STON2 gene intron SNP (Single Nucleotide Polymorphism) molecular marker related to egg laying traits of chickens and application of STON2 gene intron SNP molecular marker

Abstract

The invention discloses an STON2 gene intron SNP (Single Nucleotide Polymorphism) molecular marker related to egg laying traits of chickens and application of the STON2 gene intron SNP molecular marker, and belongs to the technical field of molecular genetics. The research finds that the STON2 gene may participate in the related process of poultry sexual precocity and egg laying, so that the reproductive performance and the egg laying character of the chicken are influenced, and four SNP loci remarkably related to the egg laying character of the chicken are found in the fifth intron region and the sixth intron region of the STON2 gene for the first time. Based on the SNP site, the invention further develops an SNP molecular marker related to the egg laying traits of the chickens, and the SNP molecular marker can be used for auxiliary breeding of high-yield laying hens.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and in particular to a STON2 gene intron SNP molecular marker associated with chicken egg-laying traits and an application thereof. Background Art

[0002] Egg production is a key economic trait for laying hens. This includes indicators such as total egg production and the longest streak. Egg production directly reflects a hen's egg-laying performance, while the longest streak refers to the longest consecutive egg-laying days. Increasing the number of consecutive days and reducing the number of days between lays can significantly increase egg production throughout the entire egg-laying cycle. Researching and applying streak performance as a breeding indicator for economic production in laying hens will help promote the development of laying hen breeding. Therefore, cultivating high-quality laying hens and improving egg-laying performance are of great significance to the development of the livestock farming industry.

[0003] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, including base transitions, transversions, insertions, and deletions. As a widely distributed and relatively stable genetic marker, SNPs offer advantages such as ease of allele frequency assessment and typing, making them widely used in population genetic diversity analysis and marker-assisted breeding. However, due to their large number (approximately 2.8 million SNPs have been identified in the chicken genome), screening for genetic markers significantly associated with functional traits from this large number of SNPs remains challenging.

[0004] STON2 is an adaptor protein involved in the endocytic machinery, participating in the recycling of synaptic vesicles. It belongs to the vesicle trafficking gene family and has an AP-2-μ subunit C-terminal homology domain (MHD). It participates in clathrin-mediated endocytosis and plays a key role in signal transduction, trafficking, and regulation. Currently, there are few reports on the expression of this gene in the chicken ovary and the association of its polymorphic sites with egg production traits such as egg number and maximum streak length. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a STON2 gene intron SNP molecular marker related to chicken egg-laying traits and its application.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, there is provided a SNP molecular marker associated with chicken egg-laying traits, comprising a first molecular marker and a second molecular marker;

[0008] The nucleotide sequence of the first molecular marker is as shown in SEQ ID NO.1, comprising a SNP1 site, a SNP2 site, and a SNP3 site. The 129th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or C; the 49th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP2 site, and its base is T or C; the 37th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP3 site, and its base is A or G;

[0009] The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.2. The 83rd base from the 5′ end of the sequence shown in SEQ ID NO.2 is the SNP4 site, and its base is G or A.

[0010] The specific nucleotides of the first molecular marker are as follows:

[0011]

[0012] Note: The nucleotides in bold shade in the sequence are SNP sites, which are represented by "n" in the sequence table.

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

[0014]

[0015] Note: The nucleotides in bold shade in the sequence are SNP sites, which are represented by "n" in the sequence table.

[0016] The present invention discovered three SNP sites significantly associated with chicken egg-laying traits in the region of intron 6 of the chicken STON2 gene, and one SNP site significantly associated with chicken egg-laying traits in the region of intron 5 of the STON2 gene; they are:

[0017] SNP1: 5_41101673 (rs317544465) corresponds to the T>C mutation at nucleotide position 39729 of the STON2 gene, located in intron 6;

[0018] SNP2: 5_41101753 (rs313213332) corresponds to the T>C mutation at nucleotide position 39649 of the STON2 gene, located in intron 6;

[0019] SNP3: 5_41101765 (rs741105703) corresponds to the A>G mutation at nucleotide position 39637 of the STON2 gene, located in intron 6;

[0020] SNP4: 5_41106166 (rs15709831) corresponds to the G>A mutation at nucleotide 35223 of the STON2 gene, located in intron 5;

[0021] The reference genome for the above physical location is GRCg6a; GenBank accession: GCA_000002315.5.

[0022] The second aspect of the present invention provides the use of the above-mentioned SNP molecular marker in chicken genetic breeding.

[0023] In the above application, the chicken genetic breeding is to select laying hens that produce a large number of eggs and have the longest continuous laying days at 43 weeks of age.

[0024] In a third aspect of the present invention, there is provided a primer pair for detecting the above-mentioned SNP molecular marker, comprising: a primer pair A for detecting a first molecular marker and a primer pair B for detecting a second molecular marker;

[0025] The nucleotide sequences of primer pair A are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. Specifically, they are as follows:

[0026] STON2-1-F: 5'-GTTAAATGTATGCAGCTACATCA-3'; (SEQ ID NO.3)

[0027] STON2-1-R: 5'-ATAGCACGGAGAAGCTTTTA-3'. (SEQ ID NO.4)

[0028] The nucleotide sequences of primer pair B are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Specific details are as follows:

[0029] STON2-2-F: 5'-TCTTTTGGTTTCTGCCTCACC-3'; (SEQ ID NO.5)

[0030] STON2-2-R: 5'-ACCCCTCAAAGATGAGCTTG-3'. (SEQ ID NO.6)

[0031] In a fourth aspect, the present invention provides a kit for detecting the above-mentioned SNP molecular markers, wherein the kit comprises the above-mentioned primer pair A and primer pair B.

[0032] The fifth aspect of the present invention provides the use of the above-mentioned primer pair and / or kit in assisting the selection of laying hen breeds with a large total egg production and a longest continuous laying period at 43 weeks of age.

[0033] A sixth aspect of the present invention provides a method for identifying egg-laying traits of laying hens, comprising the following steps:

[0034] Using the genomic DNA of the laying hen to be tested as a template, PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product A; PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product B; the amplified products are sequenced, and the egg-laying traits of the laying hen are identified based on the sequencing results.

[0035] Specifically, if the sequencing result of the amplified product A corresponds to the sequence shown in SEQ ID NO.1, and the 129th base from the 5′ end is a CC or TC genotype, the 49th base is a CC or TC genotype, and the 37th base is an AA or AG genotype; if the sequencing result of the amplified product B corresponds to the sequence shown in SEQ ID NO.2, and the 83rd base from the 5′ end is an AA or GA genotype; then it is identified as having the egg-laying trait of a large total number of eggs laid at 43 weeks of age and / or a long longest continuous laying period.

[0036] Beneficial effects of the present invention:

[0037] This study found that the STON2 gene may be involved in processes related to precocious puberty and egg production in poultry, thereby affecting the reproductive performance and egg-laying traits of chickens. Four SNPs were discovered for the first time within the fifth and sixth introns of the STON2 gene that are significantly associated with egg-laying traits in chickens. Based on these SNPs, the researchers further developed SNP molecular markers associated with egg-laying traits in chickens, which can be used to assist in the breeding of high-yielding laying hens. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :The expression of STON2 in ovarian tissues, respectively, are the ovarian tissues of Hailan Brown chicken and Zaozhuang Sunzhi chicken. Different letters indicate significant differences (P<0.05).

[0039] Figure 2 :The expression of STON2 in ovarian tissues of Zaozhuang Sunzhi chickens (peak egg-laying period) and Zaozhuang Sunzhi chickens (late egg-laying period), respectively. Different letters indicate significant differences (P<0.05).

[0040] Figure 3 : GWAS analysis results.

[0041] Figure 4 : Linkage relationship map of Langya chicken 5_41101765 locus and its adjacent SNPs. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0044] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out in accordance with conventional test methods or the operating instructions recommended by the supplier.

[0045] Hailan brown laying hens come from the Linxi Village Farm in Fan Town, Tai'an City; Langya chickens come from Shandong Jihua Poultry Breeding Co., Ltd.; Zaozhuang Sunzhi chickens come from Shandong Sunzhi Chicken Seed Technology Co., Ltd.; Jining 100-day chickens come from Jining Datang 100-day Chicken Breeding Co., Ltd.

[0046] Example 1: Analysis of STON2 gene expression in ovarian tissue

[0047] 1. Test method:

[0048] Based on the consensus sequence information of different transcripts of the chicken STON2 gene provided by NCBI (the GenBank accession numbers of the five transcripts are: XM_421302.7, XM_040702153.2, XM_046942129.1, XM_040702154.2 and XM_040702155.2), 5′-CTACCCAGTGCCAAACGAGT-3′ was designed as the upstream primer and named [STON2-F], and 5′-GGATGGCCAGAAGCTGAGTT-3′ was designed as the downstream primer and named [STON2-R].

[0049] Fluorescence quantitative PCR was used to detect the relative expression of the STON2 gene in the ovarian tissue of a high-yielding laying hen breed (Hai-Lan Brown) and a low-yielding laying hen breed (Zaozhuang Sunzhi chicken). STON2 gene expression was further compared in the ovarian tissue of Zaozhuang Sunzhi chickens during peak and late egg-laying periods. The amplification system for the fluorescence quantitative PCR assay is shown in Table 1.

[0050] Table 1: Fluorescence quantitative PCR amplification system

[0051]

[0052] The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 30 s; 40 cycles of 95°C for 10 s, 60°C for 30 s; and melting curve analysis: 95°C for 15 s, 65°C for 60 s, and 95°C for 1 s.

[0053] 2. Test results:

[0054] The expression of STON2 gene in different laying hen breeds is as follows Figure 1 The results showed that the STON2 gene was expressed in the ovarian tissues of both Hailan Brown chicken and Zaozhuang Sunzhi chicken, and the expression level in the ovarian tissue of Zaozhuang Sunzhi chicken was significantly higher than that in the ovarian tissue of Hailan Brown chicken (P<0.05).

[0055] Further comparison of STON2 expression in ovarian tissues of Zaozhuang Sunzhi chickens during the peak and late egg-laying periods revealed the following results: Figure 2 As shown in the data, the STON2 gene was expressed in the ovarian tissues of Zaozhuang Sunzhi chickens (at both the peak and late egg-laying stages), and the expression level in the ovarian tissues of Zaozhuang Sunzhi chickens at the late egg-laying stage was significantly higher than that at the peak egg-laying stage (P<0.05).

[0056] Example 2: Screening and identification of SNP molecular markers in the intron region of the STON2 gene related to egg production traits

[0057] Langya chickens with production performance records were used as the research subjects. GWAS analysis of their egg-laying traits was conducted. The results showed that molecular markers significantly associated with egg-laying traits such as age at first laying, weight at first laying, total number of eggs laid at 43 weeks of age, and longest consecutive laying days were screened in the region of chromosome 5 (Gene ID: 423390), specifically Chromosome 5, NC_052536.1 (40371593-40441976). Figure 3 ); Haploview software was used to conduct linkage disequilibrium analysis in the Langya chicken population. The D′ values ​​were all greater than 99%, indicating that there was a linkage relationship between the loci. The four most significant loci were selected for analysis in the Langya chicken population. The results were as follows: Figure 4 As shown, they are:

[0058] 5_41101673 (rs317544465) corresponds to the T>C mutation at position 39729 of intron 6 of the STON2 gene; 5_41101753 (rs313213332) corresponds to the T>C mutation at position 39649 of intron 6 of the STON2 gene; 5_41101765 (rs741105703) corresponds to the A>G mutation at position 39637 of intron 6 of the STON2 gene; 5_41106166 (rs15709831) corresponds to the G>A mutation at position 35223 of intron 5 of the STON2 gene, all of which are located on the STON2 gene.

[0059] Example 3: Association analysis between four SNP sites and egg production traits of Langya chicken

[0060] A total of 1,183 Langya chickens with production records were selected. 1-2 mL of blood was collected from the wing vein in an anticoagulant blood collection tube. Association analysis was performed on the four SNPs identified in Example 2 and their egg production traits. The details are as follows:

[0061] Genome sequencing was used to determine the genotypes of the four SNPs identified in Example 2 in the Langya chicken population. Association analysis was performed with egg production traits, including age at first laying (AFE), total eggs laid at 43 weeks of age (E43), and maximum consecutive days of laying (LCS). The results are shown in Table 2.

[0062] Table 2: Association analysis between SNP loci and egg-laying traits of Langya chicken

[0063]

[0064]

[0065] Note: AFE: Age at first laying, E43: Total number of eggs laid at 43 weeks of age, LCS: Longest consecutive days of laying. When P < 0.05, the association between each genotype and egg production traits is significantly different, otherwise it is not significant.

[0066] The results showed that the effects of locus 5_41101673 on age at first laying, total number of eggs laid at 43 weeks of age and longest consecutive days of laying were significant. The dominant allele was C, and individuals with the TC genotype had a younger age at first laying and a higher number of eggs laid. The effects of locus 5_41101753 on age at first laying, total number of eggs laid at 43 weeks of age and longest consecutive days of laying were significant. The dominant allele was C, and individuals with the TC genotype had a younger age at first laying and a higher number of eggs laid. The effects of locus 5_41101765 on age at first laying, total number of eggs laid at 43 weeks of age and longest consecutive days of laying were significant. The dominant allele was G, and individuals with the AG genotype had a younger age at first laying and a higher number of eggs laid. The effects of locus 5_41106166 on age at first laying, total number of eggs laid at 43 weeks of age and longest consecutive days of laying were significant. The dominant allele was A, and individuals with the GA genotype had a younger age at first laying and a higher number of eggs laid.

[0067] Example 4: Diplotype construction and joint analysis of four SNPs

[0068] 1. Haplotype and diplotype frequency distribution analysis:

[0069] According to the sequencing results of Langya chicken in Example 2, each individual was separated according to the order of the four SNP sites 5_41101673, 5_41101753, 5_41101765, and 5_41106166, and the alleles of each site were combined to construct two haplotypes, TTAG and CCGA, of which CCGA was the dominant haplotype; the genotype frequency distribution characteristics of the dominant haplotype and its haplotype combination (diplotype) were analyzed, and the results are shown in Tables 3-4.

[0070] Table 3: Frequency analysis of dominant haplotypes in Langya chicken population

[0071]

[0072]

[0073] Table 4: Diplotype frequency analysis in Langya chicken population

[0074]

[0075] 2. Association analysis between four SNPs diplotypes and egg production traits:

[0076] The SNPs diplotypes in the Langya chicken population were associated with egg-laying traits including age at first laying (AFE), number of eggs laid at 43 weeks (E43) and maximum consecutive number of eggs (LCS). The results are shown in Table 5.

[0077] Table 5: Association analysis between the diplotypes of four SNPs and egg production in the Langya chicken population

[0078]

[0079] Note: The values ​​in the table are the least square means ± standard errors of AFE (age at first laying), E43 (number of eggs laid at 43 weeks) and LCS (longest consecutive lay). The differences are significant at P<0.05.

[0080] Results showed that the diplotype constructed from the four SNPs was significantly associated with age at first laying, number of eggs at 43 weeks of age, and maximum consecutive laying (P = 0.000359; P = 0.000034; P = 0.000006). In Langya chickens, individuals with the diplotype H1H2 (TTAG / CCGA) were associated with a younger age at first laying and a higher number of eggs. Increasing the frequency of the H1H2 genotype in breeding populations could help accelerate sexual maturity and increase egg production.

[0081] Example 5: Application of SNP molecular markers in the intron region of the STON2 gene in laying hen breeding

[0082] Based on the four SNP sites significantly associated with chicken egg-laying traits identified in Example 2, this example designed a SNP molecular marker associated with chicken egg-laying traits. It includes a first molecular marker and a second molecular marker;

[0083] The nucleotide sequence of the first molecular marker is shown in SEQ ID NO.1, which includes SNP1 site, SNP2 site and SNP3 site. The 129th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or C; the 49th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP2 site, and its base is T or C; the 37th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP3 site, and its base is A or G.

[0084] The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.2. The 83rd base from the 5′ end of the sequence shown in SEQ ID NO.2 is the SNP4 site, and its base is G or A.

[0085] Designing a detection primer pair based on the above-mentioned SNP molecular markers, including: a primer pair A for detecting a first molecular marker and a primer pair B for detecting a second molecular marker;

[0086] The nucleotide sequences of primer pair A are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. Specifically, they are as follows:

[0087] STON2-1-F: 5'-GTTAAATGTATGCAGCTACATCA-3'; (SEQ ID NO.3)

[0088] STON2-1-R: 5'-ATAGCACGGAGAAGCTTTTA-3'. (SEQ ID NO.4)

[0089] The nucleotide sequences of primer pair B are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Specific details are as follows:

[0090] STON2-2-F: 5'-TCTTTTGGTTTCTGCCTCACC-3'; (SEQ ID NO.5)

[0091] STON2-2-R: 5'-ACCCCTCAAAGATGAGCTTG-3'. (SEQ ID NO. 6).

[0092] Another 100 Langya chickens with production performance records were selected as test subjects to verify the performance of the SNP molecular markers in the intron region of the STON2 gene. Specifically:

[0093] Using the genomic DNA of the laying hen to be tested as a template, PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product A; PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product B; the amplified products are sequenced, and the egg-laying traits of the laying hen are identified based on the sequencing results.

[0094] The identification method is as follows: if the sequencing result of the amplified product A of the laying hen to be tested corresponds to the sequence shown in SEQ ID NO.1, and the 129th base from the 5' end is a CC or TC genotype, the 49th base is a CC or TC genotype, and the 37th base is an AA or AG genotype; the sequencing result of the amplified product B corresponds to the sequence shown in SEQ ID NO.2, and the 83rd base from the 5' end is an AA or GA genotype; the total number of eggs laid and the longest consecutive laying days at 43 weeks of age are more than the sequencing result of the amplified product A, the 129th base from the 5' end of the sequence shown in SEQ ID NO.1 is a TT genotype, the 49th base is a TT genotype, and the 37th base is a GG genotype; the sequencing result of the amplified product B corresponds to the laying hen whose 83rd base from the 5' end of the sequence shown in SEQ ID NO.2 is a GG genotype.

[0095] It has been verified that the egg-laying traits of laying hens, such as the total number of eggs laid at 43 weeks of age and the longest consecutive laying days, identified using the above-mentioned SNP molecular markers, are consistent with the records of the actual production performance of laying hens, and have practical application value.

[0096] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A SNP molecular marker associated with chicken egg-laying traits, comprising a first molecular marker and a second molecular marker; The nucleotide sequence of the first molecular marker is as shown in SEQ ID NO.1, comprising a SNP1 site, a SNP2 site, and a SNP3 site. The 129th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is T or C; the 49th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP2 site, and its base is T or C; the 37th base from the 5′ end of the sequence shown in SEQ ID NO.1 is the SNP3 site, and its base is A or G; The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.

2. The 83rd base from the 5′ end of the sequence shown in SEQ ID NO.2 is the SNP4 site, and its base is G or A.

2. Use of the SNP molecular marker according to claim 1 in chicken genetic breeding.

3. The use according to claim 2, characterized in that The chicken genetic breeding is to select laying hens that produce a large number of eggs and have the longest continuous laying days at the age of 43 weeks.

4. A primer pair for detecting the SNP molecular marker according to claim 1, characterized in that: include: a primer pair A for detecting a first molecular marker and a primer pair B for detecting a second molecular marker; The nucleotide sequences of primer pair A are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively; The nucleotide sequences of primer pair B are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

5. A kit for detecting the SNP molecular marker according to claim 1, characterized in that: The kit contains the primer pair according to claim 5.

6. Use of the primer pair according to claim 4 and / or the kit according to claim 5 in assisting in the selection of laying hens with a high total egg production at 43 weeks of age and a long continuous laying period.

7. A method for identifying egg-laying traits of laying hens, characterized in that: The following steps are involved: Using the genomic DNA of the laying hen to be tested as a template, PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product A; PCR amplification is performed on A using the primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product B; the amplified products are sequenced, and the egg-laying traits of the laying hen are identified based on the sequencing results.

8. The method according to claim 7, characterized in that If the sequencing result of the amplified product A corresponds to the sequence shown in SEQ ID NO.1, and the 129th base from the 5' end is a CC or TC genotype, the 49th base is a CC or TC genotype, and the 37th base is an AA or AG genotype; if the sequencing result of the amplified product B corresponds to the sequence shown in SEQ ID NO.2, and the 83rd base from the 5' end is an AA or GA genotype; then it is identified as having the egg-laying trait of a large total number of eggs laid at 43 weeks of age and / or a long longest continuous laying period.

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