Search

CN-122012679-A - Beef cattle gestation duration typing method based on P3H3, GGTA1 and TMEM161B gene SNP and application thereof

CN122012679ACN 122012679 ACN122012679 ACN 122012679ACN-122012679-A

Abstract

The invention relates to the technical field of molecular genetic breeding, and particularly discloses a beef cattle gestation duration typing method based on P3H3, GGTA1 and TMEM161B gene SNP and application thereof, wherein the method comprises the following steps of 1) taking bovine genome DNA as a template, respectively adopting specific primer pairs P1, P2 and P3, and amplifying target fragments of P3H3 gene, GGTA1 gene and TMEM161B gene by PCR; 2) adopting extension primers P4, P5 and P6 and combining with SNaPshot single base extension reaction to respectively carry out typing analysis on the target SNP loci in the PCR amplification products obtained in the step 1), thereby realizing accurate identification of the genotypes of the SNP variation loci in the P3H3, GGTA1 and TMEM161B genes, the detection typing method based on the SNP variation of the P3H3, GGTA1 and TMEM161B genes provided by the invention is not limited by individual age and birth factors, can be implemented in the early stage of calves, is beneficial to genetic prediction of individual gestation duration traits under the condition of not depending on phenotype record, and provides effective technical support for early breeding and accurate selection of beef cattle.

Inventors

  • E GUANGXIN
  • LIU YAOLONG

Assignees

  • 西南大学

Dates

Publication Date
20260512
Application Date
20260129

Claims (10)

  1. 1. The beef cattle gestation duration typing method based on the P3H3, GGTA1 and TMEM161B gene SNP is characterized by comprising the following steps of: 0) Using bovine genome DNA as a template, respectively adopting specific primer pairs P1, P2 and P3, and amplifying target fragments of P3H3 gene, GGTA1 gene and TMEM161B gene by PCR; 1) Adopting extension primers P4, P5 and P6 and combining with SNaPshot single base extension reaction to respectively carry out typing analysis on the target SNP loci in the PCR amplification product obtained in the step 1) so as to realize accurate identification of the genotypes of the SNP variation loci in the P3H3, GGTA1 and TMEM161B genes, The primer pair P1 (P3H 3 gene) includes: Upstream primer P1-F5'-ACAGGCTCATCCATTGCTTC-3' The downstream primer R1-R5'-AGAATGAAGGCCTGTCTCTG-3'; the primer pair P2 (GGTA 1 gene) includes: upstream primer P2-F5'-CAGAACAAAGAGCCTTCTGG-3' The downstream primer P2-R5'-TGATCTCTGATTCCTCTGCC-3'; the primer pair P3 (TMEM 161B gene) includes: Upstream primer P3-F5'-CAAAGTCCTTAGATCGTTCC-3' The downstream primer P3-R5'-TGGCTCTTTTGACCAGCTAC-3'; the extension primer P4 (P3H 3 gene) is 5'-CAGGGCTCCTTCTGTCCTCG-3'; The extension primer P5 (GGTA 1 gene) is 5'-gactgactGCAGCCATTATCACTGAAGCCC-3'; The extension primer P6 (TMEM 161B gene) is 5'-gactgactgactgacACATTTCTGTCAAAATTCTCAC-3'.
  2. 2. The method for typing the gestation period of beef cattle based on the SNP of the P3H3, GGTA1 and TMEM161B genes according to claim 1, wherein SNP variation sites which are obviously related to the gestation period characters of the beef cattle in the P3H3, GGTA1 and TMEM161B genes are respectively positioned at the 103620982 th site of NC_037332.1 chromosome, the 92333432 th site of NC_037338.1 chromosome and the 87804249 th site of NC_037334.1 chromosome in the ARS-UCD version 2.0 (GCF_ 002263795.3) of a reference genome.
  3. 3. The method for typing the gestation period of beef cattle based on SNP of P3H3, GGTA1 and TMEM161B genes according to claim 1, wherein the SNP mutation type is determined according to SNaPshot single base extension typing result, wherein the mutation genotype of the P3H3 gene is G/G type, G/T type or T/T type, the mutation genotype of the GGTA1 gene is C/C type, C/A type or A/A type, and the mutation genotype of the TMEM161B gene is T/T type, T/C type or C/C type.
  4. 4. The method for typing of gestation period of beef cattle based on SNP of P3H3, GGTA1 and TMEM161B gene according to claim 1, wherein in the step 1), the PCR amplification reaction system comprises 20-50 ng/. Mu.L of template DNA 1. Mu.L and 10. Mu. Mol/L of primer pair P1 or primer pair P2 or the upstream and downstream primers corresponding to primer pair P3 of 0.5. Mu.L/2X TAQ PCR MASTER Mix 5. Mu.L and 10. Mu.L of ddH 2O.
  5. 5. The method for genotyping the gestation period of beef cattle based on SNP of P3H3, GGTA1 and TMEM161B genes according to claim 1, wherein the reaction procedure of PCR amplification comprises the steps of pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 20s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles.
  6. 6. The method for typing of gestation duration of beef cattle based on SNP of P3H3, GGTA1 and TMEM161B genes according to claim 1, wherein in the step 1), the fragment size of PCR product amplified based on the primer pair P1 is 278bp, the fragment size of PCR product amplified based on the primer pair P2 is 270bp, and the fragment size of PCR product amplified based on the primer pair P3 is 256bp.
  7. 7. Use of the method according to any one of claims 1-6 in beef cattle molecular marker-assisted selection breeding.
  8. 8. The use according to claim 7, wherein beef cattle individuals carrying a dominant genotype are significantly better in gestation duration traits than individuals carrying a minor genotype, exhibiting a significantly shortened gestation duration.
  9. 9. The use according to claim 8, wherein the length of gestation is the length of time that elapses from the start of self-fertilization or successful breeding of the cow to the end of delivery of the calf.
  10. 10. A kit for detecting SNP mutation site genotypes of beef cattle individual P3H3, GGTA1 and TMEM161B genes is characterized by comprising a primer pair P1, P2 and P3 for amplifying SNP markers related to cow gestation duration, primers P4, P5 and P6 for SNaPshot single base extension typing of the SNP sites, and related reaction system reagents required by PCR amplification reaction and SNaPshot typing reaction, The primer pair P1 (P3H 3 gene) includes: Upstream primer P1-F5'-ACAGGCTCATCCATTGCTTC-3' The downstream primer R1-R5'-AGAATGAAGGCCTGTCTCTG-3'; the primer pair P2 (GGTA 1 gene) includes: upstream primer P2-F5'-CAGAACAAAGAGCCTTCTGG-3' The downstream primer P2-R5'-TGATCTCTGATTCCTCTGCC-3'; the primer pair P3 (TMEM 161B gene) includes: Upstream primer P3-F5'-CAAAGTCCTTAGATCGTTCC-3' The downstream primer P3-R5'-TGGCTCTTTTGACCAGCTAC-3'; The extension primer P4 (P3H 3 gene) is 5'-CAGGGCTCCTTCTGTCCTCG-3'; The extension primer P5 (GGTA 1 gene) is 5'-gactgactGCAGCCATTATCACTGAAGCCC-3'; The extension primer P6 (TMEM 161B gene) is 5'-gactgactgactgacACATTTCTGTCAAAATTCTCAC-3'.

Description

Beef cattle gestation duration typing method based on P3H3, GGTA1 and TMEM161B gene SNP and application thereof Technical Field The invention relates to the technical field of molecular genetic breeding, in particular to a beef cattle gestation duration typing method based on P3H3, GGTA1 and TMEM161B gene SNP and application thereof. Background The gestation period is one of important biological characters affecting beef cattle breeding efficiency and breeding progress, and is directly related to the annual average calf-producing times of cows, the concentration of calf birth time and the health level of perinatal female calves. The overlength of gestation period not only can prolong the reproduction interval and reduce the annual average reproduction efficiency, but also can increase the occurrence risk of diseases in difficult production and perinatal period, thereby restricting the reproduction speed and the production benefit of excellent beef cattle varieties. Therefore, in the breeding practice, the individual for the seed with shorter gestation duration is accurately estimated and preferentially selected, and the method has important significance for improving the breeding efficiency of the core group, accelerating the variety breeding and improving the heredity. The length of gestation belongs to a typical quantitative trait, and the formation of the gestation is regulated by multiple genes together and is influenced by the combination of genetic background and environmental factors. The traditional breeding mode mainly depends on long-term phenotype recording and generation selection, so that the breeding period is long, the cost is high, accurate prediction and selection of gestation duration characters are difficult to realize in the early stage of calves, and the improvement of the genetic improvement efficiency is severely limited. With the continuous development of molecular biology technology and genomics research, the genetic selection technology based on molecular markers provides a new technical means for early prediction of quantitative traits. By mining molecular markers obviously related to the target characters and applying the molecular markers to breeding practice, early evaluation of individual genetic potential can be realized under the condition of not depending on phenotype data, so that the breeding period is obviously shortened, and the selection accuracy is improved. Single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNPs) are the most common, most stable form of genetic variation in the genome, meaning single base differences with a frequency greater than 1% in a population. SNP has the advantages of wide distribution, stable heredity, high flux detection and the like, and has been widely applied to genetic research of animal growth and development, reproductive performance, disease resistance and other characters. Therefore, the functional gene SNP locus obviously related to the gestation duration of the cattle is screened and used as a molecular marker for early prediction and selection, and has good application prospect. In the existing SNP detection technology, the SNaPshot single-base extension typing technology is widely adopted due to simple operation, moderate detection flux and high typing accuracy. The technology is based on fluorescence labeling single-base extension reaction, can accurately type a plurality of SNP loci in single reaction, is suitable for rapid and low-cost detection of large-scale population samples, and can effectively meet the dual requirements of detection efficiency and accuracy in molecular breeding practice. Studies have shown that a variety of functional genes are involved in biological processes such as embryo development, pregnancy maintenance, and labor initiation, and may have an important impact on pregnancy duration. The TMEM161B gene codes transmembrane protein, has a highly conserved biological function in the development process of an embryonic nervous system, and has abnormal expression which can cause embryo development disorder so as to influence the pregnancy process, the GGTA1 gene codes alpha 1, 3-galactosyltransferase and participates in glycoprotein synthesis and inflammation related signal regulation, the metabolic products of the GGTA1 gene are closely related to embryo implantation and maternal immune regulation, and the P3H3 gene belongs to a protein 3-hydroxylase family, participates in the posttranslational modification process of collagen and can play an important regulation role in reproductive tissues and embryo related cells. The genetic variation of the genes provides an important molecular basis for analyzing the difference of cow gestation duration. However, the research on molecular markers for the gestation duration traits of cattle is still limited, and a high-efficiency detection typing method which can integrate a plurality of key functional gene SNP loci simultaneously and is suitable for pr