Molecular marker primer for detecting shank circumference of cast chicken and application of molecular marker primer

Abstract

The invention belongs to the technical field of molecular markers, and particularly discloses a molecular marker primer for detecting the shank girth of a cast chicken and application of the molecular marker primer, and the molecular marker primer is characterized in that 300-day-old shank girth-related phenotype determination and whole genome re-sequencing are carried out on 297 cast chicken-Yao chicken F2 generation resource group individuals (the average sequencing depth gt of the individuals; the molecular marker Chr1: 68713248 (GRCg7b) has the advantages that the molecular marker Chr1: 68713248 (GRCg7b), which is excavated by carrying out whole genome correlation analysis on the molecular marker Chr1: 68713248 (GRCg7b), has extremely high genetic correlation with 300-day-old shin girth, and is very remarkable in genetic effect, so that the molecular marker Chr1: 68713248 (GRCg7b) can be effectively used for molecular marker-assisted selective breeding of Dast chicken thick shin girth related specialized strain breeding, individuals with the molecular marker Chr1: 68713248 (GRCg7b) sites being AA or AT genotypes are selected as breeding hens, and the breeding hens can be bred and the genetic selection progress of Dongshin chicken containing Dongshin chicken blood relationship related specialized strain Dongshin chicken can be accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and in particular to a primer for detecting molecular markers of the shank circumference of Dongtao chickens and its application. Background Art

[0002] The shank circumference of chickens is an important economic trait related to growth and carcass quality. A thicker shank circumference usually means stronger bones, which can support a greater body weight and reduce the culling rate caused by bone problems (such as lameness, fractures). At the same time, the thickness of the shank circumference is also one of the important indicators affecting carcass uniformity. Genetically, the shank circumference is a complex trait regulated by multiple genes, belonging to a trait with medium to high heritability, and has a strong phenotypic and genetic positive correlation with the body weight of broiler chickens.

[0003] For example, the application of genetic markers associated with the shank circumference of chickens in the FOXO1 gene disclosed in Chinese Patent Publication No. CN118995953A; the molecular markers of the SERCA2 gene related to chicken carcass traits and their application disclosed in Chinese Patent Publication No. CN116334235A; a SNP molecular marker related to the shank circumference of chickens and its application disclosed in Chinese Patent Publication No. CN117418016A. The similarities between the present application and these three are that they all develop relevant markers for chicken shank circumference for assisted selection breeding. The marker loci are located in the gene regions of FOXO1 (1:170460219-170524492; GRCg7b), SERCA2 (15:5319274-5360262; GRCg7b), and PLA2G7 (3:109631987-109645042; GRCg7b) respectively, and are all developed based on SNPs loci within specific gene regions without performing genome-wide significance tests.

[0004] Therefore, developing molecular marker-assisted selection related to chicken shank circumference with a large genetic effect can accelerate the genetic selection progress of chicken shank circumference traits. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a primer for detecting molecular markers of the shank circumference of Dongtao chickens and its application.

[0006] To achieve the above object, the present invention is implemented according to the following technical scheme: The first technical solution provided by the present invention is a primer for detecting molecular markers of the shank circumference of Dongtao chickens, including: Forward primer, with the sequence F: 5’-TGTGTGCTCACCCATAACCA-3’; Reverse primer, with the sequence R: 5’-AGATGGGGTACAGAGGTCGAA-3’; Among them, the specific chromosomal positions of the molecular marker sites in the genome are determined after alignment with the chicken 7.0 reference genome GRCg7b. The molecular marker is Chr1:68713248, which is located on chromosome 1 of the chicken genome. SCUBE1 At the 68713248th position within the gene region, there is an A / T mutation. The chicken is an F1 generation individual produced by using Dongtao chicken as the paternal line of the F0 generation and Guangxi Nandan Yao chicken hens as the maternal line of the F0 generation for cross-breeding, and then the F2 generation population is produced based on the F1 generation population through cross-breeding.

[0007] The second technical solution provided by the present invention is an application of the above primers in the assisted selection breeding for improving the shank circumference of Dongtao chicken, including the following steps: S1. Extract the genomic DNA of the chicken to be tested; S2. Use the primers to perform PCR amplification on the genomic DNA of the chicken to be tested. After the PCR amplification reaction program is completed, perform Sanger sequencing on the product; select individuals with the AA or AT genotype at the molecular marker Chr1:68713248 locus as breeding chickens.

[0008] Further, the PCR amplification reaction system is as follows: 500 ng of genomic DNA, 25 μL of 2X Pro TaqMaster Mix (dye plus), 1 μL of the upstream primer with a concentration of 0.2 μM, 1 μL of the downstream primer with a concentration of 0.2 μM, and add enzyme-free and sterile water to make the total reaction system 50 μl; the PCR reaction program is 94 °C for 30 s; 98 °C for 10 s, 60 °C for 30 s, 72 °C for 1 min, for 35 cycles; 72 °C for 2 min.

[0009] Compared with the prior art, through phenotypic determination and whole-genome resequencing (individual average sequencing depth > 10×) related to the shank circumference at 300 days of age for 297 individuals in the Dongtao chicken - Yao chicken F2 generation resource population, the present invention conducts a genome-wide association analysis and discovers that the locus Chr1:68713248 (GRCg7b) has a very high genetic correlation with the shank circumference at 300 days of age, and the genetic effect is very significant. Therefore, it can be effectively used for molecular marker-assisted selection breeding in the cultivation of Dongtao chicken specialized lines related to thick shanks. Selecting individuals with the AA or AT genotype at the molecular marker Chr1:68713248 (GRCg7b) locus as breeding chickens can accelerate the genetic selection progress of thick-shank chickens in specialized lines related to Dongtao chicken blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the construction process of the F2 generation full-sib family resource population of Dongtao chicken - Yao chicken.

[0011] Figure 2 Phenotypic statistics of shank circumference at 300 days of age for the F0, F1, and F2 generations of Dongtao chickens and Yaoji chickens, including both male and female populations.

[0012] Figure 3 Q-Q plot for genome-wide association analysis of shank circumference at 300 days of age in a population of 277 full-sib families of the F2 generation of Dongtao chicken - Yaoji chicken.

[0013] Figure 4 Manhattan plot for genome-wide association analysis of shank circumference at 300 days of age in a population of 277 full-sib families of the F2 generation of Dongtao chicken - Yaoji chicken.

[0014] Figure 5 Genotyping of the molecular marker locus Chr1:68713248 (GRCg7b): a is the AA genotype; b is the AT genotype; c is the TT genotype. Detailed implementation method

[0015] To make the objectives, technical solutions, and advantages of the present invention more clear and understandable, the following further details the present invention in combination with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0016] The chickens to be detected in this embodiment are F1 generation individuals produced by using Dongtao chickens as the F0 generation paternal line and Guangxi Nandan Yaoji hens as the F0 generation maternal line for crossbreeding, and then the F2 generation population is produced based on the F1 generation population through crossbreeding; the specific crossbreeding method is as follows: Using 6 Dongtao chicken roosters as the F0 generation paternal line and 12 Guangxi Nandan Yaoji hens as the F0 generation maternal line, they are crossbred in a 1:2 male-female ratio to produce 152 F1 generation individuals (61 roosters and 91 hens). Then, based on the pedigree records, the F2 generation population is produced from the F1 generation population, including 150 roosters and 147 hens, for a total of 297 individuals used for the discovery of molecular markers related to the thick shank circumference of Dongtao chickens ( Figure 1 ). In the F2 generation resource population of Dongtao chicken - Yaoji chicken constructed, the shank circumference at 300 days of age of both the F1 generation population and the F2 generation male and female populations is significantly higher than that of the purebred male and female Yaoji chicken populations, but significantly lower than that of the purebred male and female Dongtao chicken populations. This indicates that when using thick-shank Dongtao chickens as breeding materials and crossbreeding with other chicken breeds with ordinary shank circumferences to cultivate specialized lines related to thick shank circumferences, it can indeed significantly improve the shank circumference of the related line populations ( Figure 2 ).

[0017] Example 1. Discovery of the molecular marker-assisted selection locus Chr1:68713248 (GRCg7b) Taking 297 Dongtao chicken - Yao chicken F2 generation resource population individuals as the research objects, the shank circumference was measured at 300 days of age. After collecting blood samples, whole-genome resequencing was performed (average sequencing depth >10×). After excluding 20 individuals without phenotypic records, duplicate numbers, duplicate samples, and individuals without numbers, 277 individual genome data were retained, and VCFtools was used for population SNP quality control. The quality control conditions were: "--not-chr W --not-chr Z --min-alleles 2 --max-alleles 2 --maf 0.05 --max-missing 0.95", obtaining 11,434,236 high-quality autosomal SNPs for genome-wide association analysis. At the same time, based on the adjustment of the effective independent test times, Plink 1.9 was used to perform LD-pruning on autosomal SNPs (parameter "--indep-pairwise 50 5 0.5"), estimating 2,225,843 low-linkage autosomal SNP loci as the effective locus numbers, and the Bonferroni correction was adopted to perform a significance test on the genome-wide association analysis, that is, the recommended significance threshold was 4.493E-07 (1 / 2,225,843), and the genome-wide significance threshold was 2.246E-08 (0.05 / 2,225,843); Based on the obtained 11,434,236 high-quality autosomal SNPs, a genome-wide association analysis of the shank circumference at 300 days of age was performed using the mixed linear model of GCTA (PC1 - PC10 as quantitative covariates and gender as a discrete covariate). With 4.493E-07 (Bonferroni correction; 1 / 2,225,843) as the recommended significance threshold and 2.246E-08 (0.05 / 2,225,843) as the genome-wide significance threshold, the results showed that 22 and 3 SNPs respectively reached genome-wide recommendatory (<4.493E-07) and genome-wide significance (<2.246E-08) with the shank circumference at 300 days of age (see Figure 3 and Figure 4 ); The genome-wide significant loci included Chr2:58227650 (p = 9.40E-09), Chr2:58227636 (p = 1.17E-08), and Chr1:68713248 (p = 1.81E-08) (GRCg7b), and only the locus Chr1:68713248 was located at SCUBE1The intron region of the (Signal peptide, CUB domain and EGF like domain containing 1) protein-coding gene, and the locus Chr1:68713242 within this gene region reached the recommended threshold (p = 4.44E-08), while there were no protein-coding genes within 50 Kb upstream and downstream of the other 2 significant loci. Importantly, previous studies have shown that SCUBE1 can act as a cofactor for the BMP2 receptor, and BMP2 the gene plays a key role in bone formation and maintenance. Therefore, considering the correlation between the functions of the genes associated with the significant SNPs and the shank circumference trait, the locus Chr1:68713248 is the best marker locus for shank circumference in the Dongtao chicken - Yaoji chicken F2 resource population.

[0018] Further statistics were performed on the genotypes and phenotypes of the locus Chr1:68713248, and the results are shown in Table 1.

[0019] Table 1

[0020] Note: REF represents the reference allele, and ALT represents the mutant allele.

[0021] As can be seen from Table 1, the genotype AA population > genotype AT population > genotype TT population. Individuals with the A allele have a thicker shank circumference and belong to the dominant allele for shank circumference.

[0022] Example 2. Design and synthesis of primers for detecting gene molecular markers related to the shank circumference of Dongtao chickens SCUBE1 Using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design upstream and downstream primers for amplifying the locus Chr1:68713248 (GRCg7b). The upstream primer sequence is: F: 5’-TGTGTGCTCACCCATAACCA-3’ (see SEQ ID NO.1); The downstream primer sequence is: R: 5’-AGATGGGGTACAGAGGTCGAA-3’ (see SEQ ID NO.2); The upstream primer and downstream primer were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0023] Example 3. Application of primers for gene molecular markers related to the shank circumference of Dongtao chickens in the assisted selection breeding for improving the shank circumference of Dongtao chickens SCUBE1 ​​ 1) Extract the blood samples of the individual chickens to be tested, and extract genomic DNA by the phenol-chloroform method; 2) Use the above primers to perform PCR amplification on the genomic DNA of the chickens to be tested; The PCR reagents, methods and reaction procedures are all selected from Aikerui Bioengineering Co., Ltd. (Changsha, China), and the reaction system and reaction conditions are shown in Tables 2 and 3 below.

[0024] Table 2 PCR reaction system

[0025] *1: When the 2X Pro Taq Master Mix (dye plus) in the product is used for the first time, centrifuge it first and then use it to avoid loss of enzyme amount.

[0026] *2: Usually, it is recommended that the added amount of the template does not exceed 500 ng; The template dosage can be adjusted according to actual needs.

[0027] *3: The primers are usually used at a final concentration of 0.2 μM, and can be adjusted within the range of 0.2 - 1.0 μM according to the experimental results.

[0028] *4: The reaction system needs to be prepared on ice, and finally the prepared reaction solution is placed in a PCR instrument for reaction.

[0029] Table 3 PCR reaction conditions

[0030] After the PCR amplification reaction program is completed, the product is stored at 4 °C for later use, and sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. The full length of the PCR amplification sequence is as follows (see SEQ ID NO.3): GCTCTTACTG CATAGCGCCA CTAAGCCTGA TTCTGCAGGA GTATTTGAGA ACTGTCCCTGTCTCTTTAAC TGAGAACAGA TGCTGAGAGT GGTATCAGAC GCTCTTTTCT GATCTCCTTT CCCGTGTCCWGAGTTTTAAC GCATTCGTGA AGGATAAAAT GCCAAGGTTC TTATGCTGTC TTTGGAACAG TAAAAGCCAAGCATGAACTG AAAAGCTCAG ACTGTGCACT AATAAATGTT GCTTTGGTTT TCCTTTACAG CTAGCTACTTCTAATGTTTT GAAGGAGCAG AAATGGGCAA CTATTTGATT TTTTCCCAGT AATACCTCTG CAGCTTTTGCTTAGCCTCAA TTAGTGATCA ACAGTTGACA AAGATTTGGT TGAGATTTTT AACCTCAATT TTGAATAGAAAAATTCAGGC ATCACCGCAG GATTGAATTT TTAGAAGTGT ATCAGACTAC CTTTTGTAAC TGAATGTTTCTGCAGCCCAG AGTTACTCAT CTAAATCTCT GTCCAGTTAA AAACAGTTCG ACCTCTGTAC CCCATCTCTTTGCGGTAATA AAACCCTCTA ATAAAAAGAG AAAAATGACT GAAGTAGGTA AATGCAGAAG AATTAAGGATGAGGAAAGCT AATGAACTCA ACTGCAATCA CTGTGCCAGT AACAGCCTTA ACTTAAAAGT ATTGATTAAATATCTGAATA TTTACATCTT GTACCGAAAC CGCTGAAGGC AAATGATGCA TTGCAAAGCA AACAGAAGCTGACCTTTGCA CGTTTTCCCA TCAGGCTGCA GGGTAAATCC AACTGGGCAA CTGCATCGTA CCCCTGTCG。

[0031] The full length of the PCR amplified sequence is 829 bp, and the 130th base (the shaded base W; A / T mutation) therein is the SNP marker at the locus Chr1:68713248 (GRCg7b); the genotyping of the locus Chr1:68713248 (GRCg7b) is as Figure 5 shown. When the Sanger sequencing peak map of an individual is AA (see Figure 5 a in), the shank circumference of individuals of the Dongtao chicken-related hybrid strain at 300 days of age is thicker. When the sequencing peak map is AT (see Figure 5 b in), the shank circumference of individuals of the Dongtao chicken-related hybrid strain at 300 days of age is medium. When the sequencing peak map is TT (see Figure 5 c in), the shank circumference of individuals of the Dongtao chicken-related hybrid strain at 300 days of age is thinner. Therefore, when using Dongtao chickens to cross with other chicken breeds to cultivate specialized strains with thick shanks, the genetic progress of the selection of populations with thick or thin shanks can be accelerated by detecting this SNP marker and selectively retaining breeding chickens with the AA or TT genotype through directional selection.

[0032] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A primer for detecting molecular markers of Dongtao chicken shank circumference, characterized in that: include: The upstream primer had the sequence F: 5′-TGTGTGCTCACCCATAACCA-3′; Downstream primer, sequence: R: 5′-AGATGGGGTACAGAGGTCGAA-3′; The specific chromosome position of the molecular marker site in the genome was determined by comparing the chicken 7.0 reference genome GRCg7b as the reference genome. The molecular marker is Chr1:68713248, located on chromosome 1 of the chicken genome. SCUBE1 The 68713248th position in the gene region is an A / T mutation; the chicken is an F1 generation individual produced by hybridization using Dongtao chicken as the F0 generation paternal line and Guangxi Nandan Yao chicken hen as the F0 generation maternal line, and then hybridization based on the F1 generation group produces an F2 generation group.

2. Use of the primer according to claim 1 in assisted selection breeding for increasing the shank circumference of Dongtao chickens, characterized in that: The following steps are involved: S1. Extracting genomic DNA from the chicken to be tested; S2. Perform PCR amplification on the genomic DNA of the chicken to be tested using the primers. After the PCR amplification reaction program is completed, perform Sanger sequencing on the product; and select individuals with AA or AT genotype at the molecular marker Chr1:68713248 site as breeder chickens.

3. The use according to claim 2, characterized in that: The PCR amplification reaction system is: 500 ng genomic DNA, 25 μL 2X Pro Taq Master Mix (dye plus), 1 μL upstream primer with a concentration of 0.2 μM, 1 μL downstream primer with a concentration of 0.2 μM, and enzyme-free sterile water is added to make the total reaction system 50 μl; the PCR reaction program is 94°C 30s; 98°C 10s, 60°C 30s, 72°C 1min, 35 cycles; 72°C 2min.

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