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CN-122012744-A - Method for detecting ADK gene SNP of Anhui white goat and breeding application

CN122012744ACN 122012744 ACN122012744 ACN 122012744ACN-122012744-A

Abstract

The invention belongs to the technical field of molecular genetic breeding, and discloses a method for detecting an ADK gene SNP of an Anhui white goat and a breeding application. According to the method, the whole genome DNA of the Anhui white goat is used as a template, and 3 SNP loci of the seventh intron of the ADK gene can be accurately identified through specific primer PCR amplification, agarose gel electrophoresis and Sanger sequencing, wherein the SNP loci are NC_030835.1:g.15498308, NC_030835.1:g.15498501 and NC_030835.1:g.15498816 respectively. The sites have genetic polymorphism in Anhui white goat populations and are obviously related to growth characters such as weight, height, body length, chest circumference, tube circumference, jirime width and the like. The method is simple and convenient to operate, low in cost and high in accuracy, and the obtained molecular marker can be used for auxiliary breeding of Anhui white goat molecular markers, so that the fine breed breeding process is accelerated.

Inventors

  • DONG LUYI
  • ZHANG SIHUAN
  • LING YINGHUI
  • ZHANG YILIN
  • YANG RUIQIAO
  • XU HAN
  • JI KAIYUAN
  • LI SHUANG
  • ZHU LU

Assignees

  • 安徽农业大学

Dates

Publication Date
20260512
Application Date
20260324

Claims (8)

  1. 1. A method for detecting ADK gene SNP of Anhui white goat is characterized in that whole genome DNA of Anhui white goat to be detected is taken as a template, a seventh intronic fragment of the ADK gene is obtained through PCR amplification, sanger sequencing is carried out after agarose gel electrophoresis detection strip is correct, 3 SNP loci are identified, and the 3 SNP loci are NC_030835.1:g.15498308, NC_030835.1:g.15498501 and NC_030835.1:g.15498816 respectively; The primers used for PCR amplification are: an upstream primer 5'-TGATGACCTGCATTAAGCACCA-3'; and a downstream primer 5'-AGCCATAATCCGATCTGAGGTAA-3'.
  2. 2. The method for detecting the ADK gene SNP of the Anhui white goat according to claim 1, wherein the PCR amplification reaction process is as follows, wherein the PCR amplification reaction process comprises the steps of pre-denaturation at 93-95 ℃ for 3-5 min, denaturation at 95-98 ℃ for 10-30 s, renaturation at 55-60 ℃ for 30-50 s, extension at 65-72 ℃ for 1-2 min, circulation for 30-35 times, extension at 65-72 ℃ for 3-5 min and preservation at 4 ℃.
  3. 3. The method for detecting ADK gene SNP of Anhui white goat according to claim 1, wherein the PCR amplification reaction system comprises 2 XPCR Master mix 12.5 mu L, 25-50 ng of whole genome DNA of Anhui white goat to be detected, 2.5-5 pmol of each of amplified upstream primer and amplified downstream primer, and ultra-pure water added to be supplemented to 25 mu L.
  4. 4. The method for detecting the ADK gene SNP of the Anhui white goat of claim 1, wherein agarose gel electrophoresis is carried out by adopting agarose gel with mass fraction of 1.5% -2.0%, and PCR amplified product fragment size is 767bp.
  5. 5. The method for detecting the ADK gene SNP of the Anhui white goat according to claim 1, wherein the genotype judgment rule of Sanger sequencing is as follows: NC_030835.1:g.15498308 site, wherein the unimodal genotype is AA, and the interplanting genotype is AG; NC_030835.1:g.15498501 locus, the genotype at single peak is CC or GG, and the genotype at cover peak is CG; NC_030835.1:g.15498816 site, wherein the genotype at the single peak is TT or GG, and the genotype at the cover peak is TG.
  6. 6. An Anhui white goat molecular marker assisted selective breeding method is characterized in that the method for detecting the ADK gene SNP of the Anhui white goat is adopted, 3 SNP locus genotypes of the ADK gene intron seven of the Anhui white goat are detected, and the 3 SNP loci are used as molecular markers to screen individuals with excellent growth traits.
  7. 7. The method for assisting selective breeding of Anhui white goat molecular markers according to claim 6, wherein the growth traits comprise body weight, height, body length, chest circumference, tube circumference and jirimwidth.
  8. 8. The method for assisting in selective breeding by using Anhui white goat molecular markers according to claim 6, wherein Anhui white goat individuals with NC_030835.1:g.15498308 locus AA genotype, NC_030835.1:g.15498501 locus GG genotype and NC_030835.1:g.15498816 locus GG genotype are screened as a breeding core group.

Description

Method for detecting ADK gene SNP of Anhui white goat and breeding application Technical Field The invention relates to the technical field of molecular genetic breeding, in particular to a method for detecting an ADK gene SNP of an Anhui white goat and breeding application. Background Molecular marker assisted selection is a core technology of modern livestock and poultry breeding, and utilizes a DNA molecular marker closely linked with target characters to realize genotype accurate selection in early breeding, shorten the breeding period and improve the genetic improvement efficiency. Single Nucleotide Polymorphism (SNP) is a genetic marker formed by single nucleotide variation in genome, can influence important economic traits of livestock and poultry by regulating gene expression and protein function, and is a key marker for molecular breeding. Current SNP detection methods include Sanger sequencing, pyrosequencing, high resolution melting curve methods, PCR-LDR, allele-specific PCR, taqMan probe methods, SNaPshot methods, and the like. The method has the advantages that special equipment is needed for pyrosequencing, the requirement on instrument stability by a high-resolution melting curve method is high, the preparation period of the PCR-LDR in the early stage is long, the detection flux of a TaqMan probe method is low, the cost of a SNaPshot method is high, and the simplicity, the low cost and the high precision are difficult to be achieved. Adenosine kinase (ADK) genes are widely involved in adenosine metabolism and epigenetic regulation, and have been shown to be associated with angiogenesis, muscle regeneration, and neurological diseases in humans and model animals, but are poorly studied in goats. An Anhui white goat is taken as an important local meat goat variety, and lacks an ADK gene SNP molecular marker directly related to growth characters, so that the prior art cannot realize specific, low-cost and high-accuracy SNP detection aiming at a seventh intron of the ADK gene of the variety, and is difficult to meet the auxiliary breeding requirement of the molecular marker. Disclosure of Invention The invention aims to overcome the defects of the prior art, provide a method for detecting the ADK gene intronic seven-single nucleotide polymorphism of the Anhui white goat with simple and convenient operation, low cost and high accuracy and application thereof, and solve the problems of complex SNP detection means, lack of ADK gene function markers of the Anhui white goat and low breeding efficiency of the traditional SNP detection means. In order to achieve the above object, the present invention provides the following technical solutions: The method for detecting ADK gene SNP of Anhui white goat is characterized in that whole genome DNA of Anhui white goat to be detected is taken as a template, a seventh intronic fragment of the ADK gene is obtained through PCR amplification, sanger sequencing is carried out after an agarose gel electrophoresis detection strip is correct, 3 SNP loci are identified, and the 3 SNP loci are NC_030835.1:g.15498308, NC_030835.1:g.15498501 and NC_030835.1:g.15498816 respectively; The primers used for PCR amplification are: an upstream primer 5'-TGATGACCTGCATTAAGCACCA-3'; and a downstream primer 5'-AGCCATAATCCGATCTGAGGTAA-3'. Preferably, the PCR amplification reaction is carried out by pre-denaturing at 93-95 ℃ for 3-5 min, denaturing at 95-98 ℃ for 10-30 s, renaturation at 55-60 ℃ for 30-50 s, extending at 65-72 ℃ for 1-2 min, circulating for 30-35 times, extending at 65-72 ℃ for 3-5 min, and preserving at 4 ℃. Preferably, the PCR amplification reaction system comprises 2 XPCR MasterMix 12.5 mu L, 25-50 ng of the whole genome DNA of the Anhui white goat to be detected, 2.5-5 pmol of each of amplified upstream primer and amplified downstream primer, and adding ultrapure water to be supplemented to 25 mu L. Preferably, agarose gel with mass fraction of 1.5% -2.0% is adopted for agarose gel electrophoresis, and the size of PCR amplified product fragment is 767bp. Preferably, the genotype determination rule for Sanger sequencing is: NC_030835.1:g.15498308 site, wherein the unimodal genotype is AA, and the interplanting genotype is AG; NC_030835.1:g.15498501 locus, the genotype at single peak is CC or GG, and the genotype at cover peak is CG; NC_030835.1:g.15498816 site, wherein the genotype at the single peak is TT or GG, and the genotype at the cover peak is TG. Preferably, the method for auxiliary selective breeding of the Anhui white goat molecular marker adopts a method for detecting the ADK gene SNP of the Anhui white goat, detects the genotype of 3 SNP loci of the ADK gene intron seven of the Anhui white goat, takes the 3 SNP loci as molecular markers, and screens individuals with excellent growth characters. Preferably, the growth traits include body weight, height, body length, chest circumference, tube circumference, and width. Preferably, anhui white goat individu