CN106755326B - One kind molecular genetic marker relevant to duck egg-laying deseription and application - Google Patents
One kind molecular genetic marker relevant to duck egg-laying deseription and application Download PDFInfo
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- CN106755326B CN106755326B CN201611061262.4A CN201611061262A CN106755326B CN 106755326 B CN106755326 B CN 106755326B CN 201611061262 A CN201611061262 A CN 201611061262A CN 106755326 B CN106755326 B CN 106755326B
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Abstract
The present invention relates to a kind of molecular genetic marker relevant to duck egg-laying deseription and applications, belong to field of poultry breeding, and in particular to application of the relevant site AMH gene G575A of duck egg-laying deseription as molecular genetic marker in duck production performance research.The present invention obtains one section of special DNA fragmentation as molecular labeling relevant to laying duck egg-laying deseription from AMH gene, there is G575-A575 base mutation at one in the sequence of the molecular labeling, the mutational site and the 43 week old egg numbers of duck, 72 week old egg numbers are significant related (P < 0.05), by PCR amplification and by PCR product sequencing and typing, selecting GG genotype is laying duck.Detection method significant effect of the invention, method is simple and fast, and free from the influence of the external environment.
Description
Technical field
The invention belongs to field of poultry breeding, and in particular to a kind of to improve duck group's egg number using homozygous complex gene type
Method.
Background technique
For a long time, China's laying duck cultivation mostly uses families selecting mode to carry out based on group rearing, in breeding.Laying duck group
In low yield group be to influence duck group's laying rate, an important factor for influencing breeding efficiency, huge economic loss can be caused.Laying duck produces
Egg number is influenced by multiple factors such as heredity, environment, nutrition and feeding managements, mostly uses reinforcement to raise in laying duck production at present
Management is supported, the aggregate measures of nutrition regulation and enhancement of environment are aided with.To a certain extent by the above method, it can be improved laying duck
The level of laying eggs of group.However, the means that the most fundamental method still passes through genetic improvement carry out choice breeding, gradually cultivate high
The laying duck group of production and good evenness.
Traditional breeding way is mainly selected from the phenotype of character, is made some progress, and character is especially worked as
Hereditary basis it is relatively simple or complex show additive gene effect heredity when, Phenotypic Selection is highly effective
's;But the quantitative characters such as egg-laying deseription, phenotypic character by minor-polygene control, at this time according to phenotype provide for property
The measurement of shape genetic potential is mostly inaccurate, thus selects to be often inefficient even invalid；And molecular labeling auxiliary choosing
Select the choosing that this kind of low-heritability traits can be greatlyd improve and influencing the intensity and accuracy of time of selection, selection
Select effect.Marker assisted selection is exactly to pass through label to implement indirect selections to objective trait, with the proviso that label and objective trait
Tight association.It has been that current Application of Animal Genetic improvement engineering is taken that molecular marker assisted selection is combined with traditional breeding way
One of main method find the mutually chain molecule of laying duck egg-laying deseription locus as the basis of molecular marker assisted selection
Label, to improve molecular marker assisted selection, the cultivation of high laying ducks kind provides new way and method.
SNP marker refers to the DNA sequence polymorphism as caused by genome single nucleotide variations, including base transition, transversion, list
Base insertion or missing etc., are acknowledged as newest third generation DNA molecular labeling.It can be divided according to position of the SNP in gene
For gene coding region SNP (coding-region SNP cSNP), gene periphery SNP (perigenic SNP, pSNP) and
SNP (intergenic SNP, iSNP) three classes between gene.Wherein the SNP of gene coding region may cause the ammonia of coded by said gene
Base acid sequence changes, and then changes the advanced form of albumen, influences the function of the protein hormone of coded by said gene.
Anti- Miao Le Shi pipe hormone (anti-Mullerian hormone, AMH) is transforming growth factor β superfamily
One of (transforming growth factor β superfamily, TGF-P) member.AMH is a kind of pair of reproductive system
Development and its important glycoprotein, and evaluation ovarian reserve index.In jenny, AMH to granulosa cell proliferation,
Activity of aromatizing enzyme, metakentrin receptor have inhibiting effect.It adjusts ovum original by reducing FSH and increasing inhibin indirectly
The development of cell, in Follicular growth, AMH plays main effect in the follicular recruitment stage that folliculus ovarii is developed.?
In fish, mammal, AMH gene polymorphic is expressed closely related with it.
However, research of the AMH gene in laying duck is also rarely reported, so far, there is no related laying duck AMH gene as
The research of the molecular labeling of duck egg-laying deseription.
Summary of the invention
The purpose of the present invention is passing through the Partial Fragment of PCR amplification AMH gene extron subsequence, mutational site, sieve are found
A kind of molecular labeling relevant to laying duck egg number is selected, and establishes batch detector methods answering as the marker assisted selection of laying duck
With.
The invention is realized by the following technical scheme:
Applicant by PCR amplification, obtained from AMH gene one section of special DNA fragmentation as with laying duck egg-laying deseription
Relevant molecular labeling, its portion gene exon sequence is as described in sequence table SEQ ID NO:1 and Fig. 1.
In the sequence of the 892bp of sequence table SEQ ID NO:1,1 polymorphic site is shared, is mono- G → A of 575bp
Base mutation, the detection in above-mentioned site is carried out using the method for direct Sequencing.
Applicant is prepared for detecting the primer pair of above-mentioned molecular labeling base mutation, and the DNA sequence dna of the primer is as follows:
5 ' CAGGGATAGCGGGCAGTT 3 ' (see sequence table SEQ ID NO:2) of P1 forward primer,
5 ' TCGGCGAGAAGCAAAGC 3 ' of P2 reverse primer (see sequence table SEQ ID NO:3).
Applicant provide a kind of methods for preparing above-mentioned molecular labeling, are prepared according to the following steps:
Sample populations Jingjiang duck blood liquid total DNA is extracted, resulting DNA is mixed and is built into the pond DNA, is set according to reference sequences
Primer (that is: P1 and P2 shown in primer) is counted, PCR amplification is carried out, obtains the partial exon sequences of duck AMH gene, PCR product
Direct Sequencing after purification, by sequence analysis obtain as shown in SEQ ID NO:1 with nucleotide sequence shown in FIG. 1；Wherein: institute
The exon sequence for the laying duck AMH gene stated is as shown in sequence table SEQ ID NO:1.
Molecular labeling prepared by the present invention can be applied to the detection of laying duck egg-laying deseription, and the primer pair can also be applied to egg
Duck egg-laying deseription detects in program.
Compared with prior art, the present invention having the advantage that are as follows: the present invention, which is realized, carries out early stage to laying duck egg-laying deseription
Selection, detection method quickly, accurately, and are not influenced by breeding environment condition factor.
Detailed description of the invention
Fig. 1: the genotyping result for the relevant molecular labeling of laying duck egg-laying deseription that the present invention clones is shown.
Fig. 2: the agarose gel electrophoresis figure for the AMH gene extron subsequence that PCR amplification obtains, swimming lane in figure: M is
DL2000Marker。
Specific embodiment
Embodiment 1
1, the extraction of the acquisition of duck blood sample and DNA:
After the completion of the pedigree data and egg-laying test of tested duck group, using disposable syringe under duck wing in vein
About 1ml blood is extracted, injection is through high pressure sterilization and is equipped in the 1.5ml centrifuge tube of the sterile ACD anti-coagulants of 200 μ L, gently shakes
It is even, wing number is recorded, -20 DEG C save backup.
The extracting of genomic DNA extracts genomic DNA using DNA extraction kit, operates according to kit specification, has
Body step is that 10 μ L duck bloods is taken to be added in 1.5ml centrifuge tube, and 20 μ L Proteinase Ks and 0.5ml are then added into centrifuge tube
Binging buffer after vortex oscillation 15 seconds, is stored at room temperature 10 minutes.Acquired solution is added in an adsorption column, 12,
000 rpm is centrifuged 1 minute, abandons waste liquid.Adsorption column puts back to collecting pipe and 0.5ml clean buffer is added, 12,000 rpm from
The heart 1 minute, abandon waste liquid.Adsorption column puts back to collecting pipe and 0.5ml wash buffer is added again, and 12,000 rpm are centrifuged 1 point
Clock abandons waste liquid, repeats 1 step time.Adsorption column is put into a clean centrifuge tube, is added 65 DEG C to adsorption column film centre
Sterilize high purity water, and after being placed at room temperature for 2 minutes, 12,000 rpm are centrifuged 1 minute collection genomic DNA.
2, the nucleotide fragments containing site to be measured are expanded
According to duck AMH genome sequence, design includes the primer of site sequence to be measured, using round pcr, expands tested duck
The DNA of group, using SEQ ID NO:2 and SEQ ID NO:3 primer pair, PCR reaction system is 25 μ L, and wherein template DNA is
50ng, dNTPs concentration are 200 μm of ol/L, and every primer concentration is 0.4 μm of ol/ L, 3U'sTaqArchaeal dna polymerase adds deionization
Water is to 25 μ L of total volume；PCR response procedures: 94 DEG C of initial denaturation 4min；Then 94 DEG C of denaturation 30s, 64 DEG C of annealing 30s, 72 DEG C prolong
1min is stretched, totally 40 circulations；Last 72 DEG C of extensions 10min.The PCR product of acquisition, commission sharp (Wuhan) biotechnology of elder brother Thailand have
The sequencing of limit company.
3, genotype judgement and association analysis
Genotype of the site in detection group is determined according to sequencing result.As a result as shown in Figure 1, if sample is pure
If zygote, with regard to only one product peak: the peak G or the peak A；If it is heterozygote, it just will appear 2 peaks: the peak G and the peak A.
Embodiment 2(Application Example)
In the group being made of 320 Jingjiang ducks, duck AMH gene different genotype and duck egg-laying deseription (including are opened
Produce age in days, 43 weeks egg numbers, 72 weeks egg numbers) carry out the detection application of association analysis.Genotype call results show examining
In 320 individuals surveyed, GG genotype individuals occupy the majority, and have 182 individuals, and GA genotype individuals have 90, AA genotype
Body is minimum, has 48 individuals, the results are shown in Table 1 for the association analysis of different genotype and character.
By table 1 it can be concluded that the mutational site and 43 week old egg number of laying duck, significant related (the P < of 72 week old egg numbers
0.05), the gene mutation in the above-mentioned analytic explanation site with there are significant or extremely significant to be associated between above-mentioned character.
Table 1AMHThe association analysis of gene G575A base mutation and egg-laying deseription
| Character | GG type (182) | AA type (48) | GA type (90) |
| Age at first laying/day | 126.74±7.03 | 132.98±8.83 | 128.62±9.93 |
| 43 weeks egg numbers/ | 155.30±8.34a | 142.18±6.44b | 145.99±6.89b |
| 72 weeks egg numbers/ | 327.38±11.75a | 286.69±22.61c | 311.74±17.96b |
Note: indicate different Superscript letters a, b, c indicate significant difference (P< 0.05)；Content representation number of individuals in ().
SEQ ID NO:1
<110>Hubei Province's academy of agricultural science animal and veterinary research institute
<120>a kind of molecular genetic marker relevant to duck egg-laying deseription and application
<160>3
<210> 1
<211> 892
<212> DNA
<400> 1
cagggatagc gggcagttcc tgggcatgct gacccgcttc atccgccggg tgctgagccc 60
ctccagtgag ccgcccaccc agcccagctc ccaccactgg ctggacttcc agatgatgga 120
gacgctccct caccagctgc tcaacctgtc tgagacggca gcgctggagc ggctggtgca 180
gtcggaggag ccttcggtgc tgcttttccc ccaggagggc agcgccgggc tggagcagca 240
ttttggggac tggcagccag aggggaccgt gctgcagctg ctgctgggca agctgcaggc 300
agtgatccag gagctgaggg acatcccggc gttccaagcc aacatggggc ttttccagca 360
cctcctgagc ttctgctact acccgccagg gccaggcacg ggcagcgcgg gtgagcggcc 420
gcctggctct gggaagctgc acgcgctgct gctgctgaag gcgctgcaga cggtgcgagt 480
gcactggcag gagcggagga aagtcctgcg acaaaaccgc agcgcccggc accaggccca 540
ctgccggctg caggagctga ccatcgacct gcacaaccgc aagttcatcg tcatgcccac 600
cgtgtacgcc gccaacaact gcgagggtcc ctgcaagctg cccctctcca cacgtgtccc 660
cagctactac tcgcacacgg tgctgctgct gggcatgcag gagcggggct cgcccctgca 720
gcgcgctccc tgctgcgtgc ccgtccgcta ctcggaccag ctcatcatca gcgtgtccag 780
ccaggggctg gaggtgcgca agttccccaa catggtggca gaggagtgcg gctgtcggta 840
gaggctcctg cccatagccc tggagcgccc ctgcagcttt gcttctcgcc ga 892
SEQ ID NO:2
<210> 2
<211> 18
<212> DNA
<400> 2
cagggatagc gggcagtt 18
SEQ ID NO:3
<210> 3
<211> 17
<212> DNA
<400> 3
tcggcgagaa gcaaagc 17
Claims (1)
1. a kind of molecular genetic marker relevant to duck egg-laying deseription, it is characterised in that: obtain the one of 892bp from AMH gene
Section specific DNA fragment has at one in the special DNA fragmentation as molecular genetic marker relevant to duck egg-laying deseription
G575-A575 base mutation is the base mutation of mono- G → A of 575bp, using PCR method to including the special of base mutation
DNA fragmentation is expanded, primer used in the PCR amplification are as follows:
P1 forward primer 5 ' to 3 ' is CAGGGATAGCGGGCAGTT；
P2 reverse primer 5 ' to 3 ' is TCGGCGAGAAGCAAAGC.
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| CN107034297B (en) * | 2017-06-05 | 2020-04-14 | 江苏省家禽科学研究所 | Molecular marker related to growth traits of meat ducks and application thereof, nucleic acid combination and kit |
| CN108546764B (en) * | 2018-05-02 | 2021-12-24 | 浙江省农业科学院 | Molecular marker related to egg laying performance of laying ducks and application of molecular marker in breeding |
| CN108977553B (en) * | 2018-09-05 | 2021-08-31 | 湖北省农业科学院畜牧兽医研究所 | Egg duck circular RNA circ _13267 and detection reagent, method and application thereof |
| CN109182562B (en) * | 2018-11-27 | 2021-05-04 | 湖北省农业科学院畜牧兽医研究所 | miRNA apla-mir-25-42 related to follicular development of laying ducks as well as detection primer, inhibitor and application thereof |
| CN109628613B (en) * | 2019-01-18 | 2022-02-11 | 湖北省农业科学院畜牧兽医研究所 | Molecular marker related to egg laying and egg quality traits of laying ducks and application thereof |
| CN110317809B (en) * | 2019-06-27 | 2021-06-29 | 湖北省农业科学院畜牧兽医研究所 | Long-chain RNA Lnc-30215 for regulating follicular development of laying duck and application thereof |
| CN111676295A (en) * | 2020-05-28 | 2020-09-18 | 浙江省农业科学院 | Research method of gene related to feed intake regulation |
| CN115992254A (en) * | 2020-12-22 | 2023-04-21 | 广东海洋大学 | Reagent for detecting genotype of SNP locus correlated with Leizhou black duck egg laying character |
| CN114317776B (en) * | 2022-01-13 | 2023-11-14 | 江苏省家禽科学研究所 | Molecular marker combination related to duck egg laying characteristics, obtaining method and application |
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