SNP molecular marker of chicken whamm gene and its application in selection of egg traits

Abstract

This invention discloses the SNP molecular markers of the chicken WHAMM gene and their application in the selection of chickens for egg production traits, belonging to the field of molecular genetics. This invention discovered a single nucleotide polymorphism (SNP) at the g.9976 locus of intron 10 of the WHAMM gene on chicken chromosome 10. This SNP locus is highly significantly associated with the egg production trait in chickens, and the GG genotype at this SNP locus corresponds to individuals with high egg production. Based on the g.9976 locus, linkage disequilibrium analysis was performed with neighboring SNP loci, further revealing four SNPs, including the g.9976 locus, that are significantly associated with the egg production trait. This forms a haplotype combination of SNP molecular markers associated with the chicken egg production trait. Using this SNP molecular marker combination, association analysis of chicken egg production performance can be performed through SNP diploids, which is beneficial for screening high-producing egg-laying hens and guiding molecular marker-assisted breeding of egg-laying hens.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a chicken WHAMM gene SNP molecular marker and its application in the breeding of egg-producing traits. Background Technology

[0002] WHAMM, a member of the Wiskott-Aldrich syndrome protein (WASP) family, is an actin nucleation promoting factor (NPF) located in the cis-Golgi apparatus and renal tubule-vesicle membrane transport intermediates. It is a crucial regulator of membrane dynamics at the microtubule-actin cytoskeleton interface, playing a vital role in maintaining Golgi structure and promoting anterograde membrane transport (Campellone KG et al., 2008). WHAMM (a homologue of WAS proteins associated with actin, the Golgi membrane, and microtubules) participates in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation promoting factor, activating the actin-associated protein 2 / 3 complex (Arp2 / 3 complex). It is essential for spindle migration and asymmetric cell division during mouse oocyte meiosis (Huang X et al., 2013). WHAMM regulates spindle formation and influences the localization of the microtubule organizing center (MTOC) in the early stages of spindle formation. The depletion of WHAMM prevents the formation of spindle actin and chromosome alignment, which may be the cause of chromosome aneuploidy and abnormal, asymmetric division. These findings suggest that WHAMM is an important component of the actin cytoskeleton mechanism and plays a crucial role in oocyte maturation, possibly by controlling the formation of a normally long spindle through activation of spindle actin formation via the Arp2 / 3 complex (Jo YJ et al., 2021).

[0003] The WHAMM gene has only been studied in mammals so far. There has been no related exploration or analysis in poultry, and there are no reports on the correlation between chicken WHAMM gene SNP molecular markers and egg production performance. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a molecular marker for the chicken WHAMM gene SNP and its application in breeding for egg production traits. This invention has discovered that the g.9976 site of intron 10 of the chicken WHAMM gene is associated with the egg production trait in chickens and can be used for marker-assisted breeding to improve egg production performance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a chicken WHAMM gene SNP molecular marker, wherein the chicken WHAMM gene SNP molecular marker corresponds to a base G or A at position 433 from the 5′ end of the sequence shown in SEQ ID NO.1; specifically as follows:

[0007] ggatgacccttcagaaagccctgcactgcacaaacaggatgactcatccaggaggtctataaataattgcataggtaagtaggctcacattgactgatgtttttcattcttcctttc tggtattcttgagttctccctctggttctagacaatagtagcatacagtctttctcaagagaaagaagttcttagaaattcagtttaaaatcagcatgcagtaacaccacaggtcagg atcctaaatgagtaaccatggttggttgatccagtttgatatgctccacaggatcctgacttccagagtgtatgacttggtgttttgggggaaatgctgtctagttgaacattcagt ttgatattttaaaacatctgcacttgctgaagatcccgctggtgtaagaggtagaaaggtgtcagaaattccaggta / / gag[G / A]gaatttattcatgttgttcatgttggtggta agcaacccttattaccagacttattttttccccagttttacaacagttgaacagaatgactcattttgatgaggataaatcacctgtccagtcaaa agaccgctgtgggtagat.

[0008] Note: In the sequence, " / / " indicates the restriction endonuclease Bsc4I cleavage site; "[G / A]" is an SNP site, represented by "n" in the sequence listing.

[0009] The chicken WHAMM gene SNP molecular marker is located on the 10th intron of the WHAMM gene on chicken chromosome 10 (reference genome: GRCg6a(GCF_000002315.6)), and is named g.9976. The study found that this site is significantly associated with the egg production trait of chickens.

[0010] In a second aspect, the present invention provides the application of the above-mentioned chicken WHAMM gene SNP molecular marker in chicken genetic breeding.

[0011] In the above applications, chicken genetic breeding includes the selection and breeding of laying hen breeds with high total egg production at 52 weeks of age and / or the longest consecutive laying days.

[0012] Furthermore, in the above applications, individuals with the SNP marker and the GG genotype exhibited high total egg production at 52 weeks of age and a longer consecutive laying period.

[0013] A third aspect of the present invention provides primer pairs for detecting the above-mentioned chicken WHAMM gene SNP molecular markers, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. Specifically:

[0014] g.9976-F: 5′-GGATGACCCTTCAGAAAGC-3′; (SEQ ID NO.2)

[0015] g.9976-R: 5′-ATCTACCCACAGCGGTCTT-3′. (SEQ ID NO.3)

[0016] In a fourth aspect, the present invention provides a kit for detecting the above-mentioned chicken WHAMM gene SNP molecular marker, the kit comprising the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3.

[0017] A fifth aspect of the present invention provides the application of the above-mentioned primer pairs and / or kits in the assisted breeding of laying hen breeds or strains; wherein the laying hen breed or strain has the laying trait of high total egg production at 52 weeks of age and / or long consecutive laying days.

[0018] A sixth aspect of the present invention provides a method for identifying egg-laying traits in laying hens, comprising the following steps:

[0019] Using the genomic DNA of the laying hens to be tested as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products. The amplification products were digested with the restriction endonuclease Bsc4I. If two bands of 149bp and 429bp appeared, the chicken WHAMM gene SNP molecular marker corresponding to the laying hens to be tested was the GG genotype; if one band of 578bp appeared, the laying hens to be tested were the AA genotype; if three bands of 149bp, 429bp, and 578bp appeared, the laying hens to be tested were the GA genotype.

[0020] The total number of eggs laid by hens with the GG genotype at 52 weeks of age and the longest consecutive laying days were both higher than those with the AA and GA genotypes.

[0021] In the above method, the PCR amplification system is as follows: 10 μL of 2×Rapid Taq Master Mix, 1 μL each of the primers shown in SEQ ID NO.2 and SEQ ID NO.3, 1 μL of DNA template, and 7 μL of ddH2O.

[0022] The PCR amplification reaction program was as follows: 98℃ for 5 min; 95℃ for 15 s, 64℃ for 15 s, 58.3℃ for 15 s, 35 cycles; 72℃ for 10 s, 72℃ for 5 min.

[0023] In a seventh aspect, the present invention provides a combination of SNP molecular markers associated with chicken egg production traits, including: molecular marker g.8752, molecular marker rs735101591, molecular marker g.9976 and molecular marker rs315273441;

[0024] The molecular marker g.8752 has a physical position of 10_11543984 and a nucleotide polymorphism of A or G; the molecular marker rs735101591 has a physical position of 10_11544148 and a nucleotide polymorphism of A or C; the molecular marker g.9976 has a physical position of 10_11545372 and a nucleotide polymorphism of A or G; and the molecular marker rs315273441 has a physical position of 10_11546390 and a nucleotide polymorphism of A or G.

[0025] All of the above molecular markers are located on the WHAMM gene on chicken chromosome 10. Among them, molecular markers g.8752 and rs735101591 are located in the exons of the WHAMM gene, while molecular markers g.9976 and rs315273441 are located in the introns of the WHAMM gene. The mutation at the rs735101591 site caused a change in the encoded amino acid.

[0026] An eighth aspect of the present invention provides the application of the above-mentioned SNP molecular marker combination in chicken genetic breeding; the chicken genetic breeding includes the selection of laying hen breeds with high total egg production at 52 weeks of age and / or the longest consecutive laying days.

[0027] A ninth aspect of the present invention provides a method for assisting in the breeding of laying hen breeds using the above-mentioned SNP molecular marker combinations, comprising the following steps:

[0028] Based on the four molecular markers in the above SNP molecular marker combination, laying hens with the corresponding SNPs diploid type ACGG / GCGG were selected from the chicken population; the total number of eggs laid at 52 weeks of age and the longest consecutive laying days of laying hens with other SNPs diploid types were higher than those of laying hens with other SNPs diploid types.

[0029] In the above method, the SNP diploid type is ACGG / GCGG, which means that the heterozygous genotype AG is at molecular marker g.8752, the homozygous genotype CC is at molecular marker rs735101591, the homozygous genotype GG is at molecular marker g.9976, and the homozygous genotype GG is at molecular marker rs315273441.

[0030] The beneficial effects of this invention are:

[0031] (1) The present invention found a single nucleotide polymorphism at the g.9976 site of the 10th intron of the WHAMM gene on chromosome 10 of chicken. This SNP site is highly associated with the egg production trait of chickens. The GG genotype of this SNP site corresponds to high-producing individuals. By detecting this molecular marker associated with the egg production trait, the method is not only simple and fast, but also helps to breed high-producing chicken breeds, providing favorable assistance for breeding work.

[0032] (2) Based on the g.9976 site, this invention performs linkage disequilibrium analysis with neighboring SNP sites to further obtain four SNPs, including the g.9976 site, that are significantly associated with egg production traits, forming a combination of SNP molecular markers associated with chicken egg production traits. This combination of SNP molecular markers can be used to perform association analysis on the egg production performance of laying hens in the form of SNP diploids, which improves the accuracy of result judgment. Attached Figure Description

[0033] Figure 1 DNA was extracted from chicken blood and analyzed by electrophoresis; the marker in the figure is 10000bp.

[0034] Figure 2 : Electrophoresis detection results of the g.9976 target fragment; the marker in the figure is 2000bp.

[0035] Figure 3 : g.9976 target fragment enzyme digestion electrophoresis detection result; Marker.2000bp.

[0036] Figure 4 Linkage diagram of the g.9976 site and its neighboring SNPs in Jining 100-day chicken. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] As mentioned earlier, the WHAMM gene has only been studied in mammals, with no related research or analysis conducted in poultry. The inventors discovered that the WHAMM gene is expressed in small chicken follicles through transcriptome sequencing of chicken follicle tissue. Genome-wide association studies (GWAS) of Jining 100-day-old chickens revealed that this gene is a key candidate gene for the 52-week egg production trait, with the g.9976 site in intron 10 exhibiting the strongest signal intensity in the analysis results. Therefore, the WHAMM gene may be involved in oocyte-related processes in poultry, thereby affecting the reproductive and egg production performance of laying hens.

[0039] Using GRCg6a (GCF_000002315.6) from NCBI as the reference genome, the sequence fragment containing the g.9976 site is CCAGGTAGAG[G / A]. The restriction endonuclease Bsc4I recognition site is 5′-CCNNNNN / / NNGG-3′. When the base at the g.9976 site is G, the restriction endonuclease Bsc4I can digest this fragment, ultimately obtaining the genotype information for this site. Association analysis between this SNP site and Jining 100-day-old chickens with egg production records revealed a highly significant association between this site and the egg production trait in Jining 100-day-old chickens, with the GG genotype corresponding to high-producing individuals.

[0040] Furthermore, to identify other loci near the g.9976 site associated with egg production, linkage disequilibrium analysis was performed on the g.9976 site and its neighboring SNP sites in a Jining 100-day-old chicken population. This revealed four SNPs significantly associated with egg production: molecular markers g.8752, rs735101591, g.9976, and rs315273441. These were used as molecular marker combinations, and the diploids of the SNPs at the corresponding positions of these molecular marker combinations were detected to provide a more accurate analysis of the chicken's egg production trait.

[0041] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0042] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0043] Example 1: Genotyping and Allele Frequency Distribution Characteristics of the Chicken WHAMM Gene at Locus g.9976

[0044] 1. Test method:

[0045] Using GRCg6a(GCF_000002315.6) from NCBI as the reference genome, the sequence fragment at the g.9976 site is CCAGGTAGAG[G / A], and a single nucleotide polymorphism exists at this site.

[0046] Genomic DNA from different chicken breeds, including Jining 100-day chicken, Yimeng chicken, Zaozhuang Sunzhi chicken, Langya chicken, and Hy-Line Brown, was used as templates. PCR amplification was performed using g.9976-F and g.9976-R primer pairs. The specific primer pair sequences are as follows:

[0047] g.9976-F: 5′-GGATGACCCTTCAGAAAGC-3′; (SEQ ID NO.2)

[0048] g.9976-R: 5′-ATCTACCCACAGCGGTCTT-3′. (SEQ ID NO.3)

[0049] The PCR amplification system is shown in Table 1.

[0050] Table 1: PCR amplification system

[0051]

[0052] The PCR reaction program was as follows: 98℃ for 5 min; 95℃ for 15 s, 64℃ for 15 s, 58.3℃ for 15 s, 35 cycles; 72℃ for 10 s, 72℃ for 5 min.

[0053] The amplification products were digested with the restriction endonuclease Bsc4I. The digestion reaction system is shown in Table 2.

[0054] Table 2: Enzyme digestion reaction system for amplification products

[0055]

[0056] Note: “1×W” in Table 2 refers to the Bsc4I enzyme-matched reagent.

[0057] The enzyme digestion reaction conditions were: incubation at 55℃ for 90 min.

[0058] 2. Test Results:

[0059] (1) Genomic DNA detection results:

[0060] DNA was extracted from chicken blood, and its concentration and purity were determined using a UV spectrophotometer. OD260 / 280 values ​​were all within the range of 1.8–2.0, indicating good DNA purity. Whole-genome resequencing was then performed on the DNA. Results were obtained by 1% TBE agarose gel electrophoresis. Figure 1 As shown, the DNA band is single and there is no dragging phenomenon.

[0061] (2) PCR amplification results:

[0062] The theoretical length of the target fragment, including the g.9976 site, should be 578 bp. The sequence information of the amplified fragment is as follows:

[0063] ggatgacccttcagaaagccctgcactgcacaaacaggatgactcatccaggaggtctataaataattgcataggtaagtaggctcacattgactgatgtttttcattcttcctttc tggtattcttgagttctccctctggttctagacaatagtagcatacagtctttctcaagagaaagaagttcttagaaattcagtttaaaatcagcatgcagtaacaccacaggtcagg atcctaaatgagtaaccatggttggttgatccagtttgatatgctccacaggatcctgacttccagagtgtatgacttggtgttttgggggaaatgctgtctagttgaacattcagt ttgatattttaaaacatctgcacttgctgaagatcccgctggtgtaagaggtagaaaggtgtcagaaattccaggta / / gag[G / A]gaatttattcatgttgttcatgttggtggta agcaacccttattaccagacttattttttccccagttttacaacagttgaacagaatgactcattttgatgaggataaatcacctgtccagtcaaa agaccgctgtgggtagat.

[0064] Electrophoresis detection results of the amplified fragments are as follows Figure 2 As shown, the electrophoresis results are in line with expectations.

[0065] (3) Results of g.9976 locus genotyping:

[0066] When the base at g.9976 is G, Bsc4I digestion yields two digestion products of 149 bp and 429 bp lengths. Individuals showing only the characteristic 578 bp digestion band are homozygous AA individuals; those showing both 149 bp and 429 bp bands are homozygous GG individuals; and those showing bands of 149 bp, 429 bp, and 578 bp are heterozygous individuals with the GA genotype. Figure 3 ).

[0067] (4) Results of allele frequency distribution at the g.9976 locus:

[0068] Table 3 shows the statistical analysis and population genetic analysis results of the g.9976 locus in different breeds such as Jining Hundred-Day Chicken, Yimeng Chicken, Zaozhuang Sunzhi Chicken, Langya Chicken, and Hy-Line Brown.

[0069] Table 3: Genotype and Allele Frequency Distribution of g.9976

[0070]

[0071] The results showed that the dominant allele at the g.9976 locus was G in Sunzhi chicken, Langya chicken, Hy-Line Brown chicken, Yimeng chicken, and Jining 100-day chicken, and the dominant allele genotype was GG.

[0072] Example 2: Linkage disequilibrium analysis of the g.9976 site and its neighboring SNPs

[0073] To find other loci near the g.9976 site that are associated with egg production traits, 19 SNPs near the g.9976 site were screened using whole genome sequencing data, and linkage disequilibrium analysis was performed on a Jining 100-day-old chicken population using SHEsis software.

[0074] The results are as follows Figure 4 As shown, among these 20 sites, D′ is generally greater than 99%, r2 is generally smaller, and there is a linkage relationship between the sites.

[0075] Example 3: Association analysis between the g.9976 locus and its neighboring SNPs and the egg production trait.

[0076] Using Jining 100-day chickens with production performance records as the experimental subjects, the association between the g.9976 locus and 19 adjacent SNP loci and the chicken's egg production traits was analyzed.

[0077] Genotyping of Jining 100-day-old chickens at corresponding SNP loci was performed using low-depth resequencing to obtain genotypic data for each SNP locus. Association analysis was conducted between the genotypic data of Jining 100-day-old chickens and their maximum consecutive laying count (LCS) and egg production at 52 weeks (E52). All experimental data were analyzed using SPSS 17.0 software and Duncan's Multiple Range Test for comparison between different genotypes. All experimental data are expressed as mean ± standard error. P < 0.05 indicated significant difference, and P < 0.01 indicated extremely significant difference.

[0078] The results showed that only four SNP loci—g.8752, rs735101591, g.9976, and rs315273441—were significantly associated with egg production traits in chickens. The association analysis results between these four SNP loci and egg production traits are shown in Table 4.

[0079] Table 4: Association analysis of four significant SNPs in the Jining 100-day-old chicken population

[0080]

[0081] Note: The values ​​in the table are the least squares mean ± standard error of LCS (longest consecutive laying) and E52 (52-week laying count), with P < 0.05 indicating significant differences.

[0082] The physical locations in the table are determined based on the location of the locus on the chromosome in the reference genome GRCg6a (GCF_000002315.6).

[0083] The results showed that the effect of the g.8752 locus on the total egg production at 52 weeks of age was significantly different (P = 0.022), with the dominant allele being A and the AG genotype corresponding to individuals with a higher total egg production (E52: 156.158); the effect of the rs735101591 locus on the total egg production at 52 weeks of age was significantly different (P = 0.045), with the dominant allele being A and the AA genotype corresponding to individuals with a higher total egg production (E52: 145.291); the effect of the g.9976 locus on the total egg production at 52 weeks of age was significantly different (P = 0.02 ...). The effect of the total number of eggs laid at 2 weeks of age was extremely significant (P = 0.000), and the effect of the longest consecutive laying was also extremely significant (P = 0.010). The dominant allele was G, and the GG genotype corresponded to individuals with a higher total number of eggs laid (E52: 145.335). The effect of the rs315273441 locus on the total number of eggs laid at 52 weeks of age was significantly different (P = 0.031). The dominant allele was G, and the GA genotype corresponded to individuals with a higher total number of eggs laid (E52: 153.35).

[0084] Example 4: Joint analysis of the g.9976 site and its neighboring SNPs

[0085] 1. Haplotype and diploid frequency distribution analysis:

[0086] Based on the whole-genome resequencing results of three breeds—Jining Hundred-Day Chicken, Zaozhuang Sunzhi Chicken, and Yimeng Chicken—the allele information of the four SNP loci (g.8752, rs735101591, g.9976, and rs315273441) for each individual was arranged in ascending order of physical location. The haplotypes of the four loci and their frequency distribution in different breeds were constructed using the SHEsis software, as shown in Table 5.

[0087] Table 5: Frequency distribution of each haplotype in the three populations

[0088]

[0089] As shown in the table above, five haplotypes were constructed: AAGG, ACAG, ACGA, ACGG, and GCGG. ACGG and AAGG were the dominant haplotypes in Jining 100-day chickens; ACGG and ACGA were the dominant haplotypes in Zaozhuang Sunzhi chickens; and ACGG and ACGA were the dominant haplotypes in Yimeng chickens.

[0090] Taking the Jining 100-day chicken with production performance records as an example, the haplotype analysis results of 4 SNPs in its population are shown in Table 6.

[0091] Table 6: Haplotype frequency analysis of four SNPs in the Jining 100-day-old chicken population

[0092]

[0093] Note: The haplotype sequences in the table refer to the nucleotides corresponding to the four SNP sites in chicken: g.8752, rs735101591, g.9976, and rs315273441.

[0094] The frequency analysis of diploids constructed based on haplotypes is shown in Table 7.

[0095] Table 7: Diplotype frequency analysis of four SNPs in the Jining 100-day-old chicken population

[0096]

[0097]

[0098] As shown in the table above, H4H4 (ACGG / ACGG) and H1H4 (AAGG / ACGG) are the dominant diploid types in the Jining 100-day chicken population.

[0099] 2. Association analysis between the g.9976 locus and its neighboring SNPs diploid types and egg production trait.

[0100] The diploid analysis data of four SNP loci (g.8752, rs735101591, g.9976, and rs315273441) in Jining 100-day chickens were correlated with the egg production number at 52 weeks (E52) and the maximum consecutive laying count (LCS) for the egg production trait. The results are shown in Table 8.

[0101] Table 8: Association analysis of diploid types at four SNP loci with egg production traits in the Jining 100-day-old chicken population

[0102]

[0103] Note: The values ​​in the table are the least squares mean ± standard error of LCS (longest consecutive laying) and E52 (52-week laying count), with P < 0.05 indicating significant differences.

[0104] The results showed that there was a correlation between the diploid type and the number of eggs laid at 52 weeks of age and the longest consecutive laying period (P = 0.000; P = 0.014). In Jining 100-day-old chickens, individuals with the diploid type H4H5 (ACGG / GCGG) had a higher number of eggs laid at 52 weeks (E52: 162.571) and a longer consecutive laying period (LCS: 34.786).

[0105] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Chicken WHAMM The application of gene SNP molecular markers in the genetic breeding of laying hens is characterized by, The chicken WHAMM The gene SNP molecular marker is shown in SEQ ID NO.1, where the 433rd base from the 5′ end is either G or A; The genetic breeding of laying hens is as follows: auxiliary breeding of laying hen breeds with high total egg production and longest consecutive laying days at 52 weeks of age; laying hens with the GG genotype have higher total egg production and longer consecutive laying days at 52 weeks of age than laying hens with the AA or GA genotypes. The laying hens mentioned are Jining 100-day-old chickens.

2. Testing chickens WHAMM The application of a kit for gene SNP molecular markers in assisted breeding of laying hen breeds; characterized in that, The egg-laying hen breed has the egg-laying traits of high total egg production at 52 weeks of age and / or long longest consecutive laying days; The chicken WHAMM The gene SNP molecular marker is shown in SEQ ID NO.1, where the 433rd base from the 5′ end is either G or A; The kit contains primer pairs with sequences shown in SEQ ID NO.2 and SEQ ID NO.3; The laying hens mentioned are Jining 100-day-old chickens.

3. A method for assisting in the identification of egg-laying traits in laying hens, characterized in that, Includes the following steps: Using the genomic DNA of the Jining 100-day-old chicken as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification product. The amplification product was then digested with the restriction endonuclease Bsc4I. If two bands of 149bp and 429bp appeared, the chicken corresponding to the Jining 100-day-old chicken was identified. WHAMM The gene SNP molecular marker is the GG genotype; if a single band of 578 bp appears, the Jining 100-day chicken being tested is of the AA genotype; if three bands of 149 bp, 429 bp, and 578 bp appear, the Jining 100-day chicken being tested is of the GA genotype. The total egg production and longest consecutive laying days of the GG genotype Jining 100-day chickens at 52 weeks of age were higher than those of the AA and GA genotypes. The chicken WHAMM The gene SNP molecular marker is shown in SEQ ID NO.1, where the 433rd base from the 5′ end is either G or A.

4. The application of SNP molecular marker combinations in the genetic breeding of laying hens, characterized in that, The genetic breeding of laying hens is: auxiliary breeding of laying hen breeds with high total egg production at 52 weeks of age and the longest consecutive laying days; The SNP molecular marker combination consists of the molecular marker chicken WHAMM gene g.8752, the molecular marker rs735101591, the molecular marker chicken WHAMM gene g.9976, and the molecular marker rs315273441. The molecular marker chicken WHAMM gene g.8752 has a physical location of 10_11543984 and a nucleotide polymorphism of A or G; the molecular marker rs735101591 has a physical location of 10_11544148 and a nucleotide polymorphism of A or C; the molecular marker chicken WHAMM gene g.9976 has a physical location of 10_11545372 and a nucleotide polymorphism of A or G; and the molecular marker rs315273441 has a physical location of 10_11546390 and a nucleotide polymorphism of A or G. The reference genome for the physical location is GRCg6a GCF_000002315.6; In the chicken flock, laying hens with SNPs diplotypes of ACGG / GCGG at the corresponding position were selected; the total number of eggs laid at 52 weeks of age and the longest consecutive laying days of laying hens with SNPs diplotypes of ACGG / GCGG were higher than those of laying hens with other SNPs diplotypes. The laying hens mentioned are Jining 100-day-old chickens.

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