Molecular marker in the promoter region of rspo3 gene associated with egg laying traits in chicken and application thereof

Abstract

This invention discloses a molecular marker in the promoter region of the RSPO3 gene associated with egg production traits in chickens and its application, belonging to the field of molecular genetics. This invention detected three SNP loci significantly associated with egg production traits in the promoter region of the chicken RSPO3 gene: starting weight (LW), total eggs laid at 52 weeks of age (E52), and longest consecutive laying days (LCS). These SNP loci are g.-1,831, g.-2,515, and g.-2,760. Based on these three SNP loci, molecular marker combinations associated with egg production traits can be developed. Detecting these molecular marker combinations can aid in the breeding of high-producing hens.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a molecular marker for the promoter region of the RSPO3 gene, which is associated with egg production traits in chickens, and its application. Background Technology

[0002] R-spondin (Rspos) is a class of cysteine-rich secretory glycoproteins discovered in recent years. The four members of the Rspos family (Rspo1-4) share high structural similarity. Most current research indicates that the biological activity of Rspos is achieved through its regulation of the Wnt / β-catenin signaling system. Rspos activates β-catenin by binding to its receptors LGR4 / 5 / 6 and LRP5 / 6, forming a multi-ligand receptor complex with Wnt and frizzled (FZD). This complex participates in the regulation of cell proliferation and differentiation, influencing the development of tissues such as bone, muscle, and blood vessels, as well as the formation of organs such as limbs and gonads, and plays an important role in the pathogenesis of various diseases.

[0003] Studies on ovarian function have found that RSPO1 is considered an essential factor for ovarian differentiation during embryonic gonadal development. In mouse ovaries, RSPO2 promotes the transformation of primary follicles into secondary follicles by activating Wnt / β-catenin signaling, while knockout inhibits granulosa cell proliferation and differentiation. Another study found that RSPO2 is expressed in oocytes of mouse growing follicles, and RSPO2 deficiency prevents cell cycle progression in adjacent granulosa cells, leading to follicle growth arrest, indicating that RSPO2 activation of Wnt signaling is crucial for oocyte-granulosa cell signal transduction. Recent studies in pig ovaries have also shown that RSPO2 promotes granulosa cell proliferation and E2 secretion by activating Wnt / β-catenin signaling, thereby promoting oocyte maturation. Our previous studies found that the expression changes of RSPO3 mRNA during chicken pre-grade follicle development are consistent with those of Wnt4 and FSHR, and it can upregulate the expression of FSHR and β-catenin in pre-grade follicle granulosa cells. We speculate that RSPO3 may play an important regulatory role in chicken follicle development. Currently, no research has been conducted on the biological function of RSPO3 in chicken ovarian follicle development, and there are no reports on the correlation between chicken RSPO3 gene molecular markers and egg production traits. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a molecular marker in the promoter region of the RSPO3 gene related to egg production traits in chickens and its application. This invention screened three SNP sites in the promoter region of the RSPO3 gene that are significantly associated with egg production traits such as starting weight (LW), total eggs laid at 52 weeks of age (E52), and longest consecutive laying (LCS), which can be used for the breeding of high-producing hens.

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

[0006] In a first aspect, the present invention provides a molecular marker combination for the promoter region of the RSPO3 gene associated with egg production traits in chickens, including a first molecular marker, a second molecular marker, and a third molecular marker;

[0007] The nucleotide sequence of the first molecular marker is shown in SEQ ID NO.1. The 82nd base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or G.

[0008] The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.2. The 58th base from the 5' end of the sequence shown in SEQ ID NO.2 is the SNP2 site, and its base is G or A.

[0009] The nucleotide sequence of the third molecular marker is shown in SEQ ID NO.3. The 89th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP3 site, and its base is G or A.

[0010] The specific nucleotides marked as the first molecule are as follows:

[0011]

[0012] Note: The nucleotides in bold shaded areas in the sequence are SNP sites, represented by "n" in the sequence listing.

[0013] The specific nucleotides for the second molecular marker are as follows:

[0014] Note: The nucleotides in bold shaded areas in the sequence are SNP sites, represented by "n" in the sequence listing.

[0015] The specific nucleotides marked as third molecules are as follows:

[0016] Note: The nucleotides in bold shaded areas in the sequence are SNP sites, represented by "n" in the sequence listing.

[0017] In a second aspect, the present invention provides the application of the above-mentioned combination of molecular markers in the promoter region of the RSPO3 gene in chicken genetic breeding.

[0018] In the above applications, the chicken genetic breeding refers to the selection and breeding of laying hens with high initial egg weight, high total egg production at 52 weeks of age, and / or the longest consecutive laying days.

[0019] Furthermore, in the molecular marker combination of the RSPO3 gene promoter region, individuals with the TG genotype at SNP1, the GG genotype at SNP2, and the GA genotype at SNP3 exhibited egg production traits such as high initial egg weight, a large total number of eggs laid at 52 weeks of age, and / or a long consecutive laying period.

[0020] A third aspect of the present invention provides primer pairs for detecting the above-mentioned combination of molecular markers in the promoter region of the RSPO3 gene, comprising: primer pair A for detecting a first molecular marker, primer pair B for detecting a second molecular marker, and primer pair C for detecting a third molecular marker;

[0021] The nucleotide sequences of primer pair A are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. Specifically:

[0022] RSPO3-1-F: 5′-AGTTCGGGCTCGGGTTTGTTG-3′; (SEQ ID NO.4)

[0023] RSPO3-1-R: 5′-TCGGCCTGAACTTTGCCACC-3′. (SEQ ID NO.5)

[0024] The nucleotide sequences of primer pair B are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively. Specifically:

[0025] RSPO3-2-F: 5′-ACAGTGGCACTCTGGTCACAG-3′; (SEQ ID NO.6)

[0026] RSPO3-2-R: 5′-AGGATGTCGTGGTTGATACTG-3′. (SEQ ID NO.7)

[0027] The nucleotide sequences of primer pair C are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. Specifically:

[0028] RSPO3-3-F: 5′-AGGTGCTCTCAGGGTGTG-3′; (SEQ ID NO.8)

[0029] RSPO3-3-R: 5′-CTGTGCTGTCCAAGAATATCGT-3′. (SEQ ID NO.9)

[0030] In a fourth aspect, the present invention provides a kit for detecting the above-mentioned molecular marker combination of the RSPO3 gene promoter region, the kit comprising primer pair A shown in SEQ ID NO.4 and SEQ ID NO.5, primer pair B shown in SEQ ID NO.6 and SEQ ID NO.7, and primer pair C shown in SEQ ID NO.8 and SEQ ID NO.9.

[0031] In a fifth aspect, the invention provides the application of the primer pairs and / or kits in the assisted breeding of laying hen breeds; wherein the laying hen breed has laying traits such as high initial egg weight, high total egg production at 52 weeks of age, and / or long longest consecutive laying days.

[0032] A sixth aspect of the present invention provides a method for identifying egg-laying traits in laying hens, comprising the following steps:

[0033] Using the genomic DNA of the laying hens as a template, PCR amplification was performed using primer pair A shown in SEQ ID NO.4 and SEQ ID NO.5 to obtain amplification product A; PCR amplification was performed using primer pair B shown in SEQ ID NO.6 and SEQ ID NO.7 to obtain amplification product B; PCR amplification was performed using primer pair C shown in SEQ ID NO.8 and SEQ ID NO.9 to obtain amplification product C; amplification products A, B, and C were sequenced, and the egg production traits of the laying hens were identified based on the sequencing results.

[0034] Specifically, if the sequencing result of amplified product A corresponds to the TG genotype in the 82 bases starting from the 5′ end of the sequence shown in SEQ ID NO.1, it is identified as having the egg-laying trait with the longest consecutive laying days;

[0035] If the sequencing result of amplification product B corresponds to the sequence shown in SEQ ID NO.2, and the 58th base from the 5′ end is the GG genotype, then it is identified as having the egg-laying trait of high initial egg weight;

[0036] If the sequencing result of amplified product C corresponds to the GA genotype in the 89th base from the 5′ end of the sequence shown in SEQ ID NO.3, then it is identified as having the egg-laying traits of high total egg production and long consecutive laying days at 52 weeks of age.

[0037] The beneficial effects of this invention are:

[0038] This invention detected three SNP loci significantly associated with egg production traits such as starting weight (LW), total eggs laid at 52 weeks of age (E52), and longest consecutive laying days (LCS) in the promoter region of the chicken RSPO3 gene. These SNP loci are g.-1,831, g.-2,515, and g.-2,760. Based on these three SNP loci, molecular marker combinations associated with egg production traits can be developed. Detecting these molecular marker combinations can help in the breeding of high-producing hens. Attached Figure Description

[0039] Figure 1 : Comparison and analysis of RSPO3 promoter region recombinant vector with NCBI sequence.

[0040] Figure 2 Double enzyme digestion was used to verify promoter region fragments of different lengths of the RSPO3 gene; M: DL5000bp.

[0041] Figure 3 Luciferase activity of promoters of different lengths in the chicken RSPO3 gene was compared, with different letters indicating significant differences (P<0.05). Detailed Implementation

[0042] 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.

[0043] This invention uses GRCg6a (GCF 000002315.6) as the reference genome and NC_052534.1 as the reference sequence. Three SNP sites were detected within the first 3000 bp of the chicken RSPO3 gene promoter region:

[0044] g.-1,831 position (T>G), g.-2,515 position (G>A), and g.-2,760 position (G>A).

[0045] The above SNP sites were determined by using the start codon of the RSPO3 gene in the reference genome as +1.

[0046] The present invention further analyzed the correlation between the above-mentioned SNP sites and the egg production traits of chickens, and found that the g.-1,831 site (T>G) was significantly associated with the longest continuous laying trait in chickens; the g.-2,515 site (G>A) was significantly associated with the initial egg weight trait in chickens; and the g.-2,760 site (G>A) was significantly associated with the total number of eggs laid and the longest continuous laying trait in chickens at 52 weeks of age.

[0047] Based on the three SNP loci significantly associated with chicken egg production traits mentioned above, this invention designs a molecular marker combination for the RSPO3 gene promoter region related to chicken egg production traits, including a first molecular marker, a second molecular marker, and a third molecular marker; wherein:

[0048] The first molecular marker corresponds to the g.-1,831 site (T>G), and its nucleotide sequence is shown in SEQ ID NO.1. The 82nd base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or G.

[0049] The second molecular marker corresponds to the g.-2,515 site (G>A), and its nucleotide sequence is shown in SEQ ID NO.2. The 58th base from the 5′ end of the sequence shown in SEQ ID NO.2 is the SNP2 site, and its base is G or A.

[0050] The third molecular marker corresponds to the g.-2,760 site (G>A), and its nucleotide sequence is shown in SEQ ID NO.3. The 89th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP3 site, and its base is G or A.

[0051] In response to the aforementioned molecular marker combination of the RSPO3 gene promoter region related to chicken egg production traits, this invention also designed specific detection primers to detect the genotype of laying hens at the corresponding molecular markers, thereby enabling early selection and breeding of high-producing laying hens.

[0052] 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.

[0053] 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. Wherein:

[0054] The Hy-Line Brown chickens used in this embodiment of the invention are from the Linxi Village Egg Chicken Farm in Fanzhen Town, Tai'an City, Shandong Province; the Jining Hundred-Day Chickens are from Jining Datang Hundred-Day Chicken Breeding Co., Ltd.; the Zaozhuang Sunzhi Chickens are from Shandong Sunzhi Chicken Breeding Technology Co., Ltd.; and the Yimeng Chickens are from Shandong Longsheng Agricultural and Animal Husbandry Group Co., Ltd.

[0055] Example 1: Transcriptional Regulation Analysis of the RSPO3 Gene

[0056] 1. PCR amplification of RSPO3 gene promoter region fragment

[0057] Primers were designed using the chicken RSPO3 gene sequence (NC_052534.1) from NCBI and obtained through the online website Primer3Plus (Table 1), and synthesized by Ruiboxingke. Using Zaozhuang Sunzhi chicken genomic DNA as a template, the promoter region of the chicken RSPO3 gene (-2,703 bp to -168 bp) was amplified, and restriction enzyme sites and protective bases were added. The accuracy of the amplification was then verified.

[0058] Table 1: Primers for PCR amplification of RSPO3 gene promoter region fragment

[0059]

[0060] The sequence of the target fragment obtained from sequencing was compared with the RSPO3 gene promoter region sequence on the NCBI website. The sequence alignment results are as follows: Figure 1 As shown, the 99.86% agreement indicates that the recombinant vector pGL3-RSPO3-2,535 has been successfully constructed.

[0061] 2. Construct deletion vectors of different lengths in the RSPO3 gene promoter region.

[0062] Using the pGL3-RSPO3-2,535 vector as a template, DNA fragments were amplified by PCR to construct deletion vectors of different lengths in the RSPO3 gene promoter region. Positive clones were verified by enzyme digestion, and the resulting digested fragments were consistent with the expected size. Figure 2 As shown in the figure. Sequencing was then performed, and after the sequenced sequence was returned, DNAMAN was used to compare the obtained sequenced sequence with the chicken RSPO3 gene sequence. The sequencing results were identical to the sequenced sequence.

[0063] 3. Dual-luciferase activity analysis of different length fragments in the RSPO3 gene promoter region

[0064] To investigate the dual-luciferase activity of different length segments in the RSPO3 gene promoter region, a vector deleting the validated RSPO3 gene promoter region was transfected into chicken grade membrane cells. Cell status was observed, and dual-luciferase activity was measured 48 hours after transfection. The results are as follows: Figure 3 As shown in the figure. The results indicate that there are significant differences in luciferase activity among the -1,264 to -2,269 bp segments of the RSPO3 gene promoter (P<0.05). Therefore, the key promoter region of the RSPO3 gene is the -1,264 to -2,269 bp segment.

[0065] Example 2: Screening of SNPs in the promoter region of the RSPO3 gene and analysis of their allele and genotype frequencies in chicken flocks.

[0066] 1. Test method:

[0067] Sequencing analysis was performed on the promoter region of Jining 100-day-old chickens, and GWAS analysis was conducted on their egg production traits. Three SNPs potentially associated with chicken egg production traits were identified within the first 3000 bp of the RSPO3 gene promoter region: g.-1,831, g.-2,515, and g.-2,760. Among them, the g.-1,831 site is located precisely in the key promoter region identified in Example 1, and the recognition sequences of transcription factors such as USF-1 and Pax were altered before and after mutation. Based on these three SNPs, statistical analysis of allele frequencies and genotype frequencies, as well as population genetic analysis, were performed on Jining 100-day-old chickens, Zaozhuang Sunzhi chickens, and Yimeng chickens.

[0068] 2. Test Results:

[0069] The results are shown in Table 2.

[0070] Table 2: Statistical analysis of genotype and allele frequencies of the three SNPs in the first 3000 bp of the RSPO3 gene promoter region.

[0071]

[0072] Note: When HWE (P>0.05) is in the population genetic equilibrium.

[0073] The results showed that the dominant allele at the g.-1,831 locus was T in all three chickens: 100-day chicken, Zaozhuang Sunzhi chicken, and Yimeng chicken; the dominant allele at the g.-2,515 locus was G in all three chickens: 100-day chicken, Zaozhuang Sunzhi chicken, and Yimeng chicken; and the dominant allele at the g.-2,760 locus was G in all three chickens: 100-day chicken, Zaozhuang Sunzhi chicken, and Yimeng chicken.

[0074] Example 3: Association analysis of three SNPs with egg production traits

[0075] 1. Test method:

[0076] 369 Jining 100-day chickens with production performance records and whole-genome resequencing results were selected. Blood was collected from the wing veins and genomic DNA was extracted to analyze the association between the three SNPs screened in Example 2 and the egg production trait.

[0077] 2. Test Results:

[0078] The results are shown in Table 3.

[0079] Table 3: Association analysis of three SNPs related to egg production trait in Jining 100-day-old chickens

[0080]

[0081] The results showed that the effect of the g.-1,831 locus on the total number of eggs laid at 52 weeks of age was not significantly different (P=0.07), the effect on the age at first laying was not significantly different (P>0.05), the effect on the weight of eggs laid at first laying was not significantly different (P>0.05), but the effect on the longest consecutive laying period was significant (P=0.004). The TG genotype corresponds to individuals with a longer consecutive laying period, which is speculated to be related to the overdominance of heterozygotes. The effect on the weight of eggs laid at first laying was not significantly different (P>0.05), and the dominant allele was T.

[0082] The effect of the g.-2,515 locus on the total number of eggs laid at 52 weeks of age was not significant (P>0.05), nor on the age at first laying (P>0.05), but on the weight of eggs laid at first laying was significant (P=0.042). The GG genotype corresponds to individuals with larger eggs laid at first laying. The effect on the longest consecutive laying period was not significant (P>0.05), nor on the weight of eggs laid at first laying (P>0.05), and the dominant allele was G.

[0083] The effect of the g.-2,760 locus on the total number of eggs laid at 52 weeks of age was significantly different (P = 0.0027). The GA genotype corresponds to the individual with the largest total number of eggs, which is speculated to be related to the overdominance of heterozygotes. The effects on age at first laying, weight at first laying, and weight of eggs laid at first laying were not significantly different (P > 0.05), but the effect on the longest consecutive laying was extremely significant (P = 0.001). Individuals with the GA genotype correspond to a longer longest consecutive laying, and the dominant allele is G.

[0084] Therefore, it can be concluded that: the g.-1,831 locus (T>G) is significantly associated with the longest consecutive laying trait in chickens; the g.-2,515 locus (G>A) is significantly associated with the initial egg weight trait in chickens; and the g.-2,760 locus (G>A) is significantly associated with the total number of eggs laid and the longest consecutive laying trait in chickens at 52 weeks of age. Based on these three SNP loci, early selection and breeding of high-producing laying hens can be achieved.

[0085] Example 4: Application of molecular marker combinations in the promoter region of the RSPO3 gene in the breeding of laying hens

[0086] Based on the three SNP loci significantly associated with chicken egg production traits identified in Example 3, this example designs a molecular marker combination for the RSPO3 gene promoter region associated with chicken egg production traits, including a first molecular marker, a second molecular marker, and a third molecular marker; wherein:

[0087] The first molecular marker corresponds to the g.-1,831 site (T>G), and its nucleotide sequence is shown in SEQ ID NO.1. The 82nd base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or G.

[0088] The second molecular marker corresponds to the g.-2,515 site (G>A), and its nucleotide sequence is shown in SEQ ID NO.2. The 58th base from the 5′ end of the sequence shown in SEQ ID NO.2 is the SNP2 site, and its base is G or A.

[0089] The third molecular marker corresponds to the g.-2,760 site (G>A), and its nucleotide sequence is shown in SEQ ID NO.3. The 89th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP3 site, and its base is G or A.

[0090] Based on the above molecular markers, detection primers were designed, including primer pair A for detecting the first molecular marker, primer pair B for detecting the second molecular marker, and primer pair C for detecting the third molecular marker;

[0091] The nucleotide sequences of primer pair A are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. Specifically:

[0092] RSPO3-1-F: 5′-AGTTCGGGCTCGGGTTTGTTG-3′; (SEQ ID NO.4)

[0093] RSPO3-1-R: 5′-TCGGCCTGAACTTTGCCACC-3′. (SEQ ID NO.5)

[0094] The nucleotide sequences of primer pair B are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively. Specifically:

[0095] RSPO3-2-F: 5′-ACAGTGGCACTCTGGTCACAG-3′; (SEQ ID NO.6)

[0096] RSPO3-2-R: 5′-AGGATGTCGTGGTTGATACTG-3′. (SEQ ID NO.7)

[0097] The nucleotide sequences of primer pair C are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. Specifically:

[0098] RSPO3-3-F: 5′-AGGTGCTCTCAGGGTGTG-3′; (SEQ ID NO.8)

[0099] RSPO3-3-R: 5′-CTGTGCTGTCCAAGAATATCGT-3′. (SEQ ID NO.9)

[0100] Another 100-day-old chicken from Jining with production performance records was selected as the test subject to verify the performance of the above-mentioned molecular marker combination for the promoter region of the RSPO3 gene. Specifically:

[0101] Using the genomic DNA of the laying hens as a template, PCR amplification was performed using primer pair A shown in SEQ ID NO.4 and SEQ ID NO.5 to obtain amplification product A; PCR amplification was performed using primer pair B shown in SEQ ID NO.6 and SEQ ID NO.7 to obtain amplification product B; PCR amplification was performed using primer pair C shown in SEQ ID NO.8 and SEQ ID NO.9 to obtain amplification product C; amplification products A, B, and C were sequenced, and the egg production traits of the laying hens were identified based on the sequencing results.

[0102] If the sequencing result of amplified product A corresponds to the TG genotype in the 82 bases starting from the 5′ end of the sequence shown in SEQ ID NO.1, then it is identified as having the egg-laying trait with the longest consecutive laying days;

[0103] If the sequencing result of amplification product B corresponds to the sequence shown in SEQ ID NO.2, and the 58th base from the 5′ end is the GG genotype, then it is identified as having the egg-laying trait of high initial egg weight;

[0104] If the sequencing result of amplified product C corresponds to the GA genotype in the 89th base from the 5′ end of the sequence shown in SEQ ID NO.3, then it is identified as having the egg-laying traits of high total egg production and long consecutive laying days at 52 weeks of age.

[0105] Verification revealed that the egg production performance of laying hens predicted using the above-mentioned combination of molecular markers in the promoter region of the RSPO3 gene is consistent with the actual production performance records of laying hens, demonstrating practical application value.

[0106] 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. A trait related to egg production in chickens RSPO3 A combination of molecular markers for gene promoter regions, characterized in that, Including first molecular markers, second molecular markers, and third molecular markers; The nucleotide sequence of the first molecular marker is shown in SEQ ID NO.

1. The 82nd base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or G. The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.

2. The 58th base from the 5' end of the sequence shown in SEQ ID NO.2 is the SNP2 site, and its base is G or A. The nucleotide sequence of the third molecular marker is shown in SEQ ID NO.

3. The 89th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP3 site, and its base is G or A.

2. A detection method according to claim 1 RSPO3 The application of primer pairs combining molecular markers for gene promoter regions in chicken genetic breeding is characterized by, The chicken genetic breeding refers to the selection of laying hens with high initial egg weight, high total egg production at 52 weeks of age, and / or the longest consecutive laying days. The RSPO3 Among the molecular marker combinations in the gene promoter region, individuals with the TG genotype at SNP1, the GG genotype at SNP2, and the GA genotype at SNP3 exhibited high egg weight at the start of laying, a large total number of eggs laid at 52 weeks of age, and / or a long consecutive laying period. The primer pairs include: primer pair A for detecting a first molecular marker, primer pair B for detecting a second molecular marker, and primer pair C for detecting a third molecular marker; The nucleotide sequences of primer pair A are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; The nucleotide sequences of primer pair B are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; The nucleotide sequences of primer pair C are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

3. A detection method according to claim 1 RSPO3 The application of a kit for combining molecular markers in gene promoter regions in the assisted breeding of laying hen breeds, characterized by: The egg-laying hen breeds have the following egg-laying traits: high initial egg weight, high total number of eggs laid at 52 weeks of age, and / or the longest consecutive laying days. The RSPO3 Among the molecular marker combinations in the gene promoter region, individuals with the TG genotype at SNP1, the GG genotype at SNP2, and the GA genotype at SNP3 exhibited high egg weight at the start of laying, a large total number of eggs laid at 52 weeks of age, and / or a long consecutive laying period. The kit contains the primer pair of claim 2.

4. A method for identifying egg-laying traits in laying hens, characterized in that, Includes the following steps: Using the genomic DNA of the laying hens as a template, PCR amplification was performed using primers shown in SEQ ID NO.4 and SEQ ID NO.5 (pair A) to obtain amplification product A; PCR amplification was performed using primers shown in SEQ ID NO.6 and SEQ ID NO.7 (pair B) to obtain amplification product B; PCR amplification was performed using primers shown in SEQ ID NO.8 and SEQ ID NO.9 (pair C) to obtain amplification product C; amplification products A, B, and C were sequenced, and the egg production traits of the laying hens were identified based on the sequencing results. If the sequencing result of amplified product A corresponds to the TG genotype in the 82 bases starting from the 5′ end of the sequence shown in SEQ ID NO.1, then it is identified as having the egg-laying trait with the longest consecutive laying days; If the sequencing result of amplification product B corresponds to the sequence shown in SEQ ID NO.2, and the 58th base from the 5′ end is the GG genotype, then it is identified as having the egg-laying trait of high initial egg weight; If the sequencing result of amplified product C corresponds to the GA genotype in the 89th base from the 5′ end of the sequence shown in SEQ ID NO.3, then it is identified as having the egg-laying traits of high total egg production and long consecutive laying days at 52 weeks of age.

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