阅读说明：本技术 一种筛选种母鸡贮精能力性状候选位点的方法和应用 (Method for screening candidate sites of breeding hen sperm storage capacity character and application ) 是由 牟春燕 楚金雨 李世军 马云龙 赵倩倩 杨戈 李绍梅 樊世杰 周宝贵 于 2020-12-25 设计创作，主要内容包括：本发明公开了一种筛选种母鸡贮精能力性状候选位点的方法和应用,属于家禽分子标记筛选鉴定技术领域,本发明结合重测序技术和极端群体表型信息,通过计算每个位点的群体分化统计量并与全基因组关联分析相结合,对种母鸡贮精能力性状候选位点和候选基因进行筛选。通过人工构建表型梯度差异群体的方法,使实验样本的数目大幅度降低,有效缩减了研究成本；该筛选方法具有数据量丰富,检测精度高的优点。本发明基于该方法筛选得到了5个种母鸡贮精能力性状候选位点,从而有效提高了种母鸡遗传改良的效率,降低育种工作的成本,为实际的蛋鸡种禽育种工作提供理论依据。(The invention discloses a method for screening a breeding hen semen storage capability character candidate site and application, belonging to the technical field of poultry molecular marker screening and identification. The number of experimental samples is greatly reduced by a method of artificially constructing phenotype gradient difference groups, and the research cost is effectively reduced; the screening method has the advantages of abundant data volume and high detection precision. According to the invention, 5 candidate sites of the sperm storage capacity character of the hen are obtained by screening based on the method, so that the genetic improvement efficiency of the hen is effectively improved, the cost of breeding work is reduced, and a theoretical basis is provided for the actual work of breeding the hen.)

1. A method for screening candidate sites of a breeding hen sperm storing capacity character, which is characterized by comprising the following steps:

step 1, feeding egg-laying hens of the same variety and the same day age, counting the phenotype data of the longest fertilization days of each egg-laying hen every month from the initial stage to obtain the effective phenotype data of the egg-laying hens of no less than 250 feathers, and extracting all blood samples of the egg-laying hens on the last day of counting;

step 2, analyzing the phenotype data of the longest fertilization days counted in the step 1, and dividing 3 pairs of gradient subgroups according to the difference of the phenotype data, wherein the gradient subgroups comprise a highest difference gradient pair, a medium difference gradient pair and a common difference gradient pair, and the method specifically comprises the following steps: rank ordering is carried out according to the longest fertilization days, the first 50 samples and the last 50 samples are selected to form a common difference gradient pair, on the basis, the 35 samples with the largest longest fertilization days and the 35 samples with the smallest longest fertilization days are selected to form a medium difference gradient pair, and on the basis of the medium difference gradient pair, the 20 samples with the largest longest fertilization days and the 20 samples with the smallest longest fertilization days are selected to form a highest difference gradient pair;

step 3, extracting the DNA of the blood samples of 100 egg-laying hens of the common differential gradient pair in the step 2, performing re-sequencing, removing sites with deletion rate more than 20% and sites with minimum allele frequency less than 0.05 to obtain SNP site data, and respectively calculating F of the three gradient pairs in the step 2 based on the SNP site data_STStatistics and AFD statistics;

step 4, calculating F in step 3_STRank ordering the statistics and the statistics that vary in gradients for each site in the AFD statistics, where F_STDefining overlapping SNP loci corresponding to the first 1% statistic in the statistic and the AFD statistic as target character association loci;

step 5, performing whole genome correlation analysis on the re-sequencing data of the 100 samples obtained in the step 3, and taking 10 sites with the minimum P value as candidate sites;

and 6, annotating the target trait associated site obtained in the step 4 and the candidate site obtained in the step 5, taking an overlapped site of the target trait associated site and the candidate site as a sperm storage ability trait candidate site of the breeding hens, wherein a gene annotated by the sperm storage ability trait candidate site is a sperm storage ability trait candidate gene.

2. The method of claim 1, wherein phenotypic data is collected for at least 5 months in step 1 and egg breeder hens that die or have a loss of phenotype during the feeding process are eliminated.

3. The method of claim 1, further comprising: and carrying out correlation analysis on the genotype of the candidate site of the sperm storing capability character obtained by screening and phenotype data, and carrying out genotype effect estimation.

4. Use of the method according to any one of claims 1 to 3 for screening candidate sites for the fertility trait of breeding hens and/or candidate genes for the fertility trait of breeding hens.

5. The breeding hen sperm storing capacity character candidate site screened by the method of any one of claims 1 to 3, comprising:

SNP1 site: the 65489499 th site of chromosome 1 has a G & gtA base mutation;

SNP2 site: the 87998885 th site of chromosome 1 has an A > G base mutation;

SNP3 site: a G & gtA base mutation exists at the 4889482 th site of the 27 th chromosome;

SNP4 site: a T > A base mutation exists at the 4898241 th site of chromosome 27;

SNP5 site: there is a mutation of C > T bases at position 4898269 of chromosome 27.

6. The breeding hen sperm storage capacity trait candidate site of claim 5, wherein the annotated sperm storage capacity trait candidate genes comprise: an ENGCALG 00000052327 gene, an ENGCALG 00000052718 gene, an ENGCALG 00000052321 gene, an MRC2 gene and a TANC2 gene.

7. The breeding hen semen storage capability trait candidate site of claim 5, wherein the base mutation at the SNP1 site and the SNP2 site is positively correlated with the semen storage capability trait of the breeding hen.

8. The breeding hen semen storage capability trait candidate site of claim 5, wherein the base mutations at the SNP3 site, the SNP4 site and the SNP5 site are in negative correlation with the breeding hen semen storage capability trait.

9. The use of the candidate site for the breeding hen sperm storing capability character according to claim 5 in the auxiliary selection of the breeding hen sperm storing capability character.

Technical Field

The invention belongs to the technical field of poultry molecular marker screening and identification, and particularly relates to a method for screening candidate sites of sperm storage capacity of a hen and application thereof.

Background

Eggs are food and consumer goods which are necessary in daily life of people, China is a big country for egg consumption and production, and the egg yield is 3308 thousands tons in 2019, and the trend of continuous increase is kept, so that how to quickly cultivate high-quality laying hens with good egg laying performance and strong egg laying capacity becomes a problem to be solved urgently by breeding workers in the domestic poultry breeding industry at present. The sperm storage capacity of the breeding hens is in positive correlation with the height of the high-yield fertilized eggs, so that the screening of the sperm storage capacity character is one of ideas of breeding of the breeding birds (the breeding hens).

The 'Jinghong No. 1' laying hen as a laying hen complete set line independently cultivated in China has a plurality of advantages of superior production performance, outstanding reproductive performance and the like. The reproductive performance of the breeder hen of the 'Jinghong No. 1' laying hen is artificially selected with high intensity, and whether the sperm storage capacity is selected in the process and which genes control the sperm storage capacity are not accurate. Therefore, some common methods for screening candidate genes (such as a method for selecting signals) are not suitable for the traits, but the method for performing genome-wide association analysis (GWAS) by using the re-sequencing data of a large population has higher cost, and the method of gene chips is not accurate enough, so that the method for calculating the differentiation degrees of different populations by artificially constructing the differences with phenotype gradients to associate the populations with the target traits and further determining candidate sites by using GWAS as an auxiliary method is a good breakthrough point. Although the GWAS result is influenced to a certain extent by smaller population scale, the influence is compensated to a certain extent by a method for associating the GWAS result with a target character by utilizing gradient differentiation, and the obtained result is more accurate compared with a single GWAS, so that the method for mining the candidate sites and the candidate genes of the sperm storage capacity of the laying hen breeding hens has important theoretical significance and economic value for breeding the laying hen breeding hens, and has certain reference and guiding significance for screening the sperm storage capacity of the broilers and local variety breeding hens.

Disclosure of Invention

The invention aims to provide a method for screening breeding hen sperm storage capacity character candidate sites and application, the method utilizes group differentiation statistics of group whole genome SNP site markers and GWAS combined analysis to evaluate the breeding hen sperm storage capacity character candidate sites and candidate genes, and concretely comprises the following steps: by combining a re-sequencing technology and extreme population phenotype information, the method is used for mining candidate sites and genes influencing the sperm storage capacity of the laying hen breeding hens by calculating the population differentiation statistics of each site and carrying out combined analysis with GWAS, can effectively improve the genetic improvement efficiency of the laying hen breeding hens, reduces the cost of breeding work, and provides a theoretical basis for the actual laying hen breeding work.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for screening candidate sites of breeder hen sperm storage capacity characters comprises the following steps:

step 1, feeding egg-laying hens of the same variety and the same day age, counting the phenotype data of the longest fertilization days of each egg-laying hen every month from the initial stage to obtain the effective phenotype data of the egg-laying hens of no less than 250 feathers, and extracting all blood samples of the egg-laying hens on the last day of counting;

step 2, analyzing the phenotype data of the longest fertilization days counted in the step 1, and dividing 3 pairs of gradient subgroups according to the difference of the phenotype data, wherein the gradient subgroups comprise a highest difference gradient pair, a medium difference gradient pair and a common difference gradient pair, and the method specifically comprises the following steps: rank ordering is carried out according to the longest fertilization days, the first 50 samples and the last 50 samples are selected to form a common difference gradient pair, on the basis, the 35 samples with the largest longest fertilization days and the 35 samples with the smallest longest fertilization days are selected to form a medium difference gradient pair, and on the basis of the medium difference gradient pair, the 20 samples with the largest longest fertilization days and the 20 samples with the smallest longest fertilization days are selected to form a highest difference gradient pair;

step 3, extracting the DNA of the blood samples of 100 egg-laying hens of the common differential gradient pair in the step 2, performing re-sequencing, removing sites with deletion rate more than 20% and sites with minimum allele frequency less than 0.05 to obtain SNP site data, and respectively calculating F of the three gradient pairs in the step 2 based on the SNP site data_STStatistics and AFD statistics;

step 4, calculating F in step 3_STRank ordering the statistics and the statistics that vary in gradients for each site in the AFD statistics, where F_STDefining overlapping SNP loci corresponding to the first 1% statistic in the statistic and the AFD statistic as target character association loci;

step 5, performing whole genome correlation analysis on the re-sequencing data of the 100 samples obtained in the step 3, and taking 10 sites with the minimum P value as candidate sites;

and 6, annotating the target trait associated site obtained in the step 4 and the candidate site obtained in the step 5, taking an overlapped site of the target trait associated site and the candidate site as a sperm storage ability trait candidate site of the breeding hens, wherein a gene annotated by the sperm storage ability trait candidate site is a sperm storage ability trait candidate gene.

Further, phenotypic data for at least 5 months are collected in step 1 and egg breeder hens that die or have a loss of phenotype during the feeding process are eliminated.

Further, the method further comprises: and carrying out correlation analysis on the genotype of the candidate site of the sperm storing capability character obtained by screening and phenotype data, and carrying out genotype effect estimation.

The invention also provides application of the method in screening the breeding hen semen storage capability character candidate sites and/or the breeding hen semen storage capability character candidate genes.

The invention also provides a breeding hen sperm storing capability character candidate site obtained by screening the method, which comprises the following steps:

SNP1 site: the 65489499 th site of chromosome 1 has a G & gtA base mutation;

SNP2 site: the 87998885 th site of chromosome 1 has an A > G base mutation;

SNP3 site: a G & gtA base mutation exists at the 4889482 th site of the 27 th chromosome;

SNP4 site: a T > A base mutation exists at the 4898241 th site of chromosome 27;

SNP5 site: there is a mutation of C > T bases at position 4898269 of chromosome 27.

Further, the sperm storing capability character candidate genes annotated on the sperm storing capability character candidate sites of the breeding hens comprise: an ENGCALG 00000052327 gene, an ENGCALG 00000052718 gene, an ENGCALG 00000052321 gene, an MRC2 gene and a TANC2 gene.

Furthermore, the base mutation of the SNP1 site and the SNP2 site is positively correlated with the sperm storage capability of the breeding hens.

Further, the base mutations at the SNP3 site, the SNP4 site and the SNP5 site are in negative correlation with the sperm storage capacity of the breeding hens.

The invention also provides application of the breeding hen semen storage capability character candidate site in auxiliary selection of the breeding hen semen storage capability character.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the invention, the number of experimental samples is greatly reduced compared with that of a conventional whole genome association analysis method by a method for artificially constructing a phenotype gradient difference population, so that the research cost is effectively reduced.

(2) Compared with the traditional whole genome correlation analysis method using a gene chip technology, the screening method has the advantages of abundant data quantity and high detection precision, and the method has wide application range, including but not limited to the phenotypic characters which are definitely selected manually.

(3) According to the invention, based on a method combining population differentiation and whole genome association analysis, 5 breeding hen semen storage capability character candidate sites and annotated candidate genes thereof are obtained by screening, and can be used for auxiliary selection of the breeding hen semen storage capability characters.

Drawings

FIG. 1 is a diagram showing the distribution of the sperm storing ability trait associated sites of "Jinghong No. 1" hen species, in example 3 of the present invention, wherein the allele frequency difference method has a selective indication effect, and more than 0 represents that the reference allele is selected in the sub-population with high sperm storing ability, and less than 0 represents that the mutant allele is selected in the sub-population with high sperm storing ability;

FIG. 2 is a group linkage disequilibrium attenuation diagram of extreme gradients in the sperm storing ability of "Jinghong No. 1" hen breeder hens in example 4 of the present invention, in which high _3/low _3 represents the highest difference gradient pair, high _2/low _2 represents the medium difference gradient pair, and high _1/low _1 represents the normal difference gradient pair;

FIG. 3 is a Manhattan chart and a QQ chart of the association between the sperm storage ability of the Jinghong No. 1 hen and SNP in example 4 of the present invention;

FIG. 4 is a diagram showing linkage disequilibrium between three candidate loci located on chromosome 27 of the Jinghong 1 hen genome in example 5 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.

The following examples are provided to illustrate the present invention by taking the essence storage ability of "Jinghong No. 1" egg-laying chicken as an example, but are not intended to limit the scope of the present invention.

EXAMPLE 1 phenotypic assay

300 egg laying hens of the same variety and with the same age of day, Jinghong No. 1, were bred in the grand institute of Oncorhium of Huadu valley poultry, Beijing under the same conditions. Collecting phenotype data of the longest fertilization days of 'Jinghong No. 1' egg-laying hens in the initial stage every month, collecting phenotype data of 5 months, removing dead or phenotype-deficient individual laying hens in the feeding process, obtaining effective phenotype data of 251 breeding hens in total, and extracting all blood samples of the experimental group of 'Jinghong No. 1' egg-laying hens on the same day of the last statistics.

And (3) carrying out rank ordering on the 251 hens according to the longest fertilization Day (DN) value, selecting the first 50 samples and the last 50 samples to form a common difference gradient pair, taking the 35 samples with the largest longest fertilization day value and the 35 samples with the smallest longest fertilization day value on the basis to form a medium difference gradient pair, and taking the 20 samples with the largest longest fertilization day value and the 20 samples with the smallest longest fertilization day value on the basis of the medium difference gradient pair to form a highest difference gradient pair. In this example, the gradient pairs were divided according to phenotypic variation, the difference in the number of samples for adjacent gradient pairs was 15, i.e., the tolerance was 15, and the phenotypic descriptive statistics are shown in table 1, where H.1 and L.1 are the common differential gradient pair, h.2 and L.2 are the medium differential gradient pair, and H.3 and L.3 are the highest differential gradient pair.

TABLE 1 longest fertilization Days (DN) graduation Scale Profile data

Example 2 genotyping assay

(1) According to the results of example 1, the DNA of the blood sample of 100 samples of the differential gradient population was extracted by SDS-proteinase K digestion, specifically:

a) thawing the blood sample at room temperature, putting 100 μ l of the blood sample into a clean 1.5ml centrifuge tube, adding 500 μ l of sterilized water, standing for 10min, centrifuging at 8000r/min for 5min, and discarding the supernatant;

b) adding 500 μ l SET solution to resuspend the precipitate, blowing and crushing the precipitate with a gun head, centrifuging at 8000r/min for 10min, and discarding the supernatant;

c) repeating the previous step;

d) preparing a digestive juice (500. mu.l SET + 30. mu.l SDS + 10. mu.l proteinase K), adding 540. mu.l digestive juice into each tube, and blowing and resuspending the precipitate;

e) the tube containing the digested sample was inserted into a float and digested overnight in a 55 ℃ water bath (until the sample was completely digested);

f) adding 5-20 μ l 10mg/ml RNase into the digested sample, and putting in a water bath or oven at 37 ℃ for 1.5-2 h;

g) an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1) slowly inverting the centrifuge tube for 10 times by 560 μ l of the mixed solution, standing for 15min, centrifuging at 10000rpm at room temperature for 15min, and sucking the supernatant to another clean centrifuge tube by using a yellow gun head for a few times;

h) an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1) mixing the above solutions and supernatant, repeating the above extraction step, and transferring the supernatant to 1.5ml EP tube;

i) adding 1/10 volumes of 3M NaAC and 2-2.5 volumes of precooled absolute ethyl alcohol into the supernatant, slightly overturning until DNA aggregates into a mass, forming white filamentous suspension precipitate, and directly picking out the precipitate by using a gun head to a new EP tube;

j) adding 500 μ l 75% ethanol to wash the precipitate, centrifuging at 6000rpm for 5min, pouring off the residual liquid after centrifuging, washing again, completely suspending the precipitate (the suspension time is related to the size of the precipitate), centrifuging for a short time, sucking out the residual liquid, and volatilizing the ethanol at room temperature;

k) adding 20-100 μ l TE, dissolving, measuring DNA concentration, diluting, packaging, and storing at-20 deg.C or-80 deg.C for a long time without repeated freeze thawing of stock solution.

(2) Detecting SNP sites and genotypes thereof by using a whole genome re-sequencing technology, removing sites with deletion values larger than 20% and removing sites with minimum allele frequency smaller than 0.05 to obtain 1744595 high-quality SNP sites in total.

Example 3 calculation of group differentiation statistics of SNP loci for sperm storage ability traits of hens

(1) Based on the SNP sites obtained in example 2, F of the 3 difference gradient pair populations described in example 1 were calculated separately_STAnd evaluating the differentiation degree of the population locus according to the statistic and the AFD statistic.

F_STThe index is an index for measuring the differentiation degree of the population, F_STThe index may assess the degree of difference in allele frequencies at the same locus across genomes of different populations due to selection. Currently used for calculating F_STThere are many methods of indexing, and the most widely used methods are those proposed by Weir and Cockerman (Weir, B.S. and C.C. Cockerham (1984). "Estimating F-Statistics for the Analysis of the marketing structure", "Evolution", "international j ournal of organic Evolution 38(6):1358.), the basic formula is as follows:

whereinp_AiIs the frequency of the ith population allele A, n_iIs the number of samples of the ith population and s is the number of populations. F_STHas a value range of [0,1 ]]Larger numbers indicate a more constant (higher frequency) allele in the respective subpopulation, with greater degree of inter-population differentiation.

The AFD statistic is the most intuitive statistic which reflects the group differentiation degree, and the calculation formula is as follows:

wherein n represents the total number of different alleles observed at the polymorphic site, and f_iRepresenting the gene frequency of allele i in one population. In this experiment, 1 represents a subgroup with high sperm storing ability, and 2 represents a subgroup with low sperm storing ability. The positive and negative of the AFD statistic have directionality, with positive values indicating allele i is selected in the high-group subpopulation and negative values indicating allele i is selected in the low-group subpopulation.

(2) The F of each SNP site in the 3 gradient pairs described in example 1 was calculated on the basis of the above procedure_STValue, first screening for F with gradient differences_STStatistic, then F by highest difference gradient pair_STValues were rank ordered and sites corresponding to the top 1% values were defined as significant sites. Based on F_STThe method detects 1782 SNP molecular marker sites (as shown in figure 1) meeting the above conditions, which account for 0.1% of all the used molecular markers. And simultaneously calculating the AFD value of each SNP locus in 3 gradient pairs, selecting and calculating the frequency of a reference allele (ref), and calculating AFD statistics in 3 groups of extreme phenotype difference gradient pairs respectively. Firstly, positive and negative statistics with gradient difference are obtained through screening to carry out rank ordering, and sites corresponding to statistics of 0.5% before a positive value and 0.5% after a negative value of a highest difference gradient pair are defined as significant sites. 2663 total detection devices meeting the above conditions based on AFD methodThe SNP molecular marker sites (as shown in FIG. 1) account for 0.15% of all the molecular markers used. By using F_STSNP loci corresponding to the first 1% statistic with gradient change statistic detected by the statistic method and the AFD statistic method are defined as 'Jinghong No. 1' laying hen sperm storage capacity associated loci. The present invention detected 1070 associated sites in total, annotated 170 genes, as shown in table 2.

TABLE 2 Jinghong No. 1 laying hen sperm storage ability character correlation site and annotation gene

Example 4 GWAS analysis between sperm storage ability traits and SNP sites

(1) The 100 re-sequenced data obtained in example 2 were subjected to genome wide association analysis (GWAS), which was used as an aid to find sites associated with the target trait.

GWAS is a research method for searching genetic factors related to target traits by typing whole-genome high-density genetic markers (such as SNP or CNV) on large-scale population DNA samples on the whole-genome level. GWAS analysis was performed using GEMMA software, often using a mixed linear model, as follows:

y＝wα+xβ+Zu+ε

u～MVN_m(0，λτ^-1K)

ε～MVN_n(0，τ^-1I_n)

where n is the number of individuals, m is the number of families or groups, y is an nx1 quantitative trait vector, W ═ (W1, w2... wc) is a covariate (fixed effect) of the nxc matrix including the column vector of 1, α is the cx1 vector of the corresponding coefficients including the intercept, x is the nx1 vector of the marker genotype, β is the effect size of the marker, Z is the nxm load matrix, u is the mx1 vector of the random effect, ε is the nx1 vector of the error, τ is the nx1 vector of the error^-1Is the variance of the residual error, λ is the ratio between the two variance components, K is the known m × m correlation matrix, I_nIs an n × n identity matrix, and MVN represents a multivariate normal distribution.

(2) Carrying out GWAS analysis on SNP re-sequencing data of 100 individuals on the basis of the steps, adopting GEMMA software to carry out covariate-free mixed linear model analysis in the data analysis process, determining a whole genome significant critical value by using a simpleM method, and when p is less than 0.05/M_effWhen (M)_effMeaning the effective number of SNPs in one dataset calculated by the simpleM method), the SNPs are considered to reach the genome-wide significance level. The threshold value of the significance level of the whole genome in the research is 0.05/135206 ═ 3.7E-07, and one SNP locus is in the significance level (as shown in figure 3), the invention selects 10 SNP loci with the peak value on both sides of the peak value, namely the minimum P value, as candidate loci, and the positions and P values are shown in table 3:

TABLE 3 GWAS-based identified sperm storage capability trait candidate site of 'Jinghong No. 1' breeder hen

(3) After all candidate SNP sites are determined, itWhile being F_STSNP sites detected by AFD and GWAS are identified as extremely reliable target trait associated sites. 5 target trait significant association sites are screened (shown in table 4), 5 genome functional regions with selection potential in 'Jinghong No. 1' laying hen species hen are found by annotation, and the five sites are genotyped according to the genotype information of each individual in the variation file (shown in table 5). While using the plink software to calculate the degree of linkage disequilibrium attenuation in various populations with different gradient phenotypes (as shown in fig. 2), the LD curves in all sub-populations attenuated at similar rates, indicating that the gradient partitioning of the populations did not affect the identification of trait association sites.

TABLE 4 results of gradient statistics of significant association sites of sperm storage ability traits of 'Jinghong No. 1' breeder hens

TABLE 5 Gene typing results of significant association sites of sperm storage ability traits of 'Jinghong No. 1' breeder hen

Example 5 genotype Effect estimation

The genotype effect at the site associated with each SNP trait was evaluated by correlating the genotype with the phenotypic data (as shown in Table 6), and the results showed that the presence of two mutant bases on chromosome 1 increased the phenotypic value of the individual, indicating that the presence of mutant bases at these two sites beneficially affected (positively correlated) the sperm storage ability of the breeder hen, while the presence of three mutant bases on chromosome 27 decreased the phenotypic value of the individual, indicating that the presence of mutant bases at these three sites negatively affected (negatively correlated) the sperm storage ability. Since the 4889482 site of chromosome 27 is the most significant site related to the sperm storing ability of breeding hens, the annotated gene MRC2 at this site has been reported in the literature to be involved in the regulation of endometriosis (Wei, C., et al, 1-Methyl-tryptophan attributes regulation T cell differentiation product to the inhibition of estrogen-IDO1-MRC2 a xi in endometeriosis, cell Death Dis,2016.7(12): p.24e89.), indicating that this result has confidence as a candidate site. The linkage disequilibrium relationship between the chromosome 27 and the other two loci of the chromosome 27 is drawn by haploview software (as shown in figure 4), and the result shows that the three loci on the chromosome 27 are in a strong linkage relationship, so that when an individual with high semen storage capability of a breeding hen is bred, the genotype that 4889482bp locus of the chromosome 27 is GG, 4898241bp locus is TT and 4898269bp locus is CC is selected as far as possible, which provides help for accelerating the breeding process of a high-quality laying hen.

TABLE 6 estimation of the genotype effect of the longest fertilization days associated with the breeder hen' Jinghong No. 1

The above studies are conducted by taking the characteristics of the sperm storing ability of the laying hen of "Jinghong No. 1" as an example, but the present invention is not limited thereto, and the present invention is applicable to the quantitative characteristics of diploid organisms, and the methods can be performed in accordance with the examples.

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.