutation at 82 bp, PCR product length is 223 bp AGGACGGTTGGAATCTGATGGTCCTTGAGGTTCCTTCTAATCCAAGTCGTTCTATGACTCTCCATTATAAGAATTTTAGCCK(C / T)ATCTCTTTGTTTCATGTCCTGTGTATATTCTAGTATTACAGGCTTTTTTGTGTTCTTTTTTTTCCCCCCTTACTATTTACCTTCTCATGTTTTACTCCATAGATGCTCTCATCTTAATTACAGTCAACCAAACTTCCATGC。

[0019] SNP9 - A / G mutation at 77 bp, PCR product length is 246 bp TGCAGAAGGATGCAGAGAGAAATAAAAGCAGGAAGAATGTTTGAGGTCAAAAACTTGAAAAAATTCAGCATTGTATK(A / G)TATGTCTTTAGATATTTATTCAAATTATGTAAGGGAAAAAATTGGACAAAACCACTCAGTACATAAGATCTACCACCTGATACTCTTTTCATCATGATTTATACAGATATACACCTAAAACTTTCTCTTCTCCTATAAAGATCTACATTTTGCCCAAGACTAATCTTCC。

[0020] SNP10 - C / T mutation at 51 bp, PCR product length is 229 bp TGGGGGAGATGAAAACAGCTCCTCTATTTAGGACTTTTATCAGGCCTGK(C / T)CCAGGAATCTCAACAGGAGCAGCTATAGAGGCATGTGGTGAGCACTACAGAAACTACAGATACTGAAAACAGGAAAAGATGAGACTTCTAACACAGAGACATAGAACCACAAATCTGTTAAGGATGGAAAAGACTTAGACAATCATCTAGTCCAACCGTTCACCTGCCACCAATATCA.

[0021] Note: The K marked in the above sequence is the mutation site, and the mutated bases in brackets are allele mutations.

[0022] The specific method for determining the dominant genotype individuals is as follows: Firstly, the correlation between single SNP markers and body skew length was analyzed. The body skew lengths of different genotypes of the 10 SNPs loci were significantly different. SNP1 (rs312577341) had three genotypes: AA, GA, and GG. The body skew lengths of individuals with the AA genotype were significantly greater than those with the GA and GG genotypes (P < 0.05). SNP2 (rs313388213) had three genotypes: AA, GA, and GG. The body skew lengths of individuals with the AA and GA genotypes were significantly greater than those with the GG genotype (P < 0.05). ; SNP3 (rs730967108) has three genotypes: AA, AG, and GG. The body length of individuals with the GG genotype was significantly greater than that of individuals with the AA and AG genotypes (P < 0.05); SNP4 (rs315520013) has three genotypes: AA, GA, and GG. The body length of individuals with the AA and GA genotypes was significantly greater than that of individuals with the GG genotype (P < 0.05); SNP5 (rs317946632) has three genotypes: CC, TC, and TT. The body oblique length of the body was significantly greater than that of the TC and TT genotypes (P < 0.05); SNP6 (rs315619998) had three genotypes CC, GC, and GG, among which the body oblique length of the CC genotype individuals was significantly greater than that of the GC and GG genotypes (P < 0.05); SNP7 (rs317342517) had three genotypes CC, TC, and TT, among which the body oblique length of the CC genotype individuals was significantly greater than that of the TC and TT genotypes (P < 0.05); SNP8 (rs313349990) There are two genotypes, CC and CT, among which the body skew length of individuals with CC genotype is significantly greater than that of individuals with CT genotype (P < 0.05); SNP9 (rs731600098) has three genotypes, AA, AG, and GG, among which the body skew length of individuals with AA genotype is significantly greater than that of AG and GG genotypes (P < 0.05); SNP10 (rs13792170) has three genotypes, CC, CT, and TT, among which the body skew length of individuals with CC genotype is significantly greater than that of individuals with CT and TT genotypes (P < 0.05).

[0023] Haploview software was used to analyze the linkage disequilibrium (LD) of the 10 SNPs loci. The results of the linkage disequilibrium analysis showed that SNP5 and SNP6 were in a strong linkage state (R 2=94), the linkage between SNP5 and SNP6 produced three haplotypes: H1 (TG), H2 (CC) and H3 (CG), with haplotype frequencies of 0.795, 0.177 and 0.019, respectively. The other eight SNP sites were not in a strong linkage state, so only the combined genotype of SNP5 and SNP6 was analyzed for correlation with body oblique length, and the combined genotype of the other eight molecular markers was no longer analyzed for correlation with body oblique length. After the three haplotypes generated by the linkage of SNP5 and SNP6 were combined, five genotypes were generated. After association analysis, it was found that the body oblique length of the H2H2 haplotype (CCCC genotype) was significantly higher than that of other haplotype combinations.

[0024] The AA genotype at SNP1, the AA and GA genotypes at SNP2, the GG genotype at SNP3, the AA and GA genotypes at SNP4, the CCCC genotype resulting from the linkage of SNP5 and SNP6, the CC genotype at SNP7, the CC genotype at SNP8, the AA genotype at SNP9 and the CC genotype at SNP10 are the dominant genotypes for body oblique elongation and can serve as important molecular markers for molecular-assisted breeding of body oblique elongation.

[0025] Through the above technical solution, the present invention achieves the following beneficial effects: In molecular marker-assisted breeding of body skew length, the method of retaining individuals with superior genotypes at 10 SNP loci (SNP1~SNP10) and eliminating individuals with inferior genotypes at SNP1~SNP10 loci can assist in improving the selection of body skew length and accelerate the progress of generational selection of body skew length. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Manhattan plots for body-plaque-length-related molecules; Figure 2 The QQ plot is the molecular marker for body oblique length correlation; Figure 3 This is a linkage disequilibrium analysis diagram of 10 SNP molecular markers; Figure 4 Haplotype frequencies of three haplotypes were generated for SNP5 and SNP6 linkage. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Screening for Molecular Markers Significantly Associated with Body Plagiogranth 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, comb development, feed conversion rate and slaughter rate. It has undergone six generations of closed breeding. The experimental chickens were raised in Jiangsu Lihua Breeding Co., Ltd., 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.

[0029] 2. Determination of body oblique length 400 roosters from the specialized strain A of Lihua yellow-footed chicken were randomly selected and weighed after fasting for 12 hours. The foot numbers were recorded and the body oblique length of the 60-day-old roosters was measured. The specific measurement method was as follows: the distance from the shoulder joint to the ischial tuberosity on the same side was measured with a tape measure. All body oblique length measurements were made by the same person and the measured parts were basically the same.

[0030] 3. Mining of molecular markers of body plagioclase At 60 days of age, 1.5 mL of blood was collected from the subwing vein of all groups whose combs 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] y=Wα+xβ+u+

[0035] 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 ), τ-1 is the residual variance, K is the kinship matrix calculated based on SNPs using GEMMA, and In is the identity matrix.

[0036] 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 valid independent test SNPs 432529), then the p value is taken as the negative logarithm of log10, 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.

[0037] After quality inspection, genome-wide association analysis was performed on the plagioclase phenotype values ​​of 366 chickens. The results are as follows Figure 1 , Figure 2 As shown in the Manhattan plot, there are 10 SNP sites with significant genome correlation (threshold value above 5.63) on chromosomes 1, 4, 7, and 11 of chicken, and all of them are annotated as potential related SNP sites (threshold value above 5.63). The QQ plot further verifies the reliability of the GWAS results. The significantly correlated body plagioclase molecular markers screened are summarized as shown in Table 1: Table 1 Molecular markers significantly associated with body length

[0038] The physical position of the marker chromosome is referenced to the chicken whole genome (bGalGal1.mat broiler .GRCg7b).

[0039] 4. Genetic polymorphism analysis of 10 SNPs significantly associated with body 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.

[0040] As shown in Table 2, among the 10 SNP loci, except for SNP8 which has 2 genotypes, the other 9 SNP loci have 3 genotypes. After HW equilibrium detection, among the 10 SNPs loci, except for SNP3, SNP4, and SNP10 which deviate from the HW equilibrium state (P<0.05), the other 7 SNP loci are in the HW equilibrium state (P>0.05). The genetic diversity of 3 SNP loci (SNP8, SNP9, and SNP10) is low (He<0.25), and the other 7 SNP loci (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7) are moderately genetically diverse (0.25 <He<0.50)。

[0041] Table 2 Genetic polymorphism of 10 SNP loci and Hardy-Weinberg equilibrium test

[0042] 5. Analysis of dominant genotypes of body length SNP molecular markers The association analysis between polymorphic locus genotype and body skew 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: body skew length, LSD method was used to compare the significance of body skew length between different marker genotypes, P < 0.05 indicated significant difference.

[0043] First, the correlation between single SNP markers and body skew length was analyzed. The body skew lengths of different genotypes of the 10 SNPs loci were significantly different, as shown in Table 3. SNP1 (rs312577341) has three genotypes: AA, GA, and GG. The body skew length of individuals with the AA genotype was significantly greater than that of the GA and GG genotypes (P < 0.05). SNP2 (rs313388213) has three genotypes: AA, GA, and GG. The body skew length of individuals with the AA and GA genotypes was significantly greater than that of the GG genotype (P < 0.05); SNP3 (rs730967108) has three genotypes: AA, AG, and GG. The body length of individuals with the GG genotype is significantly longer than that of individuals with the AA and AG genotypes (P < 0.05); SNP4 (rs315520013) has three genotypes: AA, GA, and GG. The body length of individuals with the AA and GA genotypes is significantly longer than that of individuals with the GG genotype (P < 0.05); SNP5 (rs317946632) has three genotypes: CC, TC, and TT. The body skew length of individuals with this genotype was significantly greater than that of TC and TT genotypes (P < 0.05); SNP6 (rs315619998) had three genotypes, CC, GC, and GG, among which the body skew length of individuals with CC genotype was significantly greater than that of GC and GG genotypes (P < 0.05); SNP7 (rs317342517) had three genotypes, CC, TC, and TT, among which the body skew length of individuals with CC genotype was significantly greater than that of TC and TT genotypes (P < 0.05); SNP8 (rs313349990 ) has two genotypes, CC and CT, among which the body skew length of individuals with CC genotype was significantly greater than that of individuals with CT genotype (P < 0.05); SNP9 (rs731600098) has three genotypes, AA, AG, and GG, among which the body skew length of individuals with AA genotype was significantly greater than that of AG and GG genotypes (P < 0.05); SNP10 (rs13792170) has three genotypes, CC, CT, and TT, among which the body skew length of individuals with CC genotype was significantly greater than that of individuals with CT and TT genotypes (P < 0.05).

[0044] 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 box2 It is obtained by multiplying the D' value by 100. Figure 3 It can be seen that SNP5 and SNP6 are in a strong linkage state (R 2 =94), the linkage between SNP5 and SNP6 produced three haplotypes: H1 (TG), H2 (CC), and H3 (CG), with haplotype frequencies of 0.795, 0.177, and 0.019, respectively (e.g. Figure 4 As shown in Table 4). The other 8 SNP sites are not in a strong linkage state, so only the combined genotype of SNP5 and SNP6 is analyzed for correlation with body oblique length, and the combined genotype of the other 8 molecular markers is no longer analyzed for correlation with body oblique length. After the three haplotypes generated by the linkage of SNP5 and SNP6 are combined, 5 genotypes are generated, and the results are shown in Table 4. As can be seen from Table 4, the association analysis found that the body oblique length of the H2H2 haplotype (CCCC genotype) is significantly higher than that of other haplotype combinations.

[0045] The AA genotype at SNP1, the AA and GA genotypes at SNP2, the GG genotype at SNP3, the AA and GA genotypes at SNP4, the CCCC genotype resulting from the linkage of SNP5 and SNP6, the CC genotype at SNP7, the CC genotype at SNP8, the AA genotype at SNP9 and the CC genotype at SNP10 are the dominant genotypes for body oblique elongation and can serve as important molecular markers for molecular-assisted breeding of body oblique elongation.

[0046] In the breeding of oblique body length, the method of retaining individuals with superior genotypes and eliminating individuals with inferior genotypes at SNP1~SNP10 sites can be used to assist in improving the selection of oblique body length and accelerate the progress of generational selection of oblique body length.

[0047] Table 3 Association analysis between gene loci and body length traits (mean ± SD)

[0048] Table 4 Association analysis between haplotype combination of SNP5 and SNP6 and body length (mean ± SD)

[0049] Example 2 Verification of molecular marker-assisted breeding for plagioclase 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 body oblique 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 (rs312577341, rs313388213, rs730967108, rs315520013, rs317946632, rs315619998, rs317342517, rs313349990, rs731600098, rs13792170) were designed using primer premier software. The relevant information of primer sequences is shown in Table 5.

[0050] Table 5 Primer sequence related information

[0051] (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 chickens, and the dominant genotype individuals with larger body oblique length were retained.

[0052] 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.

[0053] 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.

[0054] 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—G / A mutation at 59bp, PCR product length 220bp AGGGAGTTCTGGATTCTTGCTAGAAATCATTATTCTCTTTTGAAACTTCTACATCTATK(G / A)TCTCCTAAGAAGTGTATGGAATGTTTTGTTTAAGAAATGTGCCACCTGTTGTGATCAGACATGCTGCCTGAAAAATGGAAATCGATTGATAAATTCAATATATCTAAATTCCTTCTTCCTTTGACTTTAAAATAAAATTAGGTCTCAATCTTTGGGTGTTG.

[0055] SNP2—A / G mutation at 161 bp, PCR product length is 210 bp CATTCCCCCTTGTCCTATCACTATCCACCCTTGTAAACAGACATTCCCTCTCCTGTTTATATGCTCCCTTCAAGTATTGGAAGGCCACAATAAGGTCTCCCCGGAGCCTTCTTTTCCAAGCTAAAAGACGAATCATACATCTTTTAAAAAAAGAGAACK(A / G)CAAACATCATGAGAAGGTGACCAATTTGGTGAGCAATTCCACCACAGAA.

[0056] SNP3—G / A mutation at 114 bp, PCR product length is 184 bp GATGCTGGGGATGCTACTGTTCTAGGGGATCCTAGATCCTCTATAAAGGCGGTGAGACGGAAAGCCCAGCTGAAGTGCCTTTTATACCGGTGCACACAGCCTGAGTAATAAACAK(G / A)GACAAGTTGGAAACTGTGATGCACTTGGAAAGTTATGACCTTATTGCTATCACAGAAACATGGTGGGATG.

[0057] SNP4-34bp G / A mutation, PCR product length is 240 bp GCCTGTTGCTCTCCATTGATTGCAGAAAGCACTK(G / A)TGTGAAACTACCTGAATTGTACTGATGGCAAAGAGAACTCAGCGTGGTTTTAACCCATATCTACCCTGACCATGCTTTAACCCAGACAATTCATGAGCACAGCATTAATTACAGGCCAAAGAGCAGTGCCAGCAGGCAGACAATCTGGCCGTGTGCTTTCATGGATAAGACTATGTTATAATATGTGTATGCTGCTGTGTGCCACT。

[0058] SNP5 - T / C mutation at 74 bp, PCR product length is 230 bp TGAGGCAAGATCAAGTGCAGCATAAAAACTAGTCTGAAAGCTCTCAACCTCCAGTTGGATGTTGGGAAAAAATK(T / C)CTTCTCTGAAAGAGTGGTCACACGCTGGAATGGGCTTCCCAGGGAGGTGGTGGGGTCACCATCTCTGGAGGTTTTCAAGAAATGTGTAAATGTGGTAGTAAGGGACATGGTTTAGCAGGGAAATATTGGTGGTAGGTGAATGGTTGGACTGGATGA。

[0059] SNP6 - G / C mutation at 110 bp, PCR product length is 192 bp AAATGGGCCAGTGTTTTCAGCTTATTTTGAGTTTCCCGTATTGGATTACCAGTTGCTGTTGGTTACTTACTTTGAAGAGGTCTTTCTTGCTGAAATTGCCCATGAAACAK(G / C)CTAAGTCTCTGATTTTGTGAATTATATCTTAGTGCAACCTTTCTCGATGATTGAGTTGCTGGCCTTGCTCAGACTTGCCTTC。

[0060] SNP7 - T / C mutation at 92 bp, PCR product length is 238 bp TTCTGTAAGCCTGCATACTGTTTATTCCTTACAGTGTAAAAAAAACCTTTTCTTTCCGACATTGCAGTTTCAGAGCTATTTCGGTCTTTCAK(T / C)CCTCATCCCCTGTGAGAAAGAAAGGTGGGAACAGAAGAAAACTTTTTTCCCTCTTCCGTACTTTAGAGAATTCTCCTGCTGAAGGGTTCTTCCTTTTTCTTACCAGCTCCATAGGAATCTGGCTAATTTCTACAGTGCAGCATCCG。

[0061] SNP8 - C / T mutation at 82 bp, PCR product length is 223 bp AGGACGGTTGGAATCTGATGGTCCTTGAGGTTCCTTCTAATCCAAGTCGTTCTATGACTCTCCATTATAAGAATTTTAGCCK(C / T)ATCTCTTTGTTTCATGTCCTGTGTATATTCTAGTATTACAGGCTTTTTTGTGTTCTTTTTTTTCCCCCCTTACTATTTACCTTCTCATGTTTTACTCCATAGATGCTCTCATCTTAATTACAGTCAACCAAACTTCCATGC。

[0062] SNP9 - A / G mutation at 77 bp, PCR product length is 246 bp TGCAGAAGGATGCAGAGAGAAATAAAAGCAGGAAGAATGTTTGAGGTCAAAAACTTGAAAAAATTCAGCATTGTATK(A / G)TATGTCTTTAGATATTTATTCAAATTATGTAAGGGAAAAAATTGGACAAAACCACTCAGTACATAAGATCTACCACCTGATACTCTTTTCATCATGATTTATACAGATATACACCTAAAACTTTCTCTTCTCCTATAAAGATCTACATTTTGCCCAAGACTAATCTTCC。

[0063] SNP10 - C / T mutation at 51 bp, PCR product length is 229 bp TGGGGGAGATGAAAACAGCTCCTCTATTTAGGACTTTTATCAGGCCTGK(C / T)CCAGGAATCTCAACAGGAGCAGCTATAGAGGCATGTGGTGAGCACTACAGAAACTACAGATACTGAAAACAGGAAAAGATGAGACTTCTAACACAGAGACATAGAACCACAAATCTGTTAAGGATGGAAAAGACTTAGACAATCATCTAGTCCAACCGTTCACCTGCCACCAATATCA.

[0064] Note: The K marked in the above sequence is the mutation site, and the mutated bases in brackets are allele mutations.

[0065] (3) Method for measuring body oblique length at 60 days of age: Use a tape measure to measure the distance from the shoulder joint to the ischial tuberosity on the same side. All body oblique length measurements are performed by the same person, and the measured locations are basically the same.

[0066] (4) Molecular marker-assisted breeding of obliquely elongated The AA genotype at SNP1, the AA and GA genotypes at SNP2, the GG genotype at SNP3, the AA and GA genotypes at SNP4, the CCCC genotype resulting from the linkage of SNP5 and SNP6, the CC genotype at SNP7, the CC genotype at SNP8, the AA genotype at SNP9 and the CC genotype at SNP10 are the dominant genotypes for body oblique elongation and can serve as important molecular markers for molecular-assisted breeding of body oblique elongation.

[0067] In molecular marker-assisted breeding of body skew 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 body skew length and accelerate the progress of generational selection of body skew length.

[0068] The molecular marker-assisted breeding verification experiment of the 5th and 6th generation body slant length of the terminal paternal line A of Lihua Huangjiao Ma Chicken showed that the operation is simple and can improve the body slant length more quickly. As shown in Table 6, after 2 generations of breeding, the body slant length of roosters in the 5th generation increased by 2.09mm compared with the 4th generation, and the 6th generation increased by 1.77mm compared with the 5th generation, with an average increase of 1.93mm per generation. Compared with the 2nd to 4th generations, the body slant length of each generation increased by about 0.75mm. The body slant 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 body slant length breeding.

[0069] Table 6 Results of the oblique length measurement of different generations of Lihua Mahuang chicken strain A

[0070] Measurement age: 60 days Example 3 Verification of molecular marker-assisted breeding for plagioclase At 77 days of age, 950 roosters of the 503 line of medium-speed yellow-feathered broiler specialized strain of Hesheng Food Group were genotyped, and the dominant genotype individuals with larger body oblique length were retained. The specific scheme and primer sequences refer to Example 2.

[0071] In molecular marker-assisted breeding of body skew 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 body skew length and accelerate the progress of generational selection of body skew length.

[0072] Molecular marker-assisted breeding was used to verify the body skew length of the 6th and 7th generations of the 503 line. As shown in Table 7, after two generations of breeding, the body skew length of roosters in the 6th generation increased by 2.20 mm compared with the 5th generation, and the 7th generation increased by 2.03 mm compared with the 6th generation, with an average increase of 2.12 mm per generation. Compared with the 3rd to 5th generations, the body skew length of each generation increased by about 0.95 mm. The body skew length of the 6th and 7th generations increased significantly, the coefficient of variation also decreased significantly, and the uniformity was significantly improved, which accelerated the progress of body skew length breeding.

[0073] Table 7 Results of the oblique length measurement of different generations of Hesheng medium-speed yellow-feathered broiler 503 line

[0074] Age at measurement: 77 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.

[0075] 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.

[0076] 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. An application of a SNP molecular marker combination in body length-assisted breeding, characterized in that: The SNP molecular marker combination includes the following 10 SNP sites SNP1 to SNP10: SNP1 corresponds to position 170308954 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 G or A polymorphism and is numbered as rs312577341; SNP2 corresponds to position 170818085 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 an A or G polymorphism and is numbered rs313388213; SNP3 corresponds to position 171080256 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 G or A polymorphism and is numbered rs730967108; SNP4 corresponds to position 171097585 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 G or A polymorphism and is numbered rs315520013; SNP5 corresponds to position 16584961 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 T or C polymorphism and is numbered as rs317946632; SNP6 corresponds to position 16586062 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 C polymorphism and is numbered as rs315619998; SNP7 corresponds to position 16615516 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 T or C polymorphism and is numbered as rs317342517; SNP8 corresponds to position 30317358 of the positive strand of chromosome 7 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 rs313349990; SNP9 corresponds to position 30409526 of the positive strand of chromosome 7 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 rs731600098; SNP10 corresponds to position 8736360 of the positive strand of chromosome 11 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI. It is a C or T polymorphism and is numbered rs13792170.

2. A breeding method for increasing body oblique 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 AA dominant genotype at SNP1, the AA and GA dominant genotypes at SNP2, the GG dominant genotype at SNP3, the AA and GA dominant genotypes at SNP4, the CCCC dominant genotype resulting from the linkage between SNP5 and SNP6, the CC dominant genotype at SNP7, the CC dominant genotype at SNP8, the AA dominant genotype at SNP9, and the CC dominant genotype 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' AGGGAGTTCTGGATTCTTGCT3' SNP1R: 5' CAACACCCAAAGATTGAGACC3' SNP2F: 5'CATTCCCCCTTGTCCTATCA3' SNP2R: 5'TTCTGTGGTGGAATTGCTCA3' SNP3F: 5' GATGCTGGGGATGCTACTGT3' SNP3R: 5' CATCCCACCATGTTTCTGTG3' SNP4F: 5' GCCTGTTGCTCTCCATTGAT3' SNP4R: 5'AGTGGCACACAGCAGCATAC3' SNP5F: 5' TGAGGCAAGATCAAGTGCAG3' SNP5R: 5'TCATCCAGTCCAACCATTCA3' SNP6F: 5' AAATGGGCCAGTGTTTTCAG3' SNP6R: 5'GAAGGCAAGTCTGAGCAAGG3' SNP7F: 5' TTCTGTAAGCCTGCATACTG3' SNP7R: 5'CGGATGCTGCACTGTAGAAA3' SNP8F: 5' AGGACGGTTGGAATCTGATG3' SNP8R: 5'GCATGGAAGTTTGGTTGACTG3' SNP9F: 5' TGCAGAAGGATGCAGAGAGA3' SNP9R: 5'GGAAGATTAGTCTTGGGCAAA3' SNP10F: 5'TGGGGGAGATGAAAACAG3' SNP10R: 5'TGATATTGGTGGCAGGTGAA3'; (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 220 bp, 210 bp, 184 bp, 240 bp, 230 bp, 192 bp, 238 bp, 223 bp, 246 bp, and 229 bp.

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