CN112852976A - Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof - Google Patents
Molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to later-stage egg-laying traits in an NCS1 gene of laying hens and application thereof, wherein the molecular marker is positioned at a partial coding region of the NCS1 gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO:1, the molecular marker totally comprises 3 SNP loci, namely three allelic mutation loci of C/T, C/G and A/G exist at the 171bp, 482bp and 567bp respectively, and a haplotype combination formed by the three allelic mutation loci can be used as a haplotype molecular marker related to the later-stage egg-laying traits of the laying hens, wherein the haplotype combination is H1H 3: the egg laying number of an individual CCG/CGG at 60 weeks is obviously higher than that of other haplotype combinations, and the selection of the haplotype combination is beneficial to improving the whole later egg laying number of the laying hen, namely, the invention provides a new molecular breeding marker for marker-assisted breeding of the later egg laying character of the laying hen, realizes the screening of the later egg laying character of the laying hen, and the screening method is simple and quick.
Description
Technical Field
The invention relates to the technical field of laying hen genetic marker screening, in particular to a molecular marker related to later-stage egg laying traits in an laying hen NCS1 gene and application thereof.
Background
The eggs are rich in various nutrient substances needed by human bodies, such as protein, fat, lecithin and the like, and are important components in the dietary structure of residents in China. The later egg laying period is a period in which the production performance of the chicken flocks is continuously reduced, the later egg laying period accounts for 50% of the whole egg laying period, the chicken flocks of about 500 days old are generally eliminated in breeding production, but the laying rate of the chicken flocks is still maintained at 60-70% at the moment, and the production performance of the chicken flocks in the later egg laying period directly influences the economic benefit of the laying hen breeding. The main reasons for the decrease of laying rate of laying hens in the later period of laying are ovary aging and hypofunction in the later period of laying hens, and the decrease of the number of follicles, the increase of dominant follicular atresia rate of development, the inhibition of follicular development and the like are mainly shown, so that the utilization period of the laying hens in the later period of laying hens is shortened, the economic value is reduced, and therefore, the increase of the laying rate of the laying hens in the later period of laying hens has important significance for the laying hens.
Single Nucleotide Polymorphism (SNP) refers to a polymorphism in a genomic DNA sequence caused by mutation of a Single Nucleotide (A, T, C or G), and includes insertion, deletion, transformation, transversion and the like of a Single base. genome-Wide Association Study (GWAS) is a genome-Wide Association analysis that uses millions of SNPs in a genome as molecular genetic markers to perform genome-Wide level control analysis and correlation analysis. The emergence of a large number of SNP markers gradually transformed the association analysis method centered on a single marker into the association analysis method based on haplotype (haplotype). Haplotypes are the SNPs that are tightly linked and that determine the same trait on the same chromosome or in a certain region, and are statistically related. The SNP Marker and the haplotype composed of the SNP Marker play an important role in the breeding of animal molecular Marker Assisted Selection (MAS) due to the distribution universality and stability, and become feasible technologies for the genetic improvement of the later-stage characters, especially the restrictive characters of the laying hens. Based on the method, SNP markers and haplotype combinations thereof related to the economic traits of the laying hens are further identified, and then the SNP markers and the haplotype combination sequences thereof are used as screening markers for auxiliary selection of the economic traits of the excellent laying hens, so that the accuracy of seed selection is greatly improved, and the method has certain significance for cultivating the laying hen varieties with the excellent economic traits.
Neuronal Calcium Sensor 1 (NCS 1) is a highly conserved Calcium binding protein involved in a variety of physiological cellular functions, including exocytosis, regulation of Calcium-permeable channels, neuroplasticity, and response to Neuronal injury. NCS1 has been shown to play an important role in adipocyte function, and a deficiency in NCS1 has been shown to contribute to obesity and type 2 diabetes in adult mice; altered expression of NCS1 is associated with neurodevelopmental and neurodegenerative diseases; tumor invasiveness can be promoted by promoting cell survival and metastasis, and the like. However, no report is found about the research of the NCS1 gene on the laying hens, and no research about the NCS1 gene of the laying hens as a molecular marker of the laying performance of the laying hens in the later period is available so far.
Disclosure of Invention
The invention aims to provide a molecular marker related to later-stage egg laying traits in an NCS1 gene of a laying hen and application thereof, and discovers a haplotype molecular marker related to later-stage egg laying performance in a partial coding region of the NCS1 gene of the laying hen for the first time, thereby providing an important reference basis for auxiliary selection of the later-stage egg laying traits of the laying hen.
One purpose of the invention is to provide a molecular marker related to later laying traits in an egg chicken NCS1 gene, wherein the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO. 1, and the molecular marker comprises:
SNP1 site: C/T polymorphic sites exist at the 171bp position in the sequence shown in SEQ ID NO. 1;
SNP2 site: the C/G polymorphic site exists at the 482bp position in the sequence shown in SEQ ID NO. 1;
SNP3 site: the A/G polymorphic site exists at the 567bp position in the sequence shown in SEQ ID NO. 1.
Further, the dominant alleles at 171bp, 482bp and 567bp in the sequence are C, C, G respectively, and the dominant genotypes are CC, CC and GG respectively.
Another object of the present invention is to provide a primer set for amplifying the molecular marker, the primer set comprising: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
The invention also aims to provide a detection method of the molecular marker related to the later laying character in the NCS1 gene of the laying hen, which specifically comprises the following steps: and (3) detecting by using the primer pair.
Further, the detection method comprises the following steps:
extracting genome DNA from blood of laying hens, amplifying by using the primer pair of claim 3 by using the genome DNA as a template, and comparing the sequence of the amplified products to obtain 3 SNP sites as described in claim 1.
The fourth object of the present invention is to provide a haplotype combination comprising the SNP sites, wherein the haplotype combination comprises the combination of SNP1-SNP3 sites.
The fifth objective of the present invention is to provide a method for constructing a haplotype combination, which comprises: firstly, detecting 3 SNP sites by adopting the detection method of the molecular marker; six haplotypes were then constructed, H1: CCG, H2: CCA, H3: CGG, H4: CGA, H5: TGG, H6: TGA; and then, establishing a haplotype combination according to the haplotype, and correlating the haplotype combination with the later laying traits of the laying hens.
The sixth purpose of the invention is to provide the application of the molecular marker, the primer pair or the haplotype combination in the auxiliary selection of the laying characteristics of the laying hens in the later period.
Further, the later-period egg laying traits of the laying hens comprise the egg laying number of 40 weeks and/or the egg laying number of 60 weeks
The seventh purpose of the invention is to provide a breeding method of late-stage high-yield laying hens, which comprises the following steps: the haplotype combination is constructed by adopting the construction method of the haplotype combination, and the haplotype combination is kept as HIH 3: individuals of CCG/CGG.
Compared with the prior art, the invention has the beneficial effects that: the invention firstly discovers a haplotype molecular marker related to the later laying performance of the laying hen in part of the coding region of the NCS1 gene of the laying hen, the sequence of the haplotype molecular marker is shown as SEQ ID NO:1, the length of the sequence is 1578bp, the sequence totally comprises 3 SNP sites, namely three allelic mutation sites of C/T, C/G and A/G exist at the 171bp, 482bp and 567bp respectively, and a haplotype combination formed by the haplotype marker can be used as the haplotype molecular marker related to the later laying character of the laying hen, wherein the haplotype combination H1H 3: the egg laying number of an individual CCG/CGG at 60 weeks is obviously higher than that of other haplotype combinations, and the selection of the haplotype combination is beneficial to improving the whole later egg laying number of the laying hen, namely, the invention provides a new molecular breeding marker for marker-assisted breeding of the later egg laying character of the laying hen, realizes the screening of the later egg laying character of the laying hen, and the screening method is simple and quick.
Drawings
FIG. 1 is a schematic diagram of a technical route of the present invention;
FIG. 2 shows the result of agarose gel detection of PCR amplification products of the NCS1 gene fragment of laying hen according to the embodiment of the present invention, wherein M is Marker, and 1-5 are the results of amplification of the NCS1 gene fragment;
FIG. 3 is a comparison chart of sequencing results of individuals with different genotypes at 3 SNP polymorphic sites of the layer chicken NCS1 gene in example 1 of the present invention: (a) the 171bp has C/T mutation site; (b) the 482bp has a C/G mutation site; (c) the 567bp has an A/G mutation site;
FIG. 4 shows the result of block analysis of the haplotype of the NCS1 gene in example 4 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 acquisition of partial coding region fragment of egg chicken NCS1 Gene and establishment of SNP detection method
1. Extraction of laying hen genome DNA
The test laying hen varieties comprise Jianghan chicken and Luo island red chicken, and samples are from poultry test fields of animal husbandry and veterinary research institute of agricultural and scientific institute in Hubei province. The extraction of the laying hen genome DNA adopts a genome DNA kit (operated according to the kit specification) produced by Beijing Baitacg biotechnology limited to extract, and the specific steps are as follows:
(1) about 1ml of blood was drawn from the inferior vein of the chicken wings using a disposable syringe, injected into a 1.5ml autoclaved centrifuge tube containing 200 μ L of sterile ACD anticoagulant, shaken gently, recorded wing number, and stored at-20 ℃ for future use. Sucking 10 μ L of anticoagulated blood, adding 500 μ L of BB2 and 10 μ L of proteinase K (20mg/mL), mixing well, and incubating at room temperature for 10 min;
(2) centrifuging for a short time, adding all the solution into a centrifugal column, centrifuging for 1min at 12000g, and discarding the effluent;
(3) adding 500 μ L of solution CB3, centrifuging at 12000g for 30s, and discarding the effluent;
(4) adding 500 μ L of WB3 solution, centrifuging at 12000g for 30s, and discarding the effluent;
(5) repeating the step 4 once;
(6) centrifuging at 12000g for 2min to completely remove the residual WB 3;
(7) placing the centrifugal column in a clean centrifugal tube, adding 50-200 μ L preheated EB or deionized water into the center of the column, standing at room temperature for 1min, centrifuging at 12000g for 1min, eluting DNA, and storing the eluted DNA at-20 deg.C for later use.
2. Obtaining partial nucleotide fragment of laying hen NCS1 gene
(1) PCR amplification
A primer pair is designed according to the sequence of the laying hen NCS1 Gene (accession number Gene ID: NC-006104.5) published by NCBI database, and the sequence of the primer pair is as follows:
an upstream primer: 5'-AGCAGTGGTGAGTAGCAG-3' (shown in SEQ ID NO: 2),
a downstream primer: 5'-GACATTGAACACGAAGGT-3' (shown in SEQ ID NO: 3).
The primers are used for carrying out PCR amplification in the genome DNA of the laying hens, the PCR reaction system is shown in table 1, and the PCR reaction conditions are shown in table 2.
TABLE 1 PCR reaction System
TABLE 2 PCR reaction conditions
And (3) carrying out agarose gel electrophoresis detection on the PCR amplification product, wherein the detection result is shown in figure 2, and the size of the fragment of the product is 1578 bp.
(2) Purification of PCR amplification products
The PCR amplification product is purified by using a Gel Extraction Kit of Shanghai Biotechnology engineering Co., Ltd, and the specific steps are as follows: cutting off gel containing target fragment from agarose gel, placing into 1.5mL centrifuge tube, adding 400 μ L sol solution, heating in 50-60 deg.C water bath until the gel is completely melted, mixing uniformly every 2min while heating, and cooling to room temperature; placing the centrifugal column into a collecting tube, transferring the mixed solution to the centrifugal column, and standing at room temperature for 2 min; centrifuging at 12000r/min for 1min, and adsorbing the DNA onto the column; pouring waste liquid in the collecting pipe, putting the centrifugal column into the same collecting pipe, adding 700 mu L of eluent, and centrifuging for 1min at 12000 r/min; pouring the waste liquid in the collecting pipe, and centrifuging for 1min at 12000 r/min; placing the column into a sterilized 1.5mL centrifuge tube, adding 40 μ L eluent or double distilled water (pH > 7.0), and standing at room temperature or 37 deg.C for 2-3 min; centrifuging at 12000r/min for 1min, wherein the liquid in the centrifuge tube is the recovered DNA fragment.
3. Detection of molecular markers by direct sequencing
And directly sending the PCR purified product obtained after purification to Beijing Okkomy Splending Biotechnology Limited to perform sequencing, and judging the genotype of the site in the detection group according to the sequencing result. Blast alignment analysis was performed using DNAStar software, and the analysis results are shown in fig. 3. The result shows that three allelic mutations of C/T, C/G and A/G exist at 171bp, 482bp and 567bp in the sequence respectively, the mutations cause the polymorphism of NCS1 gene, and the nucleotide sequences of the 3 SNP markers are shown in the sequence table SEQ ID NO: 1. The 3 polymorphic sites form 6 haplotypes, namely CCG, CCA, CGG, CGA, TGG and TGA.
Example 2 detection of polymorphism distribution of molecular markers of the invention in egg-laying hens
The polymorphism of 3 sites in the sequence shown in SEQ ID NO. 1 was detected, three genotypes were detected at the g.171bp C > T (NCS1_ SNP1), g.482bp C > G (NCS1_ SNP2) and g.567bp A > G (NCS1_ SNP3) sites, and the genotype frequency, allele frequency and distribution were shown in Table 3.
TABLE 3 layer chicken NCS1 genotype frequencies and allele frequencies
Note: genotype frequency the number in parentheses is the number of individuals of the genotype.
As can be seen from Table 3, 3 genotypes were detected at all 3 mutation sites of the sequence shown in SEQ ID NO. 1, and at positions NCS1_ SNP1 to NCS1_ SNP3, the dominant alleles were C, C, G, and the dominant genotypes were CC, and GG, respectively.
Example 3 correlation analysis and application of molecular marker and egg laying character of laying hens in later period
In order to determine whether the detected marks of the NCS1_ SNP 1-NCS 1_ SNP3 of the laying hens are related to the difference of the later laying character of the laying hens, Jianghan chicken and Luo island red chicken (the total number of samples is 220, wherein the Jianghan chicken and the Luo island red chicken are 110 respectively) are selected as test materials, the samples are collected in a poultry farm of animal husbandry and veterinary research institute of agricultural and scientific schools in Hubei province, the characters of the day of laying, the number of eggs laid at 40 weeks and the number of eggs laid at 60 weeks of each individual are recorded, polymorphism detection is carried out by using a direct sequencing method, and the correlation between different genotypes of partial coding regions of the NCS1 gene of the laying hens and the later laying character of the. The association analysis between genotype and phenotype was performed using SPSS18.0 software, using the following model:
Yij=u+Gi+Pj+eij
wherein YIj is a character observed value; u is the total average value of the characters; gi is the genotype effect; pj is the fixation effect; eij is the random error.
Correlation analysis between different genotypes of 3 mutation sites and later laying traits of laying hens is carried out in Jianghhan chickens and Luo island red chickens, and the statistical analysis result is shown in table 4.
TABLE 4 Association analysis of 3 mutation site polymorphisms of layer chicken NCS1 gene and later laying traits
Note: in the above table, the same letter indicates that the difference is not significant, the letters a, b and c indicate that the difference is significant, and n is the number of individuals of the genotype.
From the analysis results in table 4, it can be seen that the NCS1_ SNP1 to NCS1_ SNP3 site polymorphisms of the NCS1 gene have significant correlations with the day-to-date-of-birth age, the egg laying number at 40 weeks of age, and the egg laying number at 60 weeks of age (p < 0.05). Wherein the TT genotype at NCS1_ SNP1 site, the CC and CG genotype at NCS1_ SNP2 site, and the AA and GG genotype at NCS1_ SNP3 site have higher egg laying number of 40 weeks old; the CT genotype at the NCS1_ SNP1 site, the CG genotype at the NCS1_ SNP2 site and the GG genotype at the NCS1_ SNP3 site have higher egg laying number of 60 weeks old.
Example 4 correlation analysis and application of molecular marker and egg laying character of laying hens in later period
1. Construction of haplotypes and haplotype combinations
Haplotype analysis of NCS1_ SNP1 to NCS1_ SNP3 is carried out by utilizing Haploview software, the obtained genotype data of the NCS1_ SNP1 to NCS1_ SNP3 sites of all individuals are input into a PHASE program, the genotype of each individual is calculated, and meanwhile, the degree of pairwise linkage disequilibrium between the sites is calculated. The haplotype block analysis results are shown in FIG. 4.
As can be seen from FIG. 4, a total of 1 haplotype block was found by analyzing the linkage disequilibrium between the loci NCS1_ SNP1 and NCS1_ SNP3, and the haplotype block was analyzed to find 6 haplotypes in the laying hen population studied in the present invention, wherein two haplotypes exist per individual, and the sequence and number statistics of each haplotype are shown in Table 5.
TABLE 5 statistic results of SNP site haplotype of NCS1 gene
Haplotype | Sequence of | Quantity (only) | |
H1 | CCG | 260 | |
H2 | CCA | 72 | |
H3 | CGG | 35 | |
H4 | CGA | 27 | |
H5 | TGG | 41 | |
| TGA | 5 |
Haplotype combinations of the above haplotype compositions were analyzed for each individual, and a total of 11 haplotype combinations were found, as shown in Table 6.
TABLE 6 haplotype combinations of the NCS1 gene
Haplotype combinations | Sequence of | Quantity (only) |
H1H1 | CCG/CCG | 81 |
H1H2 | CCG/CCA | 46 |
H1H3 | CCG/CGG | 20 |
H1H4 | CCG/ |
5 |
H1H5 | CCG/TGG | 25 |
H2H2 | CCA/ |
10 |
H2H5 | CCA/TGG | 6 |
H3H3 | CGG/ |
5 |
H3H5 | CGG/TGG | 6 |
H4H4 | CGA/CGA | 11 |
H5H6 | TGG/ |
5 |
And rejecting part of haplotype combinations with small quantity in the sample population, and selecting 5 haplotype combinations with the largest quantity for association analysis.
2. Association analysis of haplotype combination and later-period egg-laying traits
The SPSS18.0 software was used to perform association analysis of haplotype combinations and late egg laying traits, and the results are shown in Table 7.
TABLE 7 correlation analysis results of advantageous haplotype combinations and later egg laying traits
Note: in the above table, the same letter indicates that the difference is not significant, the letters a, b and c indicate that the difference is significant, and n is the number of individuals of the genotype.
From the analysis results in Table 7, it was found that haplotype combination H1H 2: CCG/CCA and haplotype combination H1H 5: the 40-week-old egg production of CCG/TGG individuals was significantly higher than that of other haplotype-combined individuals (p <0.05), haplotype combination H1H 3: the 60 week old egg production of CCG/CGG individuals was significantly higher than that of other haplotype group individuals (p < 0.05). Combining the above analysis results, haplotype combination H1H 3: CCG/CGG has the best post-laying egg character. Therefore, in the laying hen population, by combining H1H 3: the selection and the retention of the CCG/CGG individuals are beneficial to improving the overall later laying character of the laying hens. Namely, the haplotype combination composed of the mutation sites identified by the invention can be used as a potential genetic marker for improving the later laying character of the laying hens so as to be used for the auxiliary selection of the high-yield laying hens.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Sequence listing
<110> institute of zootechnics of academy of agricultural sciences of Hubei province
<120> molecular marker related to later-stage egg laying traits in laying hen NCS1 gene and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1578
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> allele
<222> (171)..(171)
<223> y = c or t
<220>
<221> allele
<222> (482)..(482)
<223> s = c or g
<220>
<221> allele
<222> (567)..(567)
<223> r = a or g
<400> 1
agcagtggtg agtagcagcg tggcggtgag atgctggggg tgctggggga cccctcctgc 60
ttggagctca gtgcgggggc aggcagacct tggggtgcat gcagctcgga ggggaggaca 120
gcagcacacg gcagtccttt ccccgcaccc cgcatgcact ccgctccctc ycgtttgtgg 180
ctctgtgcag cggtggctct ctgcctttcc tcctgctcag cccccatggg gacgccgggg 240
ctctccgaac ggtgccacgg ggtgtacccc aatgcagccc tgaggggggc agagtctgag 300
gggcgtggac accaggacgg ggcatggagg cagggccgcc ttgcagcagc gtgctctcac 360
cccatcccct ccagctgcca aacgtgccgc cctaaaaaag ccaccgtttc cctttcatct 420
ccctatcaaa ccagtcaagg ttttctttct ttatcgcgtt ctctcatcct tctccccccc 480
tscccgatga ctcatagctg cagcagcacg aagatgtcac cggcacttca ctttcccacc 540
tcgccacccc cttcccctcc tcccccrggc cctcggcccg ctgacagatc tgggtgaggg 600
gttgtgtaat gctgctgcac acagcggggt cccagcgagg tccacgcgct gtcctcagac 660
tcacggagct gcctgacccc ctccctgcgc cctgtcagca ttggaggtgt tgcacctcct 720
tgtggctgtg ctctgtggtt ctgcctttgg ctgtgctgtc ccaggaggtg tagaagcccc 780
gctgtgcccc catttcccac gaggagtttt cacacacaca aaagcccctg cgctgcctca 840
gcttacctgg ctgcaaagct gccatcccag agcactgcac tgtggtgcct ggggctgtcg 900
tgtgattact ttttgctacg gcgtggctgc agcacgtgag tgtggaacag caggctgtgc 960
tggttggggg ctgctgtgct gggctctcat tgctgtcccc atcaaaatcc tcattctcat 1020
ccccatctcc gtctcattct catcctcgtc tccatcctca acctcatcct catccccagt 1080
atcatcccca tcccctttcc catccttttg cccatctcta tcccatcccc gtcctcattc 1140
ccatttccat cccactccca tctccgtccc catcctcatc cccgtcctca accgcttcgt 1200
catcctcatc ctcatctcca tcaccattcc catccccttt cccatcttca tgcccatccc 1260
catcctatcc ccatccgacc cctgtcccca tccccgtccc ctcatggcca atgctcaaca 1320
tggatgtggc catggagtag ttccccattc tgggcaggtg ctgtggttgg ggaccactcg 1380
atgtcaccgg tgccacttgg tgccctctgg gtcctaatat tgaaggaacc ctaaccctaa 1440
cgctccctcc ccaggtacaa gggctttatc aaggactgcc ccagtgggca gctcgatgct 1500
gccggcttcc agaagatcta caagcagttc ttcccctttg gtgacccaac caaatttgcc 1560
accttcgtgt tcaatgtc 1578
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcagtggtg agtagcag 18
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gacattgaac acgaaggt 18
Claims (10)
1. The molecular marker related to later-stage egg laying traits in the laying hen NCS1 gene is characterized in that the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO. 1, and the molecular marker comprises:
SNP1 site: C/T polymorphic sites exist at the 171bp position in the sequence shown in SEQ ID NO. 1;
SNP2 site: the C/G polymorphic site exists at the 482bp position in the sequence shown in SEQ ID NO. 1;
SNP3 site: the A/G polymorphic site exists at the 567bp position in the sequence shown in SEQ ID NO. 1.
2. The molecular marker of claim 1, wherein the dominant alleles at 171bp, 482bp and 567bp in the sequence are C, C, G respectively, and the dominant genotypes are CC, CC and GG respectively.
3. A primer pair for amplifying the molecular marker of claim 1, wherein the primer pair comprises: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
4. A method for detecting a molecular marker related to later-stage egg laying traits in an NCS1 gene of a laying hen, which is characterized by detecting by using the primer pair of claim 3.
5. The detection method according to claim 4, characterized in that it comprises:
extracting genome DNA from blood of laying hens, amplifying by using the primer pair of claim 3 by using the genome DNA as a template, and comparing the sequence of the amplified products to obtain 3 SNP sites as described in claim 1.
6. A haplotype combination comprising SNP sites in claim 1, wherein said haplotype combination is a combination comprising SNP1-SNP3 sites.
7. A method for constructing a haplotype combination, the method comprising: firstly, detecting 3 SNP sites by using the method of claim 5; six haplotypes were then constructed, H1: CCG, H2: CCA, H3: CGG, H4: CGA, H5: TGG, H6: TGA; and then, establishing a haplotype combination according to the haplotype, and correlating the haplotype combination with the later laying traits of the laying hens.
8. Use of the molecular marker of any one of claims 1-2, or the primer pair of claim 3, or the haplotype combination of claim 6 in assisted selection of laying hens for later laying traits.
9. The use of claim 8, wherein the late stage egg laying traits of the laying hens comprise 40-week-old egg laying numbers and/or 60-week-old egg laying numbers.
10. A breeding method of late-stage high-yield laying hens is characterized by comprising the following steps: the method of claim 7, wherein the haplotype combination is constructed, and the remaining haplotype combination is HIH 3: individuals of CCG/CGG.
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