CN101892312A

CN101892312A - Molecular breeding method for chicken quality

Info

Publication number: CN101892312A
Application number: CN2010101958734A
Authority: CN
Inventors: 常国斌; 陈国宏; 栾德琴; 盛中伟; 李芹; 周琼; 廖菁; 龚琳琳; 胡国顺; 陈蓉; 刘艳; 张颖; 洪军
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2010-06-10
Filing date: 2010-06-10
Publication date: 2010-11-24

Abstract

The invention belongs to the fields of animal breeding science and molecular biology and in particular relates to a molecular breeding method for chicken quality. The method comprises the following steps of: building a high-quality white-earlobe chicken basic group; collecting blood samples of the two-week-old chickens and performing multi-locus sequence typing detection on candidate genes (GARS-AIRS-GART) which influence inosinate content and on candidate genes (A-FABP-3, Ex-FABP-3) which influence intramuscular fat content; determining favorable gene types through the nucleotide sequence variation of a plurality of candidate genes; and performing marker-assisted selection, so that genetic progress is accelerated and the breeding efficiency of the chicken quality characters is improved.

Description

A kind of molecular breeding method for chicken quality

Technical field

The invention belongs to animal breeding science and biology field.

Background technology

At present, China uses the method for PCR detection technique at research chicken meat quality genes involved more, has simple to operate, advantages such as specificity is high, good reproducibility, and can directly determine the position and the character of sudden change, and therefore application is very wide in polymorphism detects.And multidigit point sequence somatotype (Multilocus Sequence Typing, MLST) be the classifying method that a kind of nucleotide sequence by a plurality of functional genes of direct mensuration is found genovariation, on the basis of regular-PCR, improved, have and save time, reduce cost, the advantage of raising the efficiency, particularly save precious laboratory sample, therefore once proposition, promptly obtain numerous investigators' favor, every field in life science, multidigit point sequence somatotype has become a maturation and important research means, yet the correlative study aspect chicken meat quality yet there are no report both at home and abroad.On the other hand; high-quality chicken is the specialty industries of China; it is protection; exploitation and the main path that utilizes China's abundant chicken genetic resources; but exist meat matter proterties Phenotypic Selection genetic progress slow in the high-quality chicken breeding; problems such as breeding efficiency is low; this patent is on the basis of existing research; with candidate gene (GARS-AIRS-GART) (the Genebank accession number: that influences imp content AY665560) with the candidate gene (A-FABP-3 that influences intramuscular fat content; Ex-FABP-3) (the Genebank accession number is respectively: AY545055; DQ078254) be research object; utilize multidigit point sequence typing method; the correlation function gene is carried out simultaneously the many types of detection in a plurality of sites; and bonding mark auxiliary breeding means; set up a cover and have the meat matter molecular breeding technical system of independent intellectual property right, for the seed selection of high-quality chicken specialized strain and supporting system provides technical support.

Summary of the invention

The objective of the invention is a plurality of candidate genes that influence chicken meat quality are carried out sequence typing simultaneously.

Technical scheme of the present invention is: at first set up high quality white eared pheasant underlying group, 2 age in week blood sample collection the candidate gene (A-FABP-3, Ex-FABP-3) of the candidate gene (GARS-AIRS-GART) that influence imp content and intramuscular fat content carried out the detection of multidigit point sequence somatotype, nucleotide sequence variation by a plurality of candidate genes is determined the beneficial gene type, carry out marker assisted selection, thereby quickening genetic progress, the breeding efficiency of raising meat matter proterties.

Description of drawings

Fig. 1 is the sex change gel electrophoresis figure of Ex-FABP gene, AA:3 wherein, 6,8,9,11; BB:1; CC:12; AB:2,10; AC:5,7; BC:4.

Fig. 2 is the sex change gel electrophoresis figure of A-FABP gene, wherein AA:5; BB:1,3; AB:2,4,6

Fig. 3 is the sex change gel electrophoresis figure of GARS-AIRS-GART gene, TT:1 wherein, 4; CC:2,3,6,7; CT:5

Fig. 4 is the schematic flow sheet of breeding method of the present invention.

Concrete technological line is as follows:

1 materials and methods

1.1. set up the high-quality chicken underlying group

Based on existing high quality white eared pheasant group, take all factors into consideration proterties such as plumage look (Huang Yu, numb plumage), body weight, set up the high-quality chicken underlying group.

1.2 test materials

The wing venous blood of collection chicken in 2 age in week, conventional phenol/chloroform method is extracted DNA, and-20 ℃ of preservations are standby.

1.3 meat matter trait molecular marker genotype detection

To the underlying group of having set up, adopt analysis technology such as PCR-SSCP and PCR-RFLP, imp content correlation candidate gene (GARS-AIRS-GART) and intramuscular fat correlation candidate gene (A-FABP-3, Ex-FABP-3) are detected, identify idiotype and measure the intramuscular fat content and the imp content of corresponding preponderant genotype, and then be identified for the seed selection group of multidigit point sequence somatotype.

Found that:

(1) the Ex-FABP gene does not all detect SNP sudden change (SNP: the polymorphism of mononucleotide) at exon, detect C3654T at introne 3, C3699T, 3 SNP sudden changes of A3700G, totally 6 kinds of genotype, with the homozygous AA that is defined as respectively of Ex-FABP-3, BB and CC type, heterozygous is defined as AB, AC and BC type (Yang Xia. chicken Ex-FABP gene mononucleotide polymorphism and with the correlative study [D] of fatty character. Sichuan Agricultural University, 2006), denaturing gel electrophoresis figure such as Fig. 1 of different genotype, and wherein the chicken kind intramuscular fat content of BB type is the highest (table 1);

The white eared pheasant Ex-FABP different genotype intramuscular fat content (unit: %) of table 1.

(2) A-FABP gene extron 3 (A-FABP-3) detects the SNP sudden change of A1763G, its pure and mild type is defined as AA, BB type respectively, heterozygous is defined as AB, (Luo Guifen, Chen Jilan, Wen Jie, Zhao Guiping, Zheng Maiqing, Sun Shiduo. the chicken A-FABP Polymorphism Analysis and with correlative study [J] .2006 of fatty character, 28 (1): 39-42 :) and wherein the chicken kind intramuscular fat content of AA type be the highest (table 2);

The white eared pheasant A-FABP different genotype intramuscular fat content (unit: %) of table 2.

(3) the GARS-AIRS-GART gene is through comparing sequencing result, and finding has 1 Nucleotide that sudden change has taken place in the fragment, and polymorphism is caused by this sudden change.The sequence of CC type is identical with sequence among the GenBank, be defined as wild-type, nucleotide diversity has taken place in-179 sites in the TT type, sport T by C, be defined as mutant, this sports silent mutation, do not cause that coded amino acid changes, (Shu Jingting, Zhang Xueyu, Ji Congliang, Wu Xinsheng, Chen Guohong. association analysis [J] .2007 (3) of chicken GARS-AIRS-GART gene 5 ' flanking sequence polymorphism and chicken IMP content: 27-30) and wherein the chicken kind creatinine content of TT type is the highest (table 3).

The white eared pheasant GARS-AIRS-GART different genotype creatinine content (unit: %) of table 3.

1.4 meat matter proterties multidigit point sequence somatotype detects

A-FABP, Ex-FABP beneficial gene (concrete gene is: A-FABP-3 and Ex-FBP-3, GARS-AIRS-GART and Ex-FABP-3) to the GARS-AIRS-GART gene beneficial gene that influences imp content and intramuscular fat content carry out multidigit point sequence somatotype.

1.4.1 design of primers

(http://www.ncbi.nlm.nih.gov) searches genes involved (A-FABP-3, Ex-FABP-3, GARS-AIRS-GART) polymorphic site nucleotide variation information in the NCBI website, utilizes Primer Premier5.0 software design Auele Specific Primer.Three's primer sequence is respectively:

Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

1.4.2 PCR condition optimizing

Be optimized selection through template concentrations, the suitableeest primer amount, magnesium ion concentration, annealing temperature, extension time, cycle index etc. during PCR is reacted, and finally establish the peak optimization reaction system.

1.4.3 pcr amplification system

The pcr amplification reaction system is 10 * PCR damping fluid, 2 μ L, 10mmol/L dNTPs 2 μ L, and 20 μ mol/L NE-F primers, 1 μ L, 20 μ mol/L NE-R/R primers, 1 μ L, Taq enzyme 1U, 15ng/ μ L dna profiling 1 μ L, cumulative volume is 25 μ L.Reaction conditions: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 1min, 58 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 5min, observe imaging down in gel imaging instrument ultraviolet lamp behind the electrophoresis.

1.5 set up high creatinine colony and high intramuscular fat colony

What utilize among the present invention is the multidigit point sequence somatotype of A-FABP-3 and Ex-FABP-3, GARS-AIRS-GART and Ex-FABP-3, according to the gene test result, the genotype of comprehensive selection Ex-FABP-3 is that the genotype of BB type and A-FABP-3 is the AA type, the genotype of selecting GARS-AIRS-GART is that the genotype of CC type and Ex-FABP-3 is that the individuality of BB type is set up high creatinine colony and high intramuscular fat colony, all the other individual eliminating.

1.6 the foundation of molecular breeding system

Idiographic flow such as Fig. 4, utilize meat matter proterties (t-inosinic acid and intramuscular fat) beneficial gene type to carry out molecule marker and the estimation of conventional breeding value, determine molecular association selectivity index (I=MS+EBV, wherein MS represents the molecule scoring, EBV is an estimated breeding value), according to the height of selectivity index, carry out seed selection ages in 12 ages in week and 22 weeks respectively, the cock finally rate of reserving seed for planting is 20%, and the hen rate of reserving seed for planting is 80%.

2 results

The result shows: the candidate gene of imp content (GARS-AIRS-GART) and influence the middle A-FABP-3 of candidate gene (A-FABP, Ex-FABP) of intramuscular fat content and Ex-FABP-3, GARS-AIRS-GART and Ex-FABP-3 gene multidigit point sequence somatotype band clear, and carried out the order-checking of Gene Partial genotype individuality, find that these several genes can be used for direct sequence and measure, the results are shown in accompanying drawing.

The present invention can carry out the while somatotype to the genes involved that influences chicken matter, by the present invention gene is carried out the analysis of multidigit point sequence, sets up the meat matter molecular breeding technical system that a cover has independent intellectual property right.

Claims

1. molecular breeding method for chicken quality is characterized in that:

(1) at first set up high quality white eared pheasant underlying group,

(2) utilize the multidigit point sequence somatotype of A-FABP-3 and Ex-FABP-3, GARS-AIRS-GART and Ex-FABP-3, according to the gene test result, the genotype of comprehensive selection Ex-FABP-3 is that the genotype of BB type and A-FABP-3 is the AA type, the genotype of selecting GARS-AIRS-GART is that the genotype of CC type and Ex-FABP-3 is that the individuality of BB type is set up high creatinine colony and high intramuscular fat colony, all the other individual eliminating;

(3) utilize above-mentioned beneficial gene type to carry out molecule marker and the estimation of conventional breeding value, determine molecular association selectivity index I=MS+EBV, wherein MS represents the molecule scoring, EBV is an estimated breeding value, height according to selectivity index, carry out seed selection in 12 ages in week and 22 ages in week respectively, the cock finally rate of reserving seed for planting is 20%, and the hen rate of reserving seed for planting is 80%.