CN106701919A - Molecular genetic maker for egg quality of laying hen and application of molecular genetic marker - Google Patents
Molecular genetic maker for egg quality of laying hen and application of molecular genetic marker Download PDFInfo
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- CN106701919A CN106701919A CN201611054617.7A CN201611054617A CN106701919A CN 106701919 A CN106701919 A CN 106701919A CN 201611054617 A CN201611054617 A CN 201611054617A CN 106701919 A CN106701919 A CN 106701919A
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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
Abstract
The invention belongs to the technical field of laying hen molecular genetic marker assisted selecting, and particularly relates to a molecular genetic marker for egg quality of a laying hen and application of the molecular genetic marker. The molecular genetic marker is a partial sequence of genes VLDLR and CTSD of the laying hen, and a VLDLR nucleotide sequence is as shown in SEQ ID No.1, and VLDLR nucleotide sequences are as shown in SEQ ID No.2-4. The molecular making method is utilized for selecting egg quality characters, so that a molecular marking method which is more effective, simpler and more convenient to implement is provided for marker assisted selecting in chicken breeding work; meanwhile, the molecular genetic marker can overcome the defects that the normal egg quality character selecting method is great in workload, is easily affected by the environment, is long in breeding period and the like, and provides an effective molecular marker breeding means for egg quality character improving, so that genetic progress of chicken is quickened.
Description
Technical field
The invention belongs to laying hen molecular genetic marker assisted Selection technical field, and in particular to a kind of laying hen egg quality
Molecular genetic marker and application.
Background technology
Egg Quality directly affects the market price of the nutritional ingredient, edibility and then influence egg of egg, while to egg
Holding time, hatching of breeding eggs rate, breakage rate etc. have a certain impact.Egg Quality is largely influenceed by inherent cause,
Genetic force is generally between 0.5~0.7, and the wherein genetic force such as egg size, eggshell strength is higher.Kind is the conventional Egg Quality of influence
Main factor, Egg Quality genetic improvement is an important directions of following laying hen breeding, is also particularly important during laying hen produces
An index.
In recent years, with deepening continuously for studying egg quality, it has been identified that largely with the gene of egg product qualitative correlation.
Very low density lipoprotein receptor (VLDLR) is an important multifunctional receptor, is played during chicken with yolk precursor is formed
Key effect.VLDLR genes play key effect in chicken breeds, including egg mother cell development and lipovitellinin deposition.
Tacken etc. is studied using VLDLR gene defection type transgenic mices, as a result shows VLDLR in VLDL- triglycerides generations
Played a role in thanking, this storage to fat cell triglycerides plays a very important role.Han etc. is in the research to poephila castanotis
Middle discovery, the expression of VLDLR gene mRNAs has key effect in oviparous species breeding.One alkali of VLDLR gene coding regions
Base site mutation (G/C) causes the cysteine (Cys) in somewhere on protein chain to sport serine (Ser), so as to have a strong impact on
VLDLR is combined with membrana follicularis, causes the egg number to drastically reduce.It is therefore contemplated that VLDLR genes are the passes for influenceing egg number
Key gene.Recently there are some researches show the single nucleotide mutation of VLDLR has a significant impact to the egg-laying deseription of duck.Lysosome tissue
Protease (Cathepsin D, CTSD) is the important member of aspartic protease family, and wide participation intracellular protein disappears
Change, at the same be also VLDLR formed yolk during pass build enzyme.CTSD contents in the yolk forming process of egg mother cell extremely
Abundant, transhipments of the VTG and VLDL from blood to ovarian follicle not only relies on VLDLR, and in this endocytic processes, CTSD is also this turn
The key enzyme of fortune process.Therefore, can be using VLDLR and CTSD genes as the candidate gene for studying poultry egg-laying deseription.
The present invention uses direct sequencing, SNP of the detection VLDLR and CTSD genes in chicken, and analyzes
Each polymorphic site different genotype and 10 correlations of egg quality, obtain the related molecular genetic marker of egg quality, are
Chicken breed selection with excellent egg quality provides reliable foundation.
The content of the invention
The present invention seeks to for relevant not enough using molecular marking technique selection egg quality research, there is provided Yi Zhong
The life early stage of chicken can start, accuracy is high, the heredity of utilization VLDLR and the CTSD gene molecule of seed selection cycle is short, low cost
The method that labelling technique selects egg quality proterties.
To achieve the above object, the technical solution adopted by the present invention is：A kind of molecular genetic marker of egg quality, it is
The partial sequence of laying hen VLDLR and CTSD gene, the genetic marker VLDLR nucleotide sequences as shown in SEQ ID No.1, institute
Genetic marker VLDLR nucleotide sequences are stated as shown in SEQ ID No.2-4；VLDLR the and CTSD genotype is respectively CC, CT
With the compound individualities of gene of TT tri-, i.e., it is described in the SEQ ID No.1 sequences in one C/T site of the 13444th presence
, in one T/C site of the 2614th presence, SEQ ID No.3 are in one C/T of the 9445th presence for SEQ ID No.2
Site, in one C/T site of the 9457th presence, in one C/T site of the 9475th presence, SEQ ID No.4 exist
11028th presence, one T/C site.
The present invention also provides a kind of specific primer for expanding the molecular genetic marker of laying hen egg quality, for expanding
Increase VLDLR genes primer as shown in SEQ ID No.5-6, the primer such as SEQ ID No.7-12 institutes for expanding CTSD genes
Show, so as to for the detection of the genetic marker, such as be expanded by PCR and obtain the genetic marker, then obtained by cloning and sequencing
To corresponding sequence, or detected by Bsm I-RFLP polymorphisms.Thus, present invention additionally comprises for expanding described point
The primer of sub- genetic marker or the probe of the identification molecular genetic marker, and the kit containing the primer or probe.
The present invention also provides a kind of molecular genetic marker of laying hen egg quality in laying hen quality trait marker assisted selection
In application.
Further, the laying hen quality trait includes that 300 age in days egg productions, 500 age in days egg productions, egg size, egg type refer to
Number, shell thickness, eggshell strength, albumen height, yolk color, hangh unit, yolk weight and/or yolk ratio.
Principle of the 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 in position adjacent on DNA sequence dna to be checked, and linked enzyme connection after hybridization
A complete probe is formed, detection is realized in conjunction with different detection modes.
The method have the benefit that：
First, the present invention is the selection according to genotype to egg quality, with accuracy is high, simple to operate, expense is low
The features such as (alternative costs are about 0.3 yuan /), it is possible to the scale detection for being automated；
Second, be able to can be started in the life of chicken early stage (after birth in 1 week) using molecule labelling method of the invention
The proterties of egg quality is selected, the generation inteval can be shortened, improve selection intensity, excellent breeder parent is selected earlier,
So as to accelerate a breed of chicken process, determining system of selection with conventional egg quality and compare per generation can shorten selection time 3 months；
3rd, egg quality proterties is selected using molecule labelling method of the invention, can be not only chicken breeding work
Marker assisted selection provides a molecule labelling method more effectively, simple and easy to do in work, while conventional egg can be overcome
Quality trait system of selection workload is big, easily affected by environment and breeding cycle long the shortcomings of, be that egg quality character improvement is carried
For a kind of effective molecular marker breeding means, so as to accelerate the genetic progress of chicken.
Brief description of the drawings
In order to illustrate more clearly about 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
Other accompanying drawings are obtained with according to these accompanying drawings.
Fig. 1 is VLDLR gene 688bp fragment amplification electrophoretograms in the present invention；
Fig. 2 is CTSD gene 323bp fragment amplification electrophoretograms in the present invention；
Fig. 3 is CTSD gene 331bp fragment amplification electrophoretograms in the present invention；
Fig. 4 is CTSD gene 340bp fragment amplification electrophoretograms in 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 whole embodiments.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
1. primer is designed
According to the DNA sequence dna (accession number of chicken VLDLR genes on GenBank：NC_006127) and CTSD genes DNA sequences
Row (accession number：NC_006092), following primer (table 1) is designed.
Chicken VLDLR and CTSD the genetic polymorphism detection primer of table 1
2.PCR is expanded
PCR reaction systems are 20 μ L, wherein 1 × Buffer (Mg containing 3mM2+) the μ L of 2.0 μ L, 2mM dNTPs 2.0,
The μ L of upstream and downstream primer each 2.0 μ L, template DNA 50ng, 1U Taq archaeal dna polymerase 0.2 of 0.5pmol/L, plus ddH2O is extremely
20.0μL.Pcr amplification reaction program：95 DEG C of predegeneration 2min；94 DEG C of denaturation 30s, 59 DEG C of annealing 1min 30s, 72 DEG C of extensions
1min, 40 circulations；Extend 10min after last 72 DEG C.PCR primer detects expanding effect through 3% agarose gel electrophoresis, and send
Sequencing company is sequenced.
3.PCR product detections
4 pairs of primers for designing synthesis all obtain the PCR primer that band is clear, specificity is good under appropriate conditions,
And it is consistent with expected purpose fragment length (Fig. 1-4), can be subsequently sequenced.
4. direct Sequencing and Genotyping
Genomic DNA with 276 chickens from seed farm is template, enters performing PCR amplification and direct Sequencing, application
DNAMAN softwares compare sequencing result.1 mutational site is found in the exons 17 of VLDLR genes.In CTSD gene extrons 3
It was found that 1 mutational site, 3 mutational sites of exon 7 discovery, 1 mutational site of the discovery of extron 9.All mutational sites are all
Do not cause the change of amino acid, be silent mutation.To the genotype in each mutational site, 2 are the results are shown in Table.
Table 2 VLDLR, CTSD gene mutation site allele and Genotyping analysis
The influence of 5.VLDLR genes, CTSD gene SNP s loci gene types to egg-laying deseription
Individual parting is carried out according to VLDLR genes, CTSD gene mutations respectively, using least-square analysis different genes
Type is to 300 age in days egg productions, 500 age in days egg productions, egg size, egg shape index, shell thickness, eggshell strength, albumen height, yolk
Color, hangh unit, yolk are heavy, yolk ratio, totally 11 influences of proterties, as a result VLDLR and CTSD genes different genotype
The significant proterties of individual difference is shown in Table 3, table 4.
The relation of the chicken VLDLR gene orders SNPs of table 3 and part egg-laying deseription
Different capitalizations of going together represent extremely significantly correlated, and different lowercase letters are significantly correlated
The relation of the chicken CTSD gene orders SNPs of table 4 and part egg-laying deseription
Different capitalizations of going together represent extremely significantly correlated, and different lowercase letters are significantly correlated
Analysis is associated to the SNPs in the extron of chicken VLDLR genes the 17th and part egg-laying deseription, is as a result found：
There are 2 kinds of genotype in C13444T sites, to hangh unit, yolk weight, the significantly correlated (P of yolk ratio<0.05)；In hangh unit
Aspect, TT types individuality average is significantly higher than CC types individuality average 2.93% (P<0.05)；In terms of yolk weight and yolk ratio, CC
Type individuality average is significantly higher than TT types individuality average and is respectively 1.33%, 1.19% (P<0.05).
Analysis is associated to the SNPs and part egg-laying deseription in chicken CTSD genes the 3rd, 7,9 extrons, is as a result found：
There are 3 kinds of genotype CC, CT, TT in 5 mutational sites, and T2614C sites have a significant impact (P to hangh unit<0.05), TT bases
Because type individuality average ratio CT genotype 3.29%, TT types high are preponderant genotype；Two sites of C9445T, C9475T are to yolk weight
Have a significant impact (P<0.05), CC genotype individuals average ratio CT genotype 1.58% (P high<0.05)；C9457T is to yolk
Weight also has a significant impact (P<0.05), CC genotype individuals average ratio TT genotype 1.03% (P high<0.05).T11028C sites
(P is had a significant impact to albumen height<0.05), CT genotype individuals average ratio TT genotype 3.62% (P high<0.05).
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein (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
Understand, 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 be explained.
It should be noted last that：Above example is only used 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>A kind of molecular genetic marker of laying hen egg quality and application
<130>Specification, claims
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 688
<212> DNA
<213>VLDLR genes（Primers F 1-R1）Extension increasing sequence
<400> 1
tgaaatgcca agagtgctca aagaagggca gcaaaaaagc tgataagatg aagcctctta 60
gcactgttct tgtagaacgt tattaagtta atgttggagc aatttgtggg cttaatgtct 120
tagtaagaag tctggaaaaa tcagctagct tgtgattaag cagcctcact actttacaaa 180
ttcttgtgca ggattcaaca taagtagtgt ggtgtctgaa gtagctgcaa gaggagcagc 240
aggagcttgg gctgttcttc ctatctgtga gtaccatgtg ttgatccttc ttgttgggat 300
aagctcacaa gttcgacttg atgcaactct aaacttaaaa tgtatgtctc tccctgaagt 360
actgctggtg acggctgcat tggctggcta cttcatgtgg cgtaattggc agcacaagaa 420
catgaaaagc atgaattttg ataatcccgt ctatctgaaa actacagaag aggacctcac 480
aattgatatt ggcagacaca gtggttcagt gggacacacc taccctgcag taagtaaact 540
gattgacatg cctgaggttc caggctagtc ctcagagtgg atgtagcgga gacctgccaa 600
gtgactttca acatggggct tgatcaggtc ttaagcaacc taatctagct gtgcatgtcc 660
ctgttcattg tagggcaatc agaaggga 688
<210> 2
<211> 323
<212> DNA
<213>CTSD genes（Primers F 2-R2）Extension increasing sequence
<400> 2
tatctctcca gctcagggtg gctttgtggg atgcactcca ttattgctgg gcacctctgt 60
tcacagggct cctttctttt tttttttttg caggcccagt attatggtga gattggcatt 120
gggacccccc cacagaagtt cactgtggtc tttgacacgg gctcctccaa cctctgggtg 180
ccgtcagtgc actgtcacct gctagacatc gcctgttgta agtgtcacag cttgtccttc 240
cctgtgcagt gccagtgcag agactaacat aatgatcctg cccctgccat gaactggaag 300
cccctagctt gcatcatcat gtt 323
<210> 3
<211> 331
<212> DNA
<213>CTSD genes（Primers F 3-R3）Extension increasing sequence
<400> 3
gttcagttct ctgtagccct tgctgtggca tctctaagga gtgtctactc tctgggcatc 60
tctccctagg gtggatgttg ccaatgggct gaccctttgc aaagggggct gcgaggccat 120
tgtggacaca ggcacttccc tcatcactgg ccccaccaag gaagtgaagg agctgcaaac 180
agccattggt gcaaaaccac tcatcaaagg ccaggtgagg ctgcttggga tgtaattgca 240
cccaaatgct tttagagatg ctggtctcct gttttctgat tcctgtctgt gctccaggaa 300
aagcccccag cacagaaagc acccttctgt c 331
<210> 4
<211> 340
<212> DNA
<213>CTSD genes（Primers F 4-R4）Extension increasing sequence
<400> 4
tgaaatctgt aaggttgagg agaggtgagg atgtttggct gaagttcttg ctcttctcta 60
ggtttctgca caaggagaga ccatctgcct gagtgggttt tctggcctgg atgtcccacc 120
acctggaggc ccactctgga tcctgggaga tgtcttcatt ggcccctact acactgtctt 180
tgaccgtgat aacgactctg ttggttttgc caaatgtgtc taagcgcctg acccccccct 240
ccctcctcac tgtctctgct acacacaaac acttgcatac acacacagga gctgtagaga 300
agttgtagag aaagattatc aggattagaa acccactcag 340
<210> 5
<211> 20
<212> DNA
<213> VLDLR-F1
<400> 5
tgaaatgcca agagtgctca 20
<210> 6
<211> 20
<212> DNA
<213> VLDLR-R1
<400> 6
tcccttctga ttgccctaca 20
<210> 7
<211> 18
<212> DNA
<213> CTSD-F2
<400> 7
tatctctcca gctcaggg 18
<210> 8
<211> 18
<212> DNA
<213> CTSD-R2
<400> 8
aacatgatga tgcaagct 18
<210> 9
<211> 18
<212> DNA
<213> CTSD-F3
<400> 9
gttcagttct ctgtagcc 18
<210> 10
<211> 18
<212> DNA
<213> CTSD-R3
<400> 10
gacagaaggg tgctttct 18
<210> 11
<211> 18
<212> DNA
<213> CTSD-F4
<400> 11
tgaaatctgt aaggttga 18
<210> 12
<211> 18
<212> DNA
<213> CTSD-R4
<400> 12
ctgagtgggt ttctaatc 18
Claims (5)
1. the molecular genetic marker of egg quality, it is the partial sequence of laying hen VLDLR and CTSD gene, it is characterised in that institute
Genetic marker VLDLR nucleotide sequences are stated as shown in SEQ ID No.1, the genetic marker VLDLR nucleotide sequences such as SEQ
Shown in ID No.2-4；VLDLR the and CTSD genotype is respectively the compound individuality of gene of CC, CT and TT tri-, i.e., described
, in one C/T site of the 13444th presence, SEQ ID No.2 are in the 2614th presence one for SEQ ID No.1 sequences
T/C sites, SEQ ID No.3 one C/T site of the 9445th presence, one C/T site of the 9457th presence,
In one C/T site of the 9475th presence, SEQ ID No.4 are in one T/C site of the 11028th presence.
2. it is used to expand the specific primer of the molecular genetic marker of laying hen egg quality described in claim 1, it is characterised in that
For expanding the primer of VLDLR genes as shown in SEQ ID No.5-6, the primer such as SEQ ID for expanding CTSD genes
Shown in No.7-12.
3. the kit of primer described in claim 2 is contained.
4. according to claim 1 the molecular genetic marker of laying hen egg quality in laying hen quality trait marker assisted selection
Application.
5. apply according to claim 4, it is characterised in that the laying hen quality trait includes 300 age in days egg productions, 500
Age in days egg production, egg size, egg shape index, shell thickness, eggshell strength, albumen height, yolk color, hangh unit, yolk weight
And/or yolk ratio.
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| CN110551827A (en) * | 2019-09-19 | 2019-12-10 | 江苏省家禽科学研究所 | SNP molecular marker related to egg shape index and application thereof |
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| CN111286543A (en) * | 2020-03-08 | 2020-06-16 | 湖北省农业科学院畜牧兽医研究所 | Haplotype marker related to egg shell quality traits in KRT14 gene and application thereof |
| CN111440878A (en) * | 2020-03-08 | 2020-07-24 | 湖北省农业科学院畜牧兽医研究所 | Haplotype marker related to quality traits of egg shells in CDH17 genes and application |
| CN111705139A (en) * | 2020-06-11 | 2020-09-25 | 广西大学 | Screening method and application of Nandan Yao egg shell color association SNP molecular marker |
| CN111518920A (en) * | 2020-06-11 | 2020-08-11 | 广西大学 | Screening method and application of SNP molecular marker associated with eggshell thickness, eggshell strength and egg shape index of Nandan Yao chicken |
| CN111705139B (en) * | 2020-06-11 | 2022-09-16 | 广西大学 | Screening method and application of Nandan Yao egg shell color association SNP molecular marker |
| CN111518920B (en) * | 2020-06-11 | 2022-09-20 | 广西大学 | Screening method and application of SNP molecular marker associated with eggshell thickness, eggshell strength and egg shape index of Nandan Yao chicken |
| CN113493843A (en) * | 2021-07-05 | 2021-10-12 | 江苏省家禽科学研究所 | SNP genetic marker influencing egg yolk weight of chicken and application thereof |
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