CN-121992116-A - Danzhou chicken molecular breeding method based on growth performance related SNP markers

CN121992116ACN 121992116 ACN121992116 ACN 121992116ACN-121992116-A

Abstract

The invention belongs to the technical field of poultry breeding, and particularly relates to a Danzhou chicken molecular breeding method based on growth performance-related SNP markers. Aiming at the problems of low breeding efficiency and insufficient precision of the existing Danzhou chickens, the method comprises the steps of measuring Danzhou chickens' growth performance indexes, collecting samples, extracting high-quality genome DNA, screening specific core SNP markers obviously related to the growth performance, constructing a joint detection system, screening dominant genotype individuals based on the system to construct core groups, obtaining new strains with stably improved growth performance through multiple generations of breeding, and finally verifying the effect through a control experiment. The invention realizes the transition from phenotype screening to genotype accurate screening, improves the breeding accuracy and efficiency, shortens the breeding period, improves the weight of the cultivated new line at 70 days old by more than 10%, reduces the feed weight ratio by more than 8%, can reduce the breeding cost, improves the industrialization value of Danzhou chickens, and has good industrial practicability.

Inventors

HOU GUANYU
Wen Taiyu
SHI LIGUANG
CAO TING
TENG YUWEI
CAI KEQI

Assignees

中国热带农业科学院热带作物品种资源研究所
海南热科源生态养殖有限公司

Dates

Publication Date: 20260508
Application Date: 20260326

Claims (8)

1. A Danzhou chicken molecular breeding method based on growth performance related SNP markers is characterized by comprising the following steps: S1, measuring and collecting growth performance indexes of Danzhou chickens, namely selecting Danzhou chickens at different ages, and measuring the growth performance core indexes of the chickens, wherein the growth performance core indexes comprise 7-day-old body weight, 21-day-old body weight, 42-day-old body weight, 70-day-old body weight, average daily gain and feed/weight ratio; S2, danzhou chicken genome DNA extraction and quality detection, wherein the improved salting-out method is adopted to extract genome DNA in a blood sample, the specific operation comprises the steps of mixing the blood sample and a lysate according to a volume ratio of 1:5, adding proteinase K to a final concentration of 50 mug/mL, carrying out water bath digestion for 2.5h at 55 ℃ and reversing and mixing for 1 time every 30min, adding saturated NaCl solution after digestion, fully oscillating and centrifuging for 15min at 12000r/min, taking supernatant, adding isopropanol with equal volume, standing for 10min, centrifuging for 10min at 10000r/min, collecting precipitate, washing the precipitate with 75% ethanol for 2 times, airing at room temperature, adding TE buffer solution for dissolving, obtaining genome DNA, detecting the purity of the DNA by adopting a Nanodrop nucleic acid detector, requiring an OD260/OD280 value to be 1.8-2.0, and detecting the integrity of the DNA by 1.2% agarose gel electrophoresis, thereby ensuring no obvious degradation; S3, screening and verifying SNP markers related to growth performance: S3.1, screening candidate genes related to chicken growth performance based on Danzhou chicken genome database, wherein the candidate genes comprise a growth hormone Gene (GH), a growth hormone receptor Gene (GHR), an insulin-like growth factor 1 gene (IGF-1), a myostatin gene (MSTN) and an insulin-like growth factor binding protein 3 gene (IGFBP-3); S3.2, designing specific primers for each candidate gene, amplifying coding region and regulatory region fragments of the candidate gene by adopting a PCR technology, wherein a PCR reaction system is 25 mu L, 10 times PCRBuffer2.5 mu L, dNTPs mixture (2.5 mmol/L) 2 mu L, 1 mu L of upstream primer (10 mu mol/L), 0.2 mu L of downstream primer (10 mu mol/L) 1 mu L, taqDNA polymerase (5U/mu L), 2 mu L of genome DNA template (50 ng/mu L) and sterile double distilled water, wherein the PCR reaction program is 95 ℃ pre-denaturation for 5min, 95 ℃ denaturation for 30S, annealing temperature is 55-62 ℃ and is optimized according to different primers, 72 ℃ extension for 45S, 35 cycles are total, and 72 ℃ final extension is 10min and 4 ℃ storage; S3.3, carrying out Sanger sequencing on the PCR amplified product, comparing the sequencing result with a reference genome sequence, and identifying SNP loci in candidate genes, and carrying out large sample typing on the identified SNP loci by adopting a SNPlex typing technology, wherein the sample size is not less than 300 Danzhou chickens; S3.4, carrying out correlation analysis on the parting result and the growth performance index measured in the step S1, adopting SPSS software to carry out single factor analysis of variance, screening SNP loci which are obviously related to at least 2 growth performance core indexes (P < 0.05) as core markers, wherein the core markers comprise g.1234A > G loci of GH genes, G sections of G sections describing what kind of prior art problems are solved by the device if the device is not segmented, g.567C > T loci of HR genes, g.890G > A loci of IGF-1 genes, g.345T > C loci of MSTN genes and g.678A > T loci of IGFBP-3 genes; s3.5, constructing a combined detection system of the core markers, and verifying the prediction accuracy of the core marker combination on the growth performance of Danzhou chickens through multi-marker polymerization effect analysis, so as to ensure that the prediction accuracy is not lower than 85%; S4, molecular marker assisted selective breeding: S4.1, selecting Danzhou chicken groups to be bred, and extracting genome DNA of each individual according to the method of the step S2; S4.2, designing specific TaqMan probes aiming at the 5 core SNP loci obtained by screening in the step S3, and carrying out typing detection on each core SNP locus by adopting a real-time fluorescent quantitative PCR technology; s4.3, determining dominant genotypes according to typing results, wherein the dominant genotypes of GH gene g.1234A > G locus are AA type, the dominant genotypes of GHR gene g.567C > T locus are CC type, the dominant genotypes of IGF-1 gene g.890G > A locus are GG type, the dominant genotypes of MSTN gene g.345T > C locus are TT type, and the dominant genotypes of IGFBP-3 gene g.678A > T locus are AA type; S4.4, screening Danzhou chicken individuals carrying 3 or more dominant genotypes simultaneously as parents, and constructing a breeding core group according to a male-female ratio of 1:8-1:10; S4.5, carrying out growth performance tracking measurement on the offspring of the core group, simultaneously verifying the genetic stability of the core SNP locus, eliminating individuals with the growth performance not reaching the standard and the dominant genotype lost, and continuously breeding for 3-5 generations to obtain a Danzhou new chicken strain with the stable and improved growth performance; S5, verifying the breeding effect, namely adopting a random control test to feed the new Danzhou chicken strain obtained by breeding and the original Danzhou chicken population which is not bred under the same feeding condition, measuring the core indexes of the growth performance of the new Danzhou chicken strain and the original Danzhou chicken population, and verifying that the weight of the new chicken strain at 70 days is improved by more than 10% compared with the original population, and the feed weight ratio is reduced by more than 8%.
2. The method according to claim 1, wherein in step S1, the different age-of-day stages include 7 days old, 21 days old, 42 days old, and 70 days old, and the number of Danzhou chicken individuals selected in each age-of-day stage is not less than 50, and the ratio of male to female is 1:1.
3. The method according to claim 1, wherein in step S2, the lysate is formulated as 10mmol/LTris-HCl (pH 8.0), 10mmol/LEDTA (pH 8.0), 100mmol/LNaCl, 1% SDS, and fresh proteinase K is added before use.
4. The method according to claim 1, wherein in step S3.2, the design criteria of the specific primer is that the primer length is 18-25 bp, the GC content is 40% -60%, hairpin structure and dimer formation are avoided, and the primer sequence is designed and verified by NCBIPrimer-BLAST tool.
5. The method according to claim 1, wherein in the step S4.2, the TaqMan probe is designed such that the probe length is 20-25 bp, the Tm value is 5-10 ℃ higher than that of the corresponding primer, the fluorescent reporter group is marked at the 5 'end, the fluorescent quenching group is marked at the 3' end, and the probe sequence specifically binds to the allele of the core SNP site.
6. The method according to claim 1, wherein in step S4.4, individuals with good health, no genetic disease and body conformation to the Danzhou chicken breeder characteristics are selected in addition to carrying the dominant genotype when constructing the breeding core group.
7. The method according to claim 1, wherein in step S4.5, the selection elimination rate of each generation is not lower than 20% during the continuous selection process, so as to ensure the genetic advantage of the core group.
8. The method of claim 1, wherein in step S5, the same feeding conditions comprise the same feed formula, feeding density, illumination time, temperature and humidity, and the feeding period is 70 days old.

Description

Danzhou chicken molecular breeding method based on growth performance related SNP markers Technical Field The invention belongs to the technical field of poultry breeding, and particularly relates to a Danzhou chicken molecular breeding method based on growth performance-related SNP markers. Background Danzhou chickens are good poultry varieties in characteristic places in Hainan regions of China, have the advantages of tender meat quality, unique flavor and the like, and occupy important positions in the high-quality poultry farming industry. The related technology of Danzhou chicken breeding refers to a technical system for screening excellent individuals and breeding strains with stable heredity and excellent growth performance through a human intervention means, and has the core functions of improving key production traits such as the growth rate, the feed conversion efficiency and the like of Danzhou chicken, shortening the breeding period, reducing the breeding cost, promoting the industrialized development and the quality upgrading of Danzhou chicken varieties and meeting the market demand for high-quality poultry products. The existing Danzhou chicken breeding mainly depends on the traditional phenotype selection technology, and the basic operation flow is that phenotype indexes such as weight, daily gain, material-weight ratio and the like of the Danzhou chicken in different growth stages are manually measured, the appearance performances such as individual health condition, body shape characteristics and the like are combined, individuals meeting the requirements are screened to form a breeding population, excellent strains are obtained through continuous breeding in multiple generations, part of early molecular breeding attempts adopt common chicken growth related SNP markers for auxiliary selection, marker screening is not carried out aiming at Danzhou chicken variety specificity, and only single or few SNP markers are adopted for typing detection. The prior art has the obvious defects that the traditional phenotype selection technology is greatly interfered by environmental factors, the phenotype index measurement period is long, the breeding efficiency is low, individuals carrying genes with excellent growth performance are difficult to accurately screen, the early molecular breeding technology lacks specific SNP markers aiming at Danzhou chickens, a multi-marker joint detection system is not constructed, the relevance between the markers and the growth performance is not strong, the breeding accuracy is not enough, the high-efficiency directional improvement of the growth performance of Danzhou chickens cannot be realized, and the requirements of industrial breeding on the accuracy and efficiency are difficult to meet. Therefore, research and development of a molecular breeding method based on Danzhou chicken specific growth performance related SNP markers becomes a key for improving the breeding efficiency and quality of Danzhou chicken. Disclosure of Invention Aiming at the problems of low traditional phenotype selection efficiency, lack of specific markers in early molecular breeding and insufficient precision in the conventional Danzhou chicken breeding technology, the invention provides a Danzhou chicken molecular breeding method based on growth performance-related SNP markers, and a multi-marker combined detection system is constructed by screening Danzhou chicken specific growth performance-related core SNP markers, so that the efficient and accurate directional breeding of Danzhou chickens is realized, the breeding period is shortened, and the breeding effect is improved. The invention provides a technical scheme for solving the technical problems, which comprises the following steps of 1. Danzhou chicken growth performance index measurement and sample collection, wherein Danzhou chicken individuals in different ages (7 days old, 21 days old, 42 days old and 70 days old) are selected, the number of the individuals selected in each age day stage is not less than 50, the male-female ratio is 1:1, the growth performance core index is measured, the growth performance core index comprises 7 days old weight, 21 days old weight, 42 days old weight, 70 days old weight, average daily gain and feed-weight ratio, meanwhile, blood samples of each Danzhou chicken individual are collected, and the obtained chicken individuals are placed in an anticoagulant tube containing EDTA, and refrigerated at-20 ℃ for standby. 2. Extracting and detecting quality of Danzhou chicken genome DNA, namely extracting genome DNA in a blood sample by adopting an improved salting-out method, wherein the specific operation comprises the steps of mixing the blood sample with a lysate according to a volume ratio of 1:5, adding proteinase K to a final concentration of 50 mug/mL, digesting for 2.5h in a 55 ℃ water bath, reversing and uniformly mixing every 30min for 1 time, wherein the lysate is prepared from 10mmol/LTris-HC