A SNP molecular marker related to chicken black belly membrane trait and application thereof

Abstract

This invention belongs to the field of animal molecular breeding technology, specifically disclosing a SNP molecular marker associated with the trait of black peritoneum in chickens and its application. Through genome-wide association analysis, this invention obtained an SNP molecular marker that is highly significantly associated with the black peritoneum trait in yellow-feathered broilers. This marker, using the chicken GRCg7b genome as a reference, is located at position 120222 on chromosome 3, and is an A / T mutation with genotypes including AA, AT, and TT. Association analysis showed that this locus was highly significantly associated with the black peritoneum phenotype observed after slaughter at 11 weeks of age. The incidence of black peritoneum in individuals with the AA genotype was significantly lower than that of individuals with the AT and TT genotypes. A is the dominant allele reducing the occurrence of black peritoneum, and T is the risk allele for black peritoneum. This invention also provides specific primers for detecting this molecular marker and a marker-assisted selection method, enabling early, non-destructive, and precise selection for the black peritoneum trait, reducing slaughter defect rates, and improving the carcass quality and economic benefits of yellow-feathered broilers.

Description

Technical Field

[0001] This invention relates to the field of animal molecular marker breeding technology, specifically to an SNP molecular marker related to the trait of chicken melanocytic peritoneum and its application. Background Technology

[0002] Hyperpigmentation of the visceral peritoneum (HVP) in chickens is characterized by varying degrees of abnormal melanin deposition in the peritoneal connective tissue. This severely affects the appearance and quality of broilers after slaughter, leading to a significant increase in the rate of substandard broilers and a marked decrease in market acceptance, which in turn causes serious economic losses to broiler production.

[0003] Studies have shown that the black peritoneum trait is heritable. Traditional breeding methods rely on post-slaughter phenotypic identification, which is a destructive, late-stage, and inefficient method, unable to conduct precise culling in the early stages of chick development, severely hindering breeding progress. With the development of genome-wide association analysis and marker-assisted selection (MAS) technologies, identifying SNP molecular markers significantly associated with black peritoneum and establishing non-destructive and precise molecular-assisted selection methods has significant application value for improving the carcass quality of yellow-feathered broilers. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing breeding technologies and provide an SNP molecular marker, detection primer and its application related to the black peritoneum trait in chickens, so as to realize early, non-destructive and accurate molecular marker-assisted breeding of the black peritoneum trait.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first technical solution provided by this invention is to provide a specific amplification primer pair for molecular markers related to the chicken melanocytic peritoneum trait and for molecular-assisted breeding applications. The forward primer sequence of the primer pair is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 2. It can be used to amplify DNA fragments containing the molecular marker site.

[0006] Forward primer F: 5'-ACCGCTCTGACTGCATGAAA-3' (SEQ ID NO: 1) Reverse primer R: 5'- GGTCAGCCATCCATCCACAA-3' (SEQ ID NO: 2) The molecular marker mentioned above is located at the 120222 base position on chromosome 3 of the chicken GRCg7b genome version. This site is an SNP with an A / T allele mutation, abbreviated as Chr3:120222, and its alleles are divided into three genotypes: AA, AT, and TT.

[0007] The second technical solution provided by this invention is the application of a molecular marker related to the chicken black peritoneum trait in marker-assisted breeding of the chicken black peritoneum trait. It includes the following steps: S1 Collect blood samples from the chickens to be tested and extract genomic DNA; S2 uses specific primer pairs for PCR amplification; Specifically, the molecular marker PCR amplification reaction system was prepared using the forward and reverse primers SEQ ID NO:1 and SEQ ID NO:2. The total system volume was 30 μL, including 100 ng of the chicken genomic DNA to be tested, 15 μL of 2X PCR Mix, and 1 μL each of 0.2 nmol / μL forward and reverse primers. Finally, deionized water was added to bring the total reaction volume to 30 μL. The PCR amplification reaction conditions were as follows: 95℃ pre-denaturation for 3 min, followed by 95℃ denaturation for 30 s, 61℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 33 cycles, with a final extension at 72℃ for 3 min.

[0008] After the S3 PCR amplification reaction is completed, the PCR products are sent to Sanger sequencing to determine the genotype; AA genotype means that only A peak is shown at the target site; TT genotype means that only T peak is shown at the target site; AT genotype means that double peaks of A and T are shown at the target site.

[0009] S4 breeding strategy: retain individuals with AA or AT genotypes and eliminate individuals with TT genotypes to rapidly reduce the incidence of melanosis in offspring.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention, through the determination of the black peritoneum trait and genome-wide association analysis of 252 yellow-feathered broiler breeders, identified the Chr3:120222 locus as a molecular marker significantly affecting the black peritoneum trait in yellow-feathered broilers. Using this molecular marker, the incidence of the black peritoneum trait can be significantly reduced through the identification and selection of dominant alleles. This eliminates the need for post-slaughter phenotypic identification, greatly reducing production and breeding costs and improving broiler carcass quality and commercial value. Attached Figure Description

[0011] Figure 1 Genome-wide association analysis QQ plot for the black peritoneum trait in 252 yellow-feathered broiler chickens; Figure 2 Manhattan plot of genome-wide association analysis for the black peritoneum trait in 252 yellow-feathered broiler chickens; Figure 3 Genotyping diagram of the molecular marker site Chr3:120222 (GRCg7b). Detailed Implementation

[0012] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0013] Unless otherwise specified, the experimental instruments, reagents, and general experimental procedures used in the following examples are all existing experimental instruments, reagents, and general experimental procedures, which can be purchased by those skilled in the art through commercial channels.

[0014] Example 1: Mining and Validation of Molecular Markers for Significantly Correlated SNPs in Melatonin Traits Using 252 yellow-feathered broiler chickens as experimental material, the phenotype of melanosis peritoneum was determined after slaughter at 11 weeks of age. Individuals were graded according to their melanosis peritoneum phenotype. The peritoneum of each individual was dissected to expose it for observation. The specific grading criteria were as follows: No melanosis: no melanin deposition in the peritoneal region; Mild melanosis: melanin deposition in the peritoneal region, with the stained area less than 40% of the observed area, and no obvious black patches; Severe melanosis: melanin deposition in the peritoneal region, with the stained area greater than 40% of the measured area, and obvious black patches observed. Phenotypic statistics were performed on normal no melanosis peritoneum (0), mild melanosis peritoneum (1), and severe melanosis peritoneum (2). Simultaneously, 100 μL of blood samples were collected from individuals in the population, and 10 μL of each sample was extracted using a blood genomic DNA extraction kit (TIANGEND P329, Beijing Tiangen). Samples that passed DNA integrity quality checks were sent to Novogene for whole-genome sequencing and genotyping analysis, with a sequencing depth of approximately 10 Gb per individual. Using the chicken reference genome (GRCg7b) as a template, whole-genome SNP genotyping analysis was performed according to the standard GATK4 analysis workflow, ultimately obtaining 6,162,868 high-quality SNPs for subsequent genome-wide association analysis. The quality-controlled genotype, phenotype, and kinship matrix, along with the first five PCA covariates, were integrated using the rMVP v1.2.0 package on the R language platform. Genome-wide association analysis of SNP genotypes and melanometra phenotypes was performed based on a GLM-Logistic model. Results are as follows: Figure 1 and Figure 2 As shown, according to the default significance test threshold standard for whole genome analysis ( Figure 2(Indicated by the red dashed line) Screening yielded several SNP loci that were highly significantly associated with the melanosis melanosis trait, including three SNP loci with large effects (Effect > 0.75): Chr3:120222, Chr3:120233, and Chr3:120240 (Table 1). Further LD linkage analysis of these three loci was performed, and the results are shown in Table 2. These three SNPs were almost completely highly linked (r² > 0.93). Therefore, the most significant locus, Chr3:120222, can be selected as a molecular marker locus significantly associated with the melanosis melanosis trait for molecular-assisted breeding.

[0015] Table 1. Association analysis of three SNP loci on chicken chromosome 3 with the melanosis phenotype.

[0016] Table 2. LD linkage analysis among three significantly associated sites (r² values)

[0017] Further statistical analysis of genotype, gene frequency, and phenotype was conducted at the Chr3:120222 locus. The results are shown in Table 3. The T allele represents the risk allele for the melanosis coli phenotype, with a prevalence of 1.77% in the normal group. The gene frequency gradually increased in the mild and severe melanosis coli groups, reaching 35.44% and 71.87%, respectively. Therefore, in molecular breeding, the A allele is the dominant allele for reducing the occurrence of melanosis coli. Selecting chickens with the AA or AT genotype and culling chickens with the TT genotype will significantly reduce the incidence of the melanosis coli phenotype in the chicken population.

[0018] Table 3. Gene frequency distribution of Chr3:120222 locus in different black-bellied chicken groups.

[0019] Example 2: Application of molecular marker Chr3:120222 in molecularly assisted breeding of chicken melanosis peritoneum trait 1. Preparation of DNA samples for testing (1) Collect 50 μL of whole blood sample from the chicken to be tested and put it into a centrifuge tube containing EDTA and mix well to ensure that the final concentration of EDTA is 1.2 mg / mL, which is the anticoagulated blood sample to be tested.

[0020] (2) Take 10 μL of the above anticoagulated blood sample into a 1.5 mL centrifuge tube and use the blood sample DNA extraction kit (brand: OMEGA; catalog number: D3392; Guangzhou Feiyang Biotechnology Co., Ltd.) to prepare chicken genomic DNA. Refer to the kit operation instructions for specific steps.

[0021] 2. Amplification and preparation of molecular marker sequences PCR amplification was performed using the following primer pairs. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Specific primer information is as follows: Forward primer F: 5'-ACCGCTCTGACTGCATGAAA-3' (SEQ ID NO: 1) Reverse primer R: 5'- GGTCAGCCATCCATCCACAA-3' (SEQ ID NO: 2) The chicken DNA sample obtained in the above steps was used as the PCR amplification template. The reaction solution was prepared as follows: the total PCR reaction volume was 30 μL, including 100 ng of chicken genomic DNA template, 1 μL each of 0.2 nmol / μL forward and reverse primers, 15 μL of 2X PCRMix (PCR general premix solution, provided by Sangon Biotech (Shanghai) Co., Ltd., catalog number B639295), and finally deionized water was added to the total reaction volume of 30 μL.

[0022] PCR reaction conditions: 95℃ pre-denaturation for 3 min, followed by 33 cycles of 95℃ denaturation for 30 s, 61℃ annealing for 30 s, and 72℃ extension for 30 s, with a final extension at 72℃ for 3 min.

[0023] PCR product detection: The fragment length of the amplified product was 358 bp, as detected by 2% agarose gel electrophoresis.

[0024] 3. Individual allele typing PCR amplification products were sent to Sanger sequencing at Sangon Biotech (Shanghai) Co., Ltd. The genotype of the individual was determined by interpreting the base type of the molecular marker site Chr3:120222 based on the sequencing peak profile. Figure 3 As shown, the AA genotype shows only a single peak of A at the target locus; the TT genotype shows only a single peak of T at the target locus; and the AT genotype shows a double peak of overlapping A and T at the target locus.

[0025] 4. Individual selection and retention Based on the results of individual genotype analysis, chickens with the AA or AT genotype were selected, while chickens with the TT genotype were culled, thereby significantly reducing the incidence of melanosis melanosis in the chicken population.

[0026] The sequence list is as follows: <1> 20 DNA forward primer ACCGCTCTGA CTGCATGAAA 20 <2> 20 DNA reverse primer GGTCAGCCAT CCATCCACAA 20 <3> 358 DNA PCR product ACCGCTCTGA CTGCATGAAA CGTGCTCTGT TACCCCTGAA TGTAACGATA ACATTGAGCT TTTATCACTT GCCACAAGGT TCCACTTTTT CTTTTTTTGA ACAGCTTCTG GTTGATCAAA ACAGCTTACA GCATACCTTG CTACAAAATC ACGCTACCAT AGACTTTCTG TTATTAGCTC AAGGTCACGG GATGGCTTAG TGGCTTGTCC AAGGAGGGCCT TCGTCTGTTG ATGTTGATGG TGCTGGGAAT CCTCATAATT TGTTTGGTTAT TCCTGTTAAG AAAATGCTCT CGAAGGTTAC CTCAGCTGTT TTGCTTGCAC AAGAAAAACG GGGAAATTGT GGATGGATGG CTGACC 358

Claims

1. A SNP molecular marker associated with the trait of chicken melanosis peritoneum, characterized in that, The molecular marker, based on the chicken GRCg7b genome version, is located at base 120222 on chromosome 3, representing an A / T allele mutation with three genotypes: AA, AT, and TT. The incidence of melanosis coli in individuals with the AA genotype is significantly lower than that in individuals with the AT and TT genotypes. A is the dominant allele that reduces the occurrence of melanosis coli, and T is the risk allele for the occurrence of melanosis coli.

2. A specific primer pair for detecting the SNP molecular marker of claim 1, characterized in that, The primer pair can specifically amplify DNA fragments containing the SNP molecular marker sites.

3. The application of the SNP molecular marker of claim 1 in marker-assisted breeding of chicken melanosis peritoneum trait.

4. The application of the primer pair of claim 2 in the detection and assisted breeding of molecular markers for the trait of chicken melanosis peritoneum.

5. The application according to claim 3 or 4, characterized in that, Includes the following steps: S1 Extract genomic DNA from the chicken to be tested; S2 uses the specific primer pair for PCR amplification; S3 PCR amplification products were genotyped using Sanger sequencing; S4 prioritizes individuals with the AA genotype at the SNP molecular marker site for breeding purposes, while excluding individuals with the TT genotype.

6. The application according to claim 5, characterized in that, PCR amplification was performed using standard systems and procedures, and the amplification products were sequenced to identify the AA, AT, and TT genotypes.

7. The application according to claim 6, characterized in that, The PCR amplification reaction system consisted of 100 ng of chicken genomic DNA to be tested, 15 μL of 2X PCR Mix, and 1 μL each of 0.2 nmol / μL forward and reverse primers. Finally, deionized water was added to bring the total reaction volume to 30 μL. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min, followed by 95℃ denaturation for 30 s, 61℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 33 cycles, with a final extension at 72℃ for 3 min.

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