CN106636367A - Molecular genetic marker related with egg laying performance of hens and application - Google Patents
Molecular genetic marker related with egg laying performance of hens and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Abstract
The invention provides a molecular genetic marker related with an egg laying performance of hens and application. An FSH (Follicle-Stimulating Hormone)-beta gene is used as the molecular genetic marker related with the egg laying performance of the hens; the molecular genetic marker is characterized in that the nucleotide sequence of the molecular genetic marker is shown as SEQ ID No. 1 or the sequence at least comprises a specific fragment of a 259<th> T>G mutated basic group. The egg laying performance of the hens are selected according to gene types and the molecular genetic marker has the characteristics of high accuracy, simplicity in operation and low cost (the selection cost is about 0.3 yuan per hen) and the like; automatic detection can be carried out; secondly, a molecular marking method provided by the invention can be used for selecting properties of the egg laying performance of the hens at an early life stage (within one week after chickens are hatched), the generation interval can be shortened, the selection intensity is improved and high-quality breeding hen parents can be selected relatively early, so that a breeding progress of the hens is accelerated; compared with a conventional determination selection method for the egg laying performance of the hens, the selection time of each generation can be shortened by 16 months.
Description
Technical field
The invention belongs to laying hen molecular genetic marker assisted Selection technical field, and in particular to a breeder egg laying performance point
Sub- genetic marker and application.
Background technology
China's place broiler variety source enriches, but compared with external commercial variety, aggregate performance egg laying performance is not good, carries
High broiler local varieties egg laying performance is always one of main target of China's poultry breeding.Traditional chicken egg laying performance selection-breeding side
Method is selected by determining the egg production phenotype of a generation (500 age in days).Egg is one long-term due to Phenotypic Selection
Cumulative process, its influence factor is again a lot, such as selection intensity, the number of selection traits, generation interval, environmental condition and nutrition
Level etc., all can have an effect to Phenotypic Selection, and its variation is in seriality, in addition the pass between the phenotype of character and its genotype
System is complicated, in most of the cases not fully coincide, thus selects often to be difficult to accurately, Same Way only be repeated several times,
Consecutive numbers could obtain remarkable result for orthoselection, the shortcomings of there is low cycle length, efficiency, high cost, be in progress slower.For
This, many researcheres are constantly exploring new more efficiently chicken egg-laying deseription selection technique, to change traditional system of selection
Deficiency.
Based on molecular genetic marker causes DNA fragmentation length polymorphism by species gene mutation, have many advantages：
(1) can not limited by space-time with the difference of direct detection DNA level；(2) marker number is abundant, polymorphism is high；(3) it is aobvious altogether
Property mark, homozygote and heterozygote can be distinguished；(4) some individual hereditary variatioies in family can be explained；(5) selecting to produce
During egg character, it is not necessary to carry out the determination test of laying eggs of 500 ages in days.As can be seen here, using DNA analysis technology, by
Find and the closely related genetic marker of chicken egg-laying deseription in DNA molecular level, Molecular Marker Information is included in breeding plan
It is to improve efficiency of selection to carry out assisted Selection, shortens the generation inteval, improves selection intensity, accelerates chicken egg-laying deseription genetic progress
Effective way.
In recent years, with deepening continuously to chicken egg-laying deseription research, it has been identified that related to egg-laying deseription in a large number
Gene, including prolactin antagonist, follicle stimulating hormone β subunits, Estrogen receptor α subunit, ovum yolk generation receptor, promoting sexual gland hormone
Releasing hormone, vasoactive intestinal peptide, insulin like growth factor and thyrotropin etc., these genes are mainly by adjusting
Corresponding hormone sensitive lipase gene affects the egg production of poultry with secretion.
Follicle stimulating hormone (Follical Stimulate Hormone, FSH) is animal anterior pituitary basophil cell point
A kind of glycoprotein promoting sexual gland hormone secreted, plays an important role in hypothalamic-pituitary-gonadal Reproductive Axis.Zhao Yaofeng
The insertion/deletion mutation of a 292bp is found that in+809 and+810 bases of the FSH β genes of pig Deng (1997), FSH β
Gene is associated analysis as the candidate gene of control litter size of pig with litter size of pig, it was demonstrated that FSH β genes show with litter size of pig
Write related.Recent studies indicate that FSH β genes also have certain relation with chicken reproductive trait, such as Hong Kunyue (2007) is adopted
Taihu Lake chicken FSH β gene pleiomorphisms are analyzed with PCR-SSCP methods and its relation with the egg laying performance laid eggs 20 weeks, find FSH β
Gene pairss Taihu Lake chicken early stage egg laying performance has a certain impact；Han Houming (2006) detects 6 in the control regions of Wenchang Chicken FSH β 5 '
Individual SNP site, with Age at first laying and the significantly correlated (P of 40 week old egg numbers<0.05)；Zhou Jun (2008) is in layer of green-shell egg exon
3 ' UTR detect A2447G, are in significantly correlated (P with Age at first laying<0.05)；Yang Huan (2011) has found the introne 1 of glad magnificent chicken
T+259G sites different genotypes individuality Age at first laying significant difference (P<0.05).
The present invention carries out FSH β genes using ligase reaction method (ligase detection reaction, LDR)
Polymorphic detection, SNP site screening and gene type, and analysis is associated with chicken egg laying performance, obtain a new chicken and produce
Egg performance correlation molecule genetic marker, for the broiler Fruit variety with excellent egg-laying deseription reliable foundation is provided.
The content of the invention
The present invention seeks to, for selecting the research of chicken egg laying performance not enough using molecular marking technique, there is provided one kind is in chicken
Life early stage can start, accuracy height, selection-breeding cycle is short, low cost utilization FSH β gene molecules Genetic Markers select
The method for selecting chicken egg laying performance.
For achieving the above object, the technical solution used in the present invention is：A kind of FSH- β genes are related as chicken egg laying performance
Molecular genetic marker, the genetic marker is the nucleotide sequence shown in SEQ ID No.1, or including at least the in the sequence
T at 259bp>The specific fragment of G mutating alkali yls, causes the single nucleotide polymorphism of FSH- β genes.
Those skilled in the art are easy to molecular genetic marker of the invention and design for expanding the molecular marker
Primer and probe, the primer sequence is as shown in SEQ ID No.2-3；Identify the probe of the molecular marker, the probe
Sequence so as to be used for the detection of the genetic marker, for example, obtains the heredity as shown in SEQ ID No.4-6 by PCR amplifications
Labelling, then corresponding sequence is obtained by cloning and sequencing, or detected by Bsm I-RFLP polymorphisms.Thus, this
The probe of the bright primer also included for expanding the molecular genetic marker or the identification molecular genetic marker, and containing
State the test kit of primer or probe.
The present invention also provides a kind of molecular genetic marker, primer or probe answering in chicken egg-laying deseription marker assisted selection
With.
Further, the genomic DNA of chicken breeding material is expanded, pcr amplification product agarose gel electrophoresiies are simultaneously reclaimed, right
The sequence that sequencing is obtained carries out polymorphism analysis, and in FSH- β gene rs259 mutational sites tri- kinds of genotype of TT, TG and GG are obtained,
Select TT genotype homozygous individual and set up breed system, copulation obtains the offspring that offspring is high egg production, can improve egg number.
The principle of the present invention：Ligase detection reaction is a kind of high specific technique of gene detection, and it is based primarily upon nucleic acid
Specific hybridization principle, a pair of hybridization probes hybridize adjacent position in DNA sequence to be checked, and linked enzyme connection after hybridization
A complete probe is formed, in conjunction with different detection modes detection is realized.
The method have the benefit that：The present invention propose the related FSH β Polymorphism Analysis of chicken egg laying performance with
Method for selecting molecular marker, can provide new way to improve Performance of Laying by molecular mark (MAS) technology.
In the selection work of chicken egg laying performance is applied to, with following beneficial effect：
First, the present invention is the selection according to genotype to chicken egg laying performance, with degree of accuracy is high, simple to operate, expense
The features such as low (alternative costs are about 0.3 yuan /), it is possible to carry out the scale detection of automatization；
Second, be able to can be started in the life of chicken early stage (in 1 week after birth) using the molecule labelling method of the present invention
The character of chicken egg laying performance is selected, the generation inteval can be shortened, improve selection intensity, excellent breeder parent is selected earlier
This, so as to accelerate a breed of chicken process, comparing per generation with conventional chicken egg-laying test system of selection can shorten selection time
16 months；
3rd, chicken egg laying performance character is selected using the molecule labelling method of the present invention, can be not only chicken breeding
Marker assisted selection provides a molecule labelling method more effectively, simple and easy to do in work, while conventional chicken can be overcome
Egg laying performance system of selection workload is big, easily affected by environment and long breeding cycle the shortcomings of, provide for the improvement of chicken egg laying performance
A kind of effective molecular marker breeding means, so as to accelerate the genetic progress of chicken.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 chicken FSH- β genes rs259 Genotyping figures of the present invention.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Embodiment
The method of utilization molecular marker assisted selection chicken egg laying performance of the present invention, is to complete following basis work
Obtain afterwards：
1. primer and probe
FSH- β genetic polymorphism detections primers and probe such as Tables 1 and 2：
The FSH- β genetic polymorphism detection primers of table 1
The FSH- β gene pleiomorphism primed probes of table 2
2. research of the Ligase detection reaction to SNP polymorphisms
TE is added to be diluted to 50pmol/ μ L as mother solution according to the molecular weight of primer and probe.Each primer when multiplex PCR, LDR
The ratios such as mother solution are mixed into the mixture solution that can be directly reacted.PCR amplification SNP sites place fragment, prepares according to following table
PCR Master Mix (20 μ L systems).
The PCR reaction systems of table 3
200 μ L PCR-buffer (10 ×), 60 μ L Mg are separately added in 1.5mL centrifuge tubes2+(100mM), 200 μ L
Dntp (20mM/each), 20 μ L Taq enzymes (5U/ μ L), 400 μ L Q-solution (4 ×), 40 μ L primer (5pM), finally
Add 980 μ L deionized waters.It is centrifuged after fully mixing, then takes 19 μ L and be divided in the PCR reaction tubes of 200 μ L, is eventually adding 1 μ L
Genomic DNA.
Following program is set on Perkin-Elmer Gene Amp PCR Systems 9600
Program：
95 DEG C of degeneration 15min, 35 circulations, each 3 temperature of circulation, 94 DEG C of 30s, 56 DEG C of 1min, 72 DEG C of 1min, finally
72 DEG C of extension 7min.After reaction terminates, take 2 μ L product and detect in 3.0% agarose gel electrophoresis, obtain expected length piece
Section.PCR primer is sequenced, following sequence (SEQ ID No.1) is obtained.
AGCAGTGGAAAGAGAAGAATGTGAACTCTGCATTACAGTGAATGCCACGTGGTGCTCAGGATACTGCTTCACAAGGG
TGAGAATCTTGAGCTTAATTCAGGCACCATAATTAAGTATTCAAGTGACGTATGAACAACCTGTTTCTTTGGTTTAT
AACAAACACATGAGCAAGATGAT/GCCTTATGAAAGAAAGGGCTGAATAACTGCTGTGTTGTTCAATAAGAAAGTAG
ATACATAGCCAAACTGAACTGTAAAGGGAAGGGTGAAAAAATGCTAGCTAAATTTGAATCTTTATTTGGCTGCATGC
ATTACAGTCATATTGTATAACTAGAGCTGAAAGTATGATAATCTGGATCTCTGCTGTCTGTTCAAT
Note：Black matrix mark site is rs259 sites.
The Ligase detection reaction of PCR primer：Equal-volume ddH is added in PCR primer2O dilutes, used as coupled reaction
Template.It is carried out according to the following table Ligature Mix (10 μ L systems).
The PCR Ligase detection reaction systems of table 4
100ul buffer (10 ×), 100 μ L Probe Mix, 5 μ are separately added in 1.5mleppendorf centrifuge tubes
L ligases, 695 μ L deionized waters.It is centrifuged after fully mixing, then takes 9 μ L and be divided in the PCR reaction tubes of 200ul, finally adds again
Enter 1 μ LPCR product.
Following program is set on Perkin-Elmer Gene Amp PCR Systems 9600：
Program：
95 DEG C of degeneration 2min, 35 circulations, each circulation has 2 temperature, and 94 DEG C of 30s, 50 DEG C of 2min put PCR reaction tubes
Enter PCR instrument and be attached reaction.
Respectively take 1 μ l LDR connection products and 1 μ l ABI GS-500ROX fluorescent tag molecule amount marks and 1 μ L deionization formyls
Amine sample solution mixes, 95 DEG C of heat denatureds 2 minutes, is quenched in ice, and 3000V is electric in 5% polyacrylamide and 5mol/L carbamide
Swimming 2.5 hours, carries out data collection, the correction of swimming lane line, the measurement of migration clip size and corrects using GENESCANTM672 softwares
Inherent molecular weight standard；Data analysiss and gene type are carried out using Genemapper softwares.
Chicken FSH- β gene rs259 Genotypings are as shown in Figure 1.
3. the association analysiss of genotype and egg-laying deseription and weight character
This test genotype and egg laying performance and weight character association analysiss adopt one factor analysis of variance, with chain point by force
Analysis software JMP10 (John ' s Mackintosh Program 10) is analyzed, and model used is as follows：Y=G+F (M)+M+e
Wherein Y is phenotypic number, and G is genetic effect, F (M) paternal line effects, and M is maternal effect, and e is stochastic effect.Tables of data
It is shown as least square means standard deviation.FSH- β gene SNP sites genotype the results are shown in Table 5 with egg-laying deseription association analysiss.
The association analysiss of the FSH- β gene rs259 loci gene types of table 5 and egg traits
Note：With a line, shoulder mark difference person represents significant difference.
Those skilled in the art of the present technique are appreciated that unless otherwise defined all terms used herein are (including technology art
Language and scientific terminology) have with art of the present invention in those of ordinary skill general understanding identical meaning.Should also
It is understood by, those terms defined in such as general dictionary should be understood that the meaning having with the context of prior art
The consistent meaning of justice, and unless defined as here, will not be with idealizing or excessively formal implication is explaining.
It should be noted last that：Above example is only to illustrative and not limiting technical scheme, although ginseng
The present invention has been described in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that：Still can be to this
Invention is modified or equivalent, any modification or partial replacement without departing from the spirit and scope of the present invention, and its is equal
Should cover in the middle of scope of the presently claimed invention.
SEQUENCE LISTING
<110>Zhejiang Academy of Agricultural Science
<120>The related molecular genetic marker of one breeder egg laying performance and application
<130>Description, claims
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 372
<212> DNA
<213>FSH- β gene amplification fragments
<400> 1
agcagtggaa agagaagaat gtgaactctg cattacagtg aatgccacgt ggtgctcagg 60
atactgcttc acaagggtga gaatcttgag cttaattcag gcaccataat taagtattca 120
agtgacgtat gaacaacctg tttctttggt ttataacaaa cacatgagca agatgatcct 180
tatgaaagaa agggctgaat aactgctgtg ttgttcaata agaaagtaga tacatagcca 240
aactgaactg taaagggaag ggtgaaaaaa tgctagctaa atttgaatct ttatttggct 300
gcatgcatta cagtcatatt gtataactag agctgaaagt atgataatct ggatctctgc 360
tgtctgttca at 372
<210> 2
<211> 19
<212> DNA
<213> rs259-F
<400> 2
agcagtggaa agagaagaa 19
<210> 3
<211> 19
<212> DNA
<213> rs259-R
<400> 3
attgaacaga cagcagaga 19
<210> 4
<211> 38
<212> DNA
<213> 259_modify
<400> 4
tcatcttgct catgtgtttg tttttttttt tttttttt 38
<210> 5
<211> 39
<212> DNA
<213> 259_T
<400> 5
tttttttttt ttttttttca gccctttctt tcataagga 39
<210> 6
<211> 41
<212> DNA
<213> 259_G
<400> 6
tttttttttt tttttttttt cagccctttc tttcataagg c 41
Claims (6)
1.FSH- β genes are used as the related molecular genetic marker of chicken egg laying performance, it is characterised in that the genetic marker is SEQ ID
Including at least the T at 259bp in nucleotide sequence shown in No.1, or the sequence>The specific fragment of G mutating alkali yls, leads
Cause the single nucleotide polymorphism of FSH- β genes.
2. it is used to expand the primer of molecular genetic marker described in claim 1, it is characterised in that the primer sequence such as SEQ ID
Shown in No.2-3.
3. it is used to identify the probe of molecular genetic marker described in claim 1, it is characterised in that the probe sequence such as SEQ ID
Shown in No.4-6.
4. containing the test kit of probe described in primer described in claim 2 and claim 3.
5. molecular genetic marker described in claim 1, probe described in primer, claim 3 described in claim 2 are laid eggs in chicken
Application in character marker assisted selection.
6. application according to claim 5, it is characterised in that the genomic DNA of amplification chicken breeding material, PCR amplifications are produced
Thing agarose gel electrophoresiies are simultaneously reclaimed, and to the sequence that sequencing is obtained polymorphism analysis are carried out, and in FSH- β genes rs259 position is mutated
Point obtains tri- kinds of genotype of TT, TG and GG, selects TT genotype homozygous individual and sets up breed system, and copulation obtains offspring to lay eggs
The high offspring of amount, can improve egg number.
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| CN107142313A (en) * | 2017-05-26 | 2017-09-08 | 湖北省农业科学院畜牧兽医研究所 | The SNP genetic marker related to quail egg-laying deseription and application |
| CN107338317A (en) * | 2017-08-24 | 2017-11-10 | 中国农业大学 | A kind of molecular labeling related to eggshell strength character and its application |
| CN107354220A (en) * | 2017-08-24 | 2017-11-17 | 中国农业大学 | A kind of molecular labeling related to chicken egg number character and its application |
| CN109182542A (en) * | 2018-10-17 | 2019-01-11 | 华南农业大学 | One kind SNP marker relevant to chicken Carcass Traits and its application |
| CN110499369A (en) * | 2018-05-17 | 2019-11-26 | 华中农业大学 | For selecting hen to store up the SNP marker and its application of smart ability |
| CN112430674A (en) * | 2020-12-23 | 2021-03-02 | 重庆市畜牧科学院 | Molecular marking method for detecting goose egg qualification rate |
| CN112592983A (en) * | 2020-12-23 | 2021-04-02 | 重庆市畜牧科学院 | SNP molecular marker method and primer for screening goose egg quality |
| CN112852977A (en) * | 2021-03-17 | 2021-05-28 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to later-stage egg laying traits in laying hen DPT gene and application thereof |
| CN113667759A (en) * | 2021-07-05 | 2021-11-19 | 江苏省家禽科学研究所 | SNP genetic marker related to egg-laying duration of chicken and application thereof |
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| CN112430674A (en) * | 2020-12-23 | 2021-03-02 | 重庆市畜牧科学院 | Molecular marking method for detecting goose egg qualification rate |
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| CN113667759B (en) * | 2021-07-05 | 2022-06-14 | 江苏省家禽科学研究所 | SNP genetic marker related to egg-laying duration of chicken and application thereof |
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