CN101948831B - Locus in GARNL1 gene associated with egg production of 300-day-old chicken and use thereof - Google Patents
Locus in GARNL1 gene associated with egg production of 300-day-old chicken and use thereof Download PDFInfo
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Abstract
The invention discloses a locus in a GARNL1 gene associated with the egg production of 300-day-old chickens and use thereof. The GARNL1 gene associated with the egg production of 300-day-old chickens has a nucleotide sequence represented by SEQIDNo.1. The locus is at the position of the 138th bp on a genetic exon 15 of the GARNL1 gene and has a C/G mutant. The locus is closely associated with the total production of the 300-day-old chickens (P is less than 0.01), and an allele C at the locus is an advantageous allele associated with high total production of the 300-day-old chickens. The locus in the GARNL1 gene associated with the egg production of the 300-day-old chickens can be used for screening chicken varieties with high reproductive performance.
Description
Technical field
The present invention relates to genetically engineered and field of genetic engineering, be specifically related to relevant with chicken 300 age in days egg productivitys
GARNL1Site on the gene and application thereof.
Background technology
China's high-quality chicken aboundresources, but the reproductive performance of the local variety of exhausted major part is generally low, and annual egg on average has only 70-90 piece, and has obstinate broodiness (yang and jiang, 2005).Reproductive performance has lowly become the bottleneck of restriction high-quality chicken industry development, and improving the high-quality chicken reproductive performance is the problem that needs emphasis to solve in the high-quality chicken breeding.Traditional breeding method is fruitful to the raising of chicken reproductive performance, through high-intensity seed selection raising for many years, has bred the supporting system of specialized commodity egg, and annual egg is more than 280 pieces.But traditional breeding method needs the situation of laying eggs of whole hen production cycle of complete documentation, and the generation interval is long, and this has also limited the breeding progress of high-quality chicken reproductive performance.If will further improve the reproductive performance of high-quality chicken, must more deep understanding be arranged to its hereditary mechanism.Thereby the GENERALIZATION OF MODERN BREEDING TECHNIQUE of applied molecular mark is advanced effectively way for improving the high-quality chicken egg productivity.DNA analysis is applied in the high-quality a breed of chicken as main research means, and main purpose is through searching and the closely-related genetic marker of high-quality chicken reproductive trait on the dna molecular level, is used for early stage seed selection, thereby accelerates breeding process.
The reproductive trait of chicken is the proterties of a complicacy, and it is low to have heritability, is prone to characteristics such as affected by environment.Through researching and analysing, shown that a plurality of genes have participated in the reproductive behavior of chicken, as behavior is considered to oestrogenic hormon, progesterone and prolactin antagonist results of interaction (Gruaz et al, 1993 with regard to nest to chicken is neuroendocrine; Yasin et al, 1995; E1 Halawani et al; 2000); And gonadotropin releasing hormone (Gonadotrophin Releasing Hormone GnRH) is considered to control the crucial neuropeptide hormone of laying eggs, and chicken genome research progress provides possibility for the hereditary basis of poultry propagation proterties.(Quantitative trait locus, QTL) location is extensively applied in the research of chicken reproductive performance for candidate gene method and QTL.The existing research of the reproductive trait of chicken has been positioned to the different dyeing body region, but because most QTL Position Research adopts all is hybridization colony, thereby limited the direct application of the localized result of QTL in commercial colony.
GARNL1The present research of gene is considerably less, and the people is last
GARNL1The research of gene shows, it and neural phenotypes and family's fahr disease (Familial idiopathic basal ganglia calcification, IBGC; Claim Fahr disease again) be correlated with (Schwarzbraun etc., 2004), and grow the court of a feudal ruler stagnant relevant (Shimojima etc., 2009) with brain; Research on chicken is detected in Chen etc. (2006,2007) through subtractive hybridization cDNA library screening and egg productivity genes involved, and the result shows
GARNL1Gene differential expression in same strain high yield and low yield group is extremely remarkable, and this research has shown chicken
GARNL1Gene on the mRNA level with the relation of chicken reproductive performance.
Up to now, as yet not with chicken
GARNL1Gene is as the molecule marker of chicken reproductive trait.
Summary of the invention
The objective of the invention is to the deficiency that exists in the seed selection according to existing high-quality reproductive performance chicken, provide one relevant with chicken 300 age in days egg productivitys
GARNL1Site on the gene, this site can be used as molecule marker, is used for seed selection high-quality reproductive performance chicken.
It is above-mentioned relevant with chicken 300 age in days egg productivitys that a further object of the invention is to provide
GARNL1The application in the site on the gene.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
Relevant with chicken 300 age in days egg productivitys
GARNL1Site on the gene, said site are positioned at chicken
GARNL1A C at 138bp place on the gene extron 15 → G sudden change, said
GARNL1The nucleotide sequence of gene extron 15 is shown in SEQ ID NO:1.
GARNL1Last the 138th bp place has 1 base mutation on the gene extron 15, but does not cause the variation of restriction enzyme site.We use the method for mass spectrum somatotype to study the polymorphum in this site.
The present invention is relevant with chicken 300 age in days egg productivitys
GARNL1Site on the gene is as molecule marker; This site allelotype is that the individual total egg productivity of 300 ages in days of CC type wants the utmost point to be significantly higher than GC type and GG type P＜0.01; Thereby allele C is the total egg productivity advantage of 300 ages in days allelotrope, and the concrete steps that can be used for screening the chicken breed with high-quality reproductive performance are following:
(1) from chicken blood sample to be measured, extracts the chicken genomic dna;
(2) chicken that provides according to NCBI dbSNP DB
GARNL1Site sequence on the gene extron 15 goes out the nucleotide fragments that SNP to be measured belongs to by Shenzhen Huada Genetic Technology Co., Ltd according to the sequences Design primer amplification, utilizes MALDI-TOF-MS mass spectrometric detection platform to carry out gene type.The principle of mass spectrum somatotype is to utilize the principle that the flight time of sample molecule in electric field be directly proportional with the specific charge of molecule, the flight time through the test sample molecule, record molecular weight, and detect the SNP site;
(3), obtain the genotype of testing sample according to the resulting collection of illustrative plates of gene type.
In the above-mentioned steps (2), said primer is shown in SEQ ID NO:2 ~ 3.
Compared with prior art, the present invention has following beneficial effect:
The present invention be labeled as the chicken reproductive performance particularly the seed selection of the total egg productivity of 300 ages in days a kind of new molecule marker is provided.The experiment proof
GARNL1The total egg productivity of gene pairs chicken 300 ages in days has certain influence, can be as the genetic marker of candidate gene or 300 age in days egg productivitys.
Description of drawings
Fig. 1 is the techniqueflow chart of seed selection high-quality reproductive performance chicken breed of the present invention;
Fig. 2 is homozygote CC mass spectrum somatotype collection of illustrative plates result;
Fig. 3 is heterozygote GC mass spectrum somatotype collection of illustrative plates result;
Fig. 4 is homozygote GG mass spectrum somatotype collection of illustrative plates result.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
Embodiment 1
1, the extraction of the collection of chicken blood sample and DNA:
After 300 age in days reproductive traits are measured and are accomplished; Adopt disposable syringe under chicken wings, to extract about 1 mL blood the vein; Injection is through autoclaving and about 200 μ L, 2% aseptic EDTA is housed (Ethylene diamine tetraacetic acid EDTA) in the 1.5 mL centrifuge tubes of antithrombotics, shakes up gently; Record wing number ,-80 ℃ of preservations are subsequent use.
Phenol-chloroform extraction method (this pool F difficult to understand etc., 1998) is adopted in the extracting of genomic dna:
(1) gets whole blood 30 μ L and place 1.5 mL centrifuge tubes, add 470 μ L, 1 * SET damping fluid, 12.5 μ L, 20% SDS and 6 μ L10 mg/mL Proteinase Ks respectively, be put in 55 ℃ of water-baths after mixing and spend the night;
(2) take out sample in the 1.5mL centrifuge tube, add the saturated phenol of 500 μ L, jog 20 min, centrifugal 10 min of 10,000 rpm;
(3) get supernatant, add the saturated phenol of 500 μ L once more, jog 20 min, centrifugal 10 min of 10,000 rpm;
(4) get supernatant, add 500 μ L chloroform-primary isoamyl alcohol (23:1) jogs, 20 min, centrifugal 10 min of 10,000 rpm;
(5) get supernatant, add 1 mL ice absolute ethyl alcohol (20 ℃), oscillate, pour out ethanol behind centrifugal 10 min of 10,000 rpm with deposit D NA;
(6) clean DNA once with 1 mL, 75% ethanol, outwell ethanol, place oven dry in 50 ℃ of loft drier; (7) treat to add the distilled water dissolving after 300 μ L sterilize behind the DNA complete drying, spend the night with dissolving DNA in 50 ℃ of water-baths;
(8) deposit in-20 ℃ of refrigerators preserve subsequent use.
2, segmental acquisition of purpose and mass spectrum somatotype
(1) amplification contains the nucleotide fragments in site to be measured
According to sequence, entrust Shenzhen Huada Genetic Technology Co., Ltd's design primer, amplify the nucleotide fragments in site to be measured.This primer sequence is shown in SEQ ID NO:2 ~ 3.
(2) to site to be measured, design wall scroll special primer
The primer nucleotide sequence is shown in SEQ ID NO:4.
When annealing, the base of 3 ' end of special primer just combines with the previous base in site to be measured.
(3) single (two) base is extended the SNP site
In reaction system, add ddNTP and DNA polysaccharase, when a certain ddNTP with site to be measured base complementrity and when combining, the chain extension reaction termination obtains extending the sample analytes of a base.
(4) mass spectrometric detection, automatic parting direction
With putting into mass spectrometric valve tube behind sample analytes for preparing and the wafer matrix cocrystallization, excite with (10-9s) light laser of instantaneous nanosecond; Because substrate molecule is through energy that radiation absorbed; Cause energy to be accumulated and rapidly heat production and make the host crystal distillation; The nucleic acid molecule desorption also changes metastable state ion into, and mostly the ion of generation is single charge ion, in accelerating field, obtains identical kinetic energy; And then in a non-electric field drift region, separate according to its mass-to-charge ratio rate, flight arrives detector in the vacuum tubule.The ion that MALDI produces through the flight time (Time-of-Flight, TOF) detector detects, mass of ion is more little, just arrives more soon.
3, genotype is judged and association analysis
Judge the genotype of this site in detecting colony according to mass spectral result.
Embodiment 2
To chicken
GARNL1Gene locus different genotype and chicken reproductive trait carry out the detection of association analysis and use.Chicken
GARNL1 gene locus analytical results is as shown in table 1.Can obtain by table 1 that the influence to chicken 300 age in days egg productivitys (EN300) has reached utmost point level of signification (P＜0.01) between genotype.
The association analysis of table 1 GARNL1 gene rs14532787 site and chicken 300 age in days egg productivitys
Remarks:
1: least square mean standard error; () interior numeral number of individuals; Same letter representes that difference is not remarkable in the table, and different capitalizations represent that difference is extremely remarkable; * representes utmost point significant correlation (P＜0.01)
Shown in last table, the individual total egg productivity of 300 ages in days of CC type wants the utmost point to be significantly higher than GC type and GG type (P＜0.01), thereby allele C is the total egg productivity advantage of 300 an ages in days allelotrope.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>site and the application thereof on the GARNL1 gene relevant with chicken 300 age in days egg productivitys
<130>
<160> 4
<170> PatentIn?version?3.2
<210> 1
<211> 239
<212> DNA
< 213>artificial sequence
<400> 1
actcttatag?tagcttggat?caaagcaaat?ctaaacgtgt?acatctctcg?agaactttgg 60
gatgacttac?tgtctgtcat?gtcatcactg?acatactggg?aagagctggc?cactgagtgg 120
tcactgacaa?tggagacsc?taactaaagt?gttagctaga?aacttgtata?gtctggacct 180
cagtgacctg?ccattggata?agttaagcga?gcagaagcag?aaaaaacaca?aaggcaaag 238
<210> 2
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<212> DNA
< 213>artificial sequence
<400> 2
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<210> 3
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< 213>artificial sequence
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acgttggatg?cactgaggtc?cagactatac 30
<210> 4
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tcactgacaa?tggagac 17
Claims (2)
1. relevant with chicken 300 age in days egg productivitys
GARNL1The application in the site on the gene is characterized in that said relevant with chicken 300 age in days egg productivitys
GARNL1Site on the gene is as molecule marker; This site allelotype is that the individual total egg productivity of 300 ages in days of CC type wants the utmost point to be significantly higher than GC type and GG type P＜0.01; Thereby allele C is the total egg productivity advantage of 300 ages in days allelotrope, is used to screen the chicken breed with high-quality reproductive performance;
Said screening method comprises the steps:
(1) from chicken blood sample to be measured, extracts the chicken genomic dna;
(2) according to chicken
GARNL1The C/G mutational site at 138bp place on the gene extron 15, the design primer amplification carries out gene type after going out the nucleotide fragments at SNP to be measured place;
(3), obtain the genotype of testing sample according to the resulting collection of illustrative plates of gene type;
In the step (2), said primer is shown in SEQ ID NO:2 ~ 3;
Wherein, said site is positioned at chicken
GARNL1The 138bp place C → G sudden change is said on the gene extron 15
GARNL1The nucleotide sequence of gene extron 15 is shown in SEQ ID NO:1.
2. said relevant with chicken 300 age in days egg productivitys according to claim 1
GARNL1The application in the site on the gene is characterized in that in the step (2) that said gene type is accomplished through MALDI-TOF-MS mass spectrometric detection platform.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103275986B (en) * | 2013-05-30 | 2014-02-26 | 扬州大学 | Jinghai yellow chicken laying-number molecular marker and application thereof |
| CN104342489B (en) * | 2013-08-02 | 2016-08-31 | 中国农业大学 | A kind of method detecting chicken beard gene type |
| CN103789444B (en) * | 2014-02-26 | 2014-10-15 | 扬州大学 | Molecular genetic marker of 300-day-old egg-laying number of Jinghai yellow chicken and application of molecular genetic marker |
| CN104977397A (en) * | 2015-06-30 | 2015-10-14 | 柳州市宏华牧业有限责任公司 | Breeding hen reserving and picking method |
| CN105779607B (en) * | 2016-04-13 | 2019-02-01 | 南昌师范学院 | Application of the chicken GnRHR-2 gene in terms of as the molecular labeling of chicken reproductive trait |
| CN106701919A (en) * | 2016-11-25 | 2017-05-24 | 浙江省农业科学院 | Molecular genetic maker for egg quality of laying hen and application of molecular genetic marker |
| CN111647669B (en) * | 2020-07-24 | 2021-11-16 | 南昌师范学院 | Method for detecting correlation between GARNL1 gene and cock comb and pork lobe character and application |
| CN114622020B (en) * | 2022-03-30 | 2022-09-27 | 华南农业大学 | KLHL31 gene molecular marker related to chicken growth traits and application thereof |
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