Molecular marker primer of ACBD6 gene for detecting chicken lateral crown branch defects and application of molecular marker primer

Abstract

The invention belongs to the technical field of molecular markers, and particularly discloses a primer of a molecular marker of an ACBD6 gene for detecting chicken lateral crown branch defects and application of the primer of the molecular marker of the ACBD6 gene for detecting the chicken lateral crown branch defects. Phenotype determination and whole genome re-sequencing related to 300-day-old chicken lateral crown branches are carried out on 1496 pure-line Qingyuan partridge chicken individuals, whole genome association analysis is carried out, and the molecular marker of the ACBD6 gene for detecting the chicken lateral crown branch defects is obtained. The excavated Chr8: 5934516 (GRCg7b) (rs737527050) site has extremely high genetic correlation (p = 3.49207e-10) with the lateral crown branch character phenotype, so that the excavated Chr8: 5934516 (GRCg7b) (rs737527050) site can be effectively used for molecular marker-assisted selective breeding of the lateral crown branch defect of the chicken; individuals with the molecular marker Chr8: 5934516 site as the GG genotype are selected as breeding hens, the cockscomb lateral branch defect alleles in breeding populations and the crown lateral branch occurrence rate of subsequent generations of populations can be rapidly reduced, and meanwhile the carcass uniformity of breeding core populations can be indirectly improved.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to a method for detecting defects in the lateral crown branch of chickens. ACBD6 Primers for molecular markers of genes and their applications. Background Art

[0002] The comb is an important secondary sexual characteristic of chickens and a key indicator of chicken health and carcass uniformity. Side sprigs on the chicken comb refer to extra, irregular protrusions or growths extending from the main structure of the comb. In the broiler and layer chicken industries, they are generally considered a defect, especially in breeds where a well-defined comb shape is required. The appearance of side sprigs is usually caused by genetic factors and is generally an undesirable trait during breeding selection. Genetically, side sprigs are considered a qualitative trait controlled by a single gene or a few genes, but they exhibit a complex inheritance pattern, manifesting as dominant, incompletely dominant, or semi-dominant in different population genetic backgrounds, indicating that there may be regulatory genes and genetic markers with significant genetic effects on this trait.

[0003] Currently, marker-assisted selection for chicken comb-related traits mainly focuses on several common comb-type variations, such as the molecular marker for rose comb in Silkie chickens based on KASP technology and its application disclosed in Chinese Patent Publication No. CN114854880A; primers, kits, and applications for identifying chicken rose comb genotypes using the whole blood method disclosed in Chinese Patent Publication No. CN113604580A; primers, kits, and detection methods for detecting chicken rose comb locus genes disclosed in Chinese Patent Publication No. CN108570506A; a method for detecting chicken rose comb traits disclosed in Chinese Patent Publication No. CN102041310A; a molecular marker related to chicken antler comb traits and its application disclosed in Chinese Patent Publication No. CN116397034A; and a molecular marker related to chicken "antler" combs, its typing method, and its application disclosed in Chinese Patent Publication No. CN111394473A. No patent technology related to molecular detection of chicken lateral comb traits has been found.

[0004] Therefore, conducting genome-wide association analysis on this trait and identifying genes related to chicken lateral crown branches and their significantly associated molecular markers for selection assistance is of great significance for rapidly eliminating this defective trait in chicken breeding. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a method for detecting defects in the lateral crown branch of chickens. ACBD6 Primers for molecular markers of genes and their applications.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a method for detecting defects in the lateral crown branch of chickens. ACBD6 Primers for gene molecular markers include: The upstream primer has the sequence F: 5'-AGCTGAAGTACCCCCAGTGA-3'; The downstream primer has the sequence R: 5'-GGCTGCGTTAGCGGGATTA-3'; The specific chromosomal location of the molecular marker was determined by alignment with the chicken 7.0 reference genome GRCg7b. The molecular marker is Chr8:5934516, located on chromosome 8 of the Qingyuan Ma chicken. ACBD6 The 5934516th position within the gene region is a C / A mutation.

[0007] The second technical solution provided by this invention is an application of the above-mentioned primers in assisted selection breeding to reduce lateral crown branch defects in Qingyuan Ma chickens, comprising the following steps: S1. Extract genomic DNA from the Qingyuan Ma chickens to be tested; S2. Using the primers, PCR amplification was performed on the genomic DNA of the Qingyuan Ma chicken to be tested. After the PCR amplification reaction was completed, the PCR amplification product was obtained. S3. Use the downstream primers to perform Sanger sequencing on the PCR amplification products; select individuals with the GG genotype at the molecular marker Chr8:5934516 site as breeding chickens.

[0008] Further, the PCR amplification reaction system consisted of: 500 ng genomic DNA, 25 μL 2X Pro TaqMaster Mix (dye plus), 1 μL of 0.2 μM upstream primer, 1 μL of 0.2 μM downstream primer, and enzyme-free sterile water added to a total reaction volume of 50 μL. The PCR reaction program was: 94 ℃ for 30 s; 98 ℃ for 10 s, 60 ℃ for 30 s, 72 ℃ for 1 min, for 35 cycles; and 72 ℃ for 2 min.

[0009] Compared with existing technologies, this invention, through phenotypic determination and whole-genome resequencing (average sequencing depth >10×) of 300-day-old purebred Qingyuan Ma chickens, and genome-wide association analysis, revealed that the Chr8:5934516(GRCg7b)(rs737527050) locus has a very high genetic correlation with the lateral crown phenotype (p = 3.49207e-10). Therefore, it can be effectively used for molecular marker-assisted selection breeding of chickens with lateral crown defects. Selecting individuals with the GG genotype at the Chr8:5934516 molecular marker locus as breeding chickens can rapidly reduce the incidence of crown lateral defects in the breeding population and its subsequent generations, while also indirectly improving the carcass uniformity of the core breeding population. Attached Figure Description

[0010] Figure 1 Genome-wide association analysis of lateral crown and non-lateral crown phenotypic traits in a population of 1496 purebred Qingyuan Ma chickens at 300 days of age: a is the QQ plot; b is the Manhattan plot.

[0011] Figure 2 Genotyping of molecular marker site Chr8:5934516: a is TT genotype; b is TG genotype; c is GG genotype. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0013] Example 1: Detection of defects in the lateral crown of chickens ACBD6 Mining of the molecular marker site Chr8:5934516 (GRCg7b)(rs737527050) Phenotypic analysis was performed on 1501 purebred Qingyuan Ma chickens at 300 days of age for lateral crown (n = 43) and non-lateral crown (n = 1458) phenotypic traits. Blood samples were collected from each individual for whole-genome resequencing (average sequencing depth >10×). After removing 5 individuals with a deletion rate greater than 0.05 (43 individuals with lateral crown and 1453 individuals without lateral crown), genomic data from 1496 individuals were retained. Population SNP quality control was performed using VCFtools with the following conditions: "--not-chr W --not-chr Z --min-alleles 2 --max-alleles 2 --maf 0.015 --max-missing0.95", yielding 15,175,078 high-quality autosomal SNPs. Furthermore, based on adjustments for the number of valid independent tests, LD-pruning (parameter "--indep-pairwise") was performed on the autosomal SNPs using Plink 1.9. Using the formula 50 5 0.5", 3,682,389 low-linked autosomal SNPs were estimated as the effective number of loci. Bonfroni correction was used to perform significance testing on genome-wide association analysis, with a suggested significance threshold of 2.715628e-07 (1 / 3,682,389) and a genome-wide significance threshold of 1.357814e-08 (0.05 / 3,682,389). Based on 15,175,078 high-quality autosomal SNPs, a genome-wide association analysis was performed on lateral canopy branches using a generalized linear model of PLINK2 (PC1, PC2, and PC3 as covariates; Firth logistic regression was used as a backup method to address data sparsity or segregation issues). A significance threshold of 1.357814e-08 (Bonferroni corrected; 0.05 / 3682389) was used. The results are as follows: Figure 1 As shown, Figure 1 Genome-wide association analysis of lateral crown and non-lateral crown phenotypic traits in a population of 1496 purebred Qingyuan Ma chickens at 300 days of age: a) Quantile-quantile plot, representing the goodness of fit between observed and expected values; b) Manhattan plot. Figure 1 As shown in 'a', six SNPs were significantly associated with 300-day-old lateral canopy branches. Figure 1 As indicated by b, all six significant loci are located within a 58 kb genomic region on chromosome 8 (8:5876910-5934637; GRCg7b), which contains... ACBD6 ( Acyl-CoA Binding Domain Containing 6 )and XPR1 ( Xenotropic and Polytropic Retrovirus Receptor 1Two protein-coding genes, including ) and others; located in ACBD6 The locus Chr8:5934516 (GRCg7b) (rs737527050) within the intron region of the gene showed the highest genetic correlation with lateral canopy branches. p = 3.49207e-10), the genetic effect at this locus is a typical additive effect.

[0014] Then, the incidence of lateral crown branches at 300 days of age corresponding to different genotypes of locus Chr8:5934516 (GRCg7b) (rs737527050) was statistically analyzed, and the results are shown in Table 1.

[0015] Table 1

[0016] Table 1 shows that, in both lateral crown and non-lateral crown populations, the incidence of lateral crown branches is as follows: genotype AA (33.33%) > genotype CA (5.91%) > genotype CC (1.72%). Individuals with the A allele have a higher incidence of lateral crown branches, while individuals with the C allele have a lower incidence. Therefore, eliminating allele A at this locus can significantly reduce the incidence of lateral crown branch defects in chickens.

[0017] Example 2: Detection of lateral crown branch defects in Qingyuan Ma chickens ACBD6 Design and synthesis of primers for molecular markers of genes Using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), we designed the Chr8:5934516 (GRCg7b) (rs737527050) site and amplified the upstream and downstream primers: The upstream primer sequence is F: 5'-AGCTGAAGTACCCCCAGTGA-3' (see SEQ ID NO.1); The downstream primer sequence is R: 5'-GGCTGCGTTAGCGGGATTA-3' (see SEQ ID NO.2); The upstream and downstream primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0018] Example 3: Detection of lateral crown branch defects in Qingyuan Ma chickens ACBD6 Application of molecular marker primers for genes in assisted selection breeding to reduce lateral crown branch defects in Qingyuan Ma chickens 1) Blood samples were extracted from individual Qingyuan Ma chickens to be tested, and genomic DNA was extracted using the phenol-chloroform method; 2) The genomic DNA of the Qingyuan Ma chicken to be tested was amplified by PCR using the upstream and downstream primers shown in SEQ ID NO.1 and SEQ ID NO.2. The PCR reagents, methods and reaction procedures were selected from Aikerui Biotechnology Co., Ltd. (Changsha, China). The reaction system and reaction conditions are shown in Tables 2 and 3 below.

[0019] Table 2

[0020] *1: When using the 2X Pro Taq Master Mix (dye plus) for the first time, centrifuge it first to avoid enzyme loss.

[0021] *2: Generally, it is recommended that the amount of template added should not exceed 500 ng; the amount of template used can be adjusted according to actual needs.

[0022] *3: Primers are usually used at a final concentration of 0.2 μM, which can be adjusted within the range of 0.2 ~ 1.0 μM according to experimental results.

[0023] *4: The reaction system needs to be prepared on ice, and finally the prepared reaction solution is placed in the PCR instrument for reaction.

[0024] Table 3

[0025] After the PCR amplification reaction was completed, the PCR amplification products were subjected to Sanger sequencing. The upstream primer is F: 5'-AGCTGAAGTACCCCCAGTGA-3' (NCBI online alignment result of primers: physical location is Chr8:5933867-5933886). Downstream primer: R:5'-GGCTGCGTTAGCGGGATTA-3' (NCBI online alignment result of primers: physical location is Chr8:5934746-5934728); Considering that the upstream primer is 630 bp away from the Chr8:5934516 site, which is close to the low-quality end region of Sanger sequencing, while the downstream primer is 230 bp away from the Chr8:5934516 site, which belongs to the high-quality region of Sanger sequencing, the downstream primer was selected for Sanger sequencing. The full length of the Sanger sequence is as follows: AATCTGATGA TACCGGCAGC GGGCTGGCTT CCAAACGGCA GGGGGACGGG GAGAGGGGTGCTGGGGGTGC TGGCTGCCAC CCGACTGCCC AGTGGCCCCC GCTTCTGAAC CAGCAGCGAG CCCATGGGGCTTGCTCTGCT CCGGGGAGTGGACT GGC GTGGTGGTTTGTCAGAAAAGAGCTTGTTTCACCCCTTGGGATGKCAGAGCGTATCACATAGCTCAATGGG ACCTTAAAGCCCATTCAATTCCATCCCAGCGCATGGTCCACACACAGG CAGCTCCTCTGCTGTGCTCGGAAATGGAAG CGGACGTCTGGCAGCAGCATTTGCTATCTGCCAAGTTTGCTGGAAACGTTTGTAAACTGCCTTTTTTTTTTTTTTTTTTT TCCTCCTTAAAAAAATGTTTTAAAAACTCTGAAGTTGAATTTCCAGTTGTGCAGCACTCCTGGGCTTCTCCCTCAGTC TGCATCTTAGATTTCATCCCTTCATTCCTTCCATTCATTT TCCCAATGGTTAATTCCCAACTTTGGGAAA ATTGAATTAA TATTAATTGGTCATTGAAAA ACTCCACCACTTGTGGCTTCCTTCCGGATTAAAAACAAAATAAACTT TAATCAACAA GAAAAAAACCACAATAAGCCCAACATGGTTAACCACC TTAAACCACCAA AACCCTTTTTAAACTT TCCAAGGGAATCCTCGAATTCCAACAA CAAGCCTTAA TTTTATCCTA TGGCCAAAGT GAGATTAGTG ATTTACAGAAAAACCCCAAG GGCAAAGCACTATGCTAATA CATTTTGCAA CTGTTCCCCA TTAGTCACTG GGGGTAACTTCACCTAAA(SEQ.

[0026] The full-length sequence of the Sanger downstream primer sequencing is 858 bp. The 204th base, the shaded base K (T / G mutation - A / G mutation during forward primer sequencing), is the site Chr8:5934516 (GRCg7b) (rs737527050) - SNP marker. The genotyping of the molecular marker site Chr8:5934516 is as follows: Figure 2 As shown; by Figure 2 It can be seen that when an individual's Sanger sequencing peak is TT (positive complementary genotype is AA) (see...) Figure 2 In the a) group, the frequency of lateral branches on the comb was high (33.33%). When the sequencing peak was TG (positive complementary genotype was AC) (see [reference needed]). Figure 2 (b) The frequency of lateral branches on the comb in individuals was moderate (5.91%). When the sequencing peak was GG (positive complementary genotype was CC) (see [reference]). Figure 2 In the c) group, the frequency of comb lateral branch occurrence in individuals was low (1.72%). Therefore, in chicken breeding, by detecting this SNP marker, and by selectively selecting chickens with the GG genotype at the molecular marker Chr8:5934516 locus or culling chickens with the TT genotype, the incidence of comb lateral branch defective alleles and subsequent generations in the breeding population can be rapidly reduced. At the same time, it can also indirectly improve the carcass uniformity of the core breeding population.

[0027] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for detecting defects in chicken lateral crown branches ACBD6 A primer for molecular marker of a gene, characterized in that include: Upstream primer, sequence F: 5′-AGCTGAAGTACCCCCAGTGA-3′; Downstream primer, sequence: R: 5′-GGCTGCGTTAGCGGGATTA-3′; The specific chromosome location 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 Chr8:5934516, which is located in the chromosome 8 of Qingyuan Ma chicken. ACBD6 Position 5934516 within the gene region is a C / A mutation.

2. Use of the primer according to claim 1 in assisted selection breeding for reducing lateral crown branch defects in Qingyuan Ma chicken, characterized in that: The following steps are involved: S1. Extracting genomic DNA from the Qingyuan Silkie chicken to be tested; S2. performing PCR amplification on the genomic DNA of the Qingyuan Ma chicken to be tested using the primers, and obtaining a PCR amplification product after the PCR amplification reaction procedure is completed; S3. Perform Sanger sequencing on the PCR amplification product using the downstream primers; select individuals with the GG genotype at the molecular marker Chr8:5934516 site as breeder chickens.

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

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