Tshr gene molecular marker related to egg laying traits and use thereof

Abstract

This invention discloses a molecular marker for the TSHR gene associated with egg production traits and its application, belonging to the field of molecular genetics. This invention obtained four molecular markers significantly associated with egg production traits in chickens, located on chromosome 5 at Chromosome 5, NC_006092.5:(40967852-41029739). These markers are found in the chicken TSHR gene, specifically within the region of Chromosome 5, NC_006092.5:(40967852-41029739). These markers can be used in breeding high-producing hens.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics. ， Specifically, this involves a TSHR gene molecular marker related to egg production traits and its application. Background Technology

[0002] Egg production performance effectively reflects the reproductive performance of chickens. As an important source of animal protein, global egg consumption has increased significantly over the past few decades. Understanding the genetic mechanisms of egg production traits in chickens has important practical and economic significance. It is necessary to study functional genes or molecular markers related to egg production traits in chickens for use in egg-laying hen breeding.

[0003] The TSHR gene (thyroid-stimulating hormone receptor gene) encodes a receptor protein primarily involved in the regulation of thyroid function. This receptor is expressed on thyroid cells and binds to thyroid-stimulating hormone (TSH), thereby regulating the synthesis and secretion of thyroid hormones (THs). Studies have shown that THs and their receptors are widely present in ovarian tissue, participating in the regulation of ovarian steroid production and follicle development, thus affecting female reproductive function. TSHR, as a core component of the thyroid hormone signaling pathway, plays a crucial role in the regulation of thyroid function. It is known that mutations in exon G558A of TSHR, as a "domestication gene," can directly affect the photoperiod response and seasonal reproductive characteristics of animals. However, molecular markers of the intron region of the TSHR gene associated with egg production in chickens are still rarely reported. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a molecular marker of the TSHR gene related to egg production traits and its application. This invention obtains four molecular markers significantly associated with egg production traits such as age at first laying (AFE), total eggs laid at 43 weeks of age (E43), and longest consecutive laying days (LCS) in chickens from the chicken TSHR gene located on chromosome 5 at position Chromosome 5, NC_006092.5:(40967852-41029739). These markers can be used in the breeding practice of high-producing laying 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 combination of molecular markers for TSHR genes associated with egg production traits, 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, which includes SNP1 and SNP2 sites. The 286th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is A or G; the 1158th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP2 site, and its base is A or T.

[0008] The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.2. The 155th base from the 5′ end of the sequence shown in SEQ ID NO.2 is an SNP3 site, and its base is A or G.

[0009] The nucleotide sequence of the third molecular marker is shown in SEQ ID NO.3. The 147th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP4 site, and its base is G or C.

[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]

[0015]

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

[0017] The specific nucleotides marked as third molecules are as follows:

[0018]

[0019] Note: The nucleotides in bold shaded areas in the sequence are SNP sites, represented by "n" in the sequence listing.

[0020] In a second aspect, the present invention provides the application of the above-mentioned TSHR gene molecular marker combination in chicken genetic breeding.

[0021] In the above applications, the chicken genetic breeding refers to the selection and breeding of laying hens that start laying early, have a high total egg production at 43 weeks of age, and have the longest consecutive laying days.

[0022] Furthermore, in the TSHR gene molecular marker combination, individuals with the AG genotype at SNP1, the TA genotype at SNP2, the AG genotype at SNP3, and the GC genotype at SNP4 exhibit the egg-laying traits of early onset of egg production, high total egg production at 43 weeks of age, and the longest consecutive laying period.

[0023] A third aspect of the present invention provides primer pairs for detecting the above-mentioned combination of TSHR gene molecular markers, 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;

[0024] The nucleotide sequences of primer pair A are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. Specifically:

[0025] TSHR-1-F: 5′-ACTTTCTGCCTTGCCTCCTTAC-3′; (SEQ ID NO.4)

[0026] TSHR-1-R: 5′-TGGAAGACACCAAACGTTCAG-3′. (SEQ ID NO.5)

[0027] The nucleotide sequences of primer pair B are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively. Specifically:

[0028] TSHR-2-F: 5′-AGTCAGAGTGGGTGAGACTTAG-3′; (SEQ ID NO. 6)

[0029] TSHR-2-R: 5′-GATGAACTTCAGATAAGTTCAGAT-3′. (SEQ ID NO.7)

[0030] The nucleotide sequences of primer pair C are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. Specifically:

[0031] TSHR-3-F: 5′-AGACGATAAGTTCCAATGAAC-3′; (SEQ ID NO.8)

[0032] TSHR-3-R: 5′-GTCAATTGAAAGGATTCTGTGAT-3′. (SEQ ID NO.9)

[0033] In a fourth aspect, the present invention provides a kit for detecting the above-mentioned TSHR gene molecular marker combination, 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.

[0034] 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 early onset of egg production, high total egg production at 43 weeks of age, and longest consecutive laying days.

[0035] A sixth aspect of the present invention provides a method for identifying egg-laying traits in laying hens, comprising the following steps:

[0036] 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.

[0037] Specifically, if the sequencing result of amplification product A corresponds to the AG genotype at position 286 from the 5' end of the sequence shown in SEQ ID NO.1, and the TA genotype at position 1158; the sequencing result of amplification product B corresponds to the AG genotype at position 155 from the 5' end of the sequence shown in SEQ ID NO.2; and the sequencing result of amplification product C corresponds to the GC genotype at position 147 from the 5' end of the sequence shown in SEQ ID NO.3, then the product is identified as having the egg-laying traits of early onset of laying, high total number of eggs laid at 43 weeks of age, and longest consecutive laying days.

[0038] The beneficial effects of this invention are:

[0039] (1) In this invention, four SNP loci that are significantly associated with egg production traits such as age at first laying (AFE), total number of eggs laid at 43 weeks of age (E43), and longest consecutive laying days (LCS) were detected in the 8th intron and 3′UTR region of the TSHR gene on chromosome 5 of chickens. These SNP loci are 5_41012839, 5_41013711, 5_41016884, and 5_41023899. Based on these four SNP loci, molecular marker combinations related to egg production traits can be developed. Detecting these molecular marker combinations can help in the selection of high-producing egg-laying chicken breeds and provide beneficial assistance to breeding work.

[0040] (2) Based on the four SNP loci 5_41012839, 5_41013711, 5_41016884 and 5_41023899, this invention further obtained diploids that are significantly associated with the age of first laying of chickens, the total number of eggs laid at 43 weeks of age and the longest consecutive laying days, which improves the accuracy of result judgment and can be used in the breeding practice of chickens with precocious puberty and high egg production. Attached Figure Description

[0041] Figure 1 Manhattan plot of genome-wide association analysis under the MLM model for egg production traits.

[0042] Figure 2 LD linkage disequilibrium plot of genome-wide association analysis under the MLM model for egg production traits. Detailed Implementation

[0043] 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.

[0044] As mentioned earlier, understanding the genetic mechanisms of egg production traits in chickens has significant practical and economic implications, and it is necessary to study functional genes or molecular markers related to egg production traits for use in egg-laying hen breeding. However, due to the vast number of SNP variant sites in the chicken genome, screening for SNP sites that are significantly associated with egg production traits is quite challenging.

[0045] Using the chicken TSHR gene (Gene ID: 428900) as a reference, this invention detected four SNP sites associated with chicken egg production traits in the 8th intron region of the chicken TSHR gene, namely:

[0046] SNP1 site: physical location 5_41012839, corresponding to the sequence shown in SEQ ID NO.1, where the 286th base from the 5′ end has an A>G mutation.

[0047] SNP2 site: physical location 5_41013711, corresponding to the sequence shown in SEQ ID NO.1, where the 1158th base from the 5′ end has an A>T mutation.

[0048] SNP3 site: physical location 5_41016884, corresponding to the sequence shown in SEQ ID NO.2, where the 155th base from the 5′ end has an A>G mutation.

[0049] SNP4 site: physical location 5_41023899, corresponding to the 147th base from the 5′ end of the sequence shown in SEQ ID NO.3, where there is a G>C mutation.

[0050] This invention has found that the above four SNP loci are significantly associated with egg production traits such as age at first laying, total number of eggs laid at 43 weeks of age, and longest consecutive laying days in chickens. These loci can be used in breeding practices for chickens with precocious puberty and high egg production.

[0051] 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.

[0052] 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:

[0053] The Langya chicken used in this embodiment of the invention comes from Shandong Jihua Poultry Breeding Co., Ltd.

[0054] Example 1: Screening and identification of molecular markers in the intron region of the TSHR gene associated with egg production traits

[0055] Using Langya chickens with production performance records as the research subject, GWAS analysis was performed on their egg production traits. The results showed that a signal associated with the total number of eggs laid at 43 weeks of age was screened on chromosome 5. Figure 1 Further analysis revealed that the significant locus associated with E43 (total egg production at 43 weeks of age) was located at position GGA5, between 40.989 Mb and 41.024 Mb. A total of 59 significant loci were extracted. Linkage disequilibrium analysis was performed on the Langya chicken population using Haploview software. All loci showed D′ greater than 99%, indicating linkage relationships between all loci. The results are as follows: Figure 2 As shown. Based on the p-value, the four most significant loci were selected, all located on the TSHR gene, namely:

[0056] 5_41012839 corresponds to the A>G mutation at nucleotide 39830 of intron 8 of the TSHR gene; 5_41013711 corresponds to the A>T mutation at nucleotide 40702 of intron 8 of the TSHR gene; 5_41016884 corresponds to the A>G mutation at nucleotide 43875 of intron 8 of the TSHR gene; and 5_41023899 corresponds to the G>C mutation at nucleotide 50890 of the 3′UTR of the TSHR gene. Among these, 5_41012839 is the most significant, with a P value of 4.12E-09.

[0057] Example 2: Association analysis of four SNPs with egg production traits

[0058] 1. Test method:

[0059] Another 1200 Langya chicken hens were selected as experimental subjects. All chickens were hatched in the same batch and raised in the same pen and environment during the brooding and rearing stages, with uniform lighting and feeding management methods.

[0060] Hens were housed individually, and their age at first laying (AFE), total egg production at 43 weeks of age (E43), and longest consecutive laying days (LCS) for each hen were measured and recorded. After recording, blood samples were collected from all hens via the subwing vein, anticoagulated with EDTA, and stored at -20°C for subsequent genomic DNA extraction.

[0061] Using the chicken TSHR gene (Gene ID: 428900) as a reference, three pairs of primers were designed for the detection of the four SNP sites screened in Example 1. The primers are as follows:

[0062] The upstream primer, named [TSHR-1-F], is sequence 5′-ACTTTCTGCCTTGCCTCCTTAC-3′, and the downstream primer, named [TSHR-1-R], is sequence 5′-TGGAAGACACCAAACGTTCAG-3′. These primers are used to detect the A>G mutation at nucleotide 39830 of intron 8 of the TSHR gene corresponding to 5_41012839 and the A>T mutation at nucleotide 40702 of intron 8 of the TSHR gene corresponding to 5_41013711.

[0063] The upstream primer, named [TSHR-2-F], is sequence 5′-AGTCAGAGTGGGTGAGACTTAG-3′, and the downstream primer, named [TSHR-2-R], is sequence 5′-GATGAACTTCAGATAAGTTCAGAT-3′. These primers are used to detect the A>G mutation at nucleotide 43875 of intron 8 of the TSHR gene corresponding to 5_41016884.

[0064] The upstream primer, named [TSHR-3-F], is composed of sequence 5′-AGACGATAAGTTCCAATGAAC-3′, and the downstream primer, named [TSHR-3-R], is composed of sequence 5′-GTCAATTGAAAGGATTCTGTGAT-3′. These primers are used to detect the G>C mutation at nucleotide 50890 of the 3′UTR of the TSHR gene corresponding to gene 5_41023899.

[0065] The genotypes of the four SNP loci selected in Example 1 of Langya chicken were determined by gene sequencing, and the genotype data of the corresponding SNP loci were obtained. The genotype data of Langya chicken were correlated with the egg production data of Langya chicken.

[0066] 2. Test Results:

[0067] The results are shown in Table 1.

[0068] Table 1: Association analysis of four significant SNP sites with egg production traits in the Langya chicken population

[0069]

[0070]

[0071] The results showed that the effects of locus 5_41012839 on age at first egg production, total egg production at 43 weeks of age, and longest consecutive laying days were significantly different. The dominant allele was A, and individuals with the AG genotype corresponded to a younger age at first egg production and a higher number of eggs produced. The effects of locus 5_41013711 on age at first egg production, total egg production at 43 weeks of age, and longest consecutive laying days were significantly different. The dominant allele was T, and individuals with the TA genotype corresponded to a younger age at first egg production and a higher number of eggs produced. The effects of locus 5_41016884 on age at first egg production, total egg production at 43 weeks of age, and longest consecutive laying days were significantly different. The dominant allele was A, and individuals with the AG genotype corresponded to a younger age at first egg production and a higher number of eggs produced. The effects of locus 5_41023899 on age at first egg production, total egg production at 43 weeks of age, and longest consecutive laying days were significantly different. The dominant allele was G, and individuals with the GC genotype corresponded to a younger age at first egg production and a higher number of eggs produced.

[0072] Example 3: Construction and joint analysis of diploid types of four SNPs

[0073] 1. Haplotype and diploid frequency distribution analysis:

[0074] Based on the sequencing results of Langya chickens in Example 2, each individual was analyzed by splitting the alleles at the four SNP loci (5_41012839, 5_41013711, 5_41016884, and 5_41023899) and combining them to construct seven haplotypes. After removing haplotypes with a frequency less than 0.001, four haplotypes were obtained: ATAG, GAGC, GAAG, and AAGG. Among them, ATAG and GAGC were the dominant haplotypes. Genotype frequency distribution characteristics of the dominant haplotypes and their combinations (diplotypes) were analyzed, and the results are shown in Tables 2-3.

[0075] Table 2: Frequency analysis of dominant haplotypes in the Langya chicken population

[0076]

[0077] Table 3: Diploid Frequency Analysis in Langya Chicken Population

[0078]

[0079]

[0080] 2. Association analysis of four SNP diploid types with egg production trait

[0081] Association analysis was performed on SNPs diploid types in the Langya chicken population with laying traits, age at first laying (AFE), number of eggs laid at 43 weeks (E43), and maximum consecutive laying count (LCS). The results are shown in Table 4.

[0082] Table 4: Association analysis of four SNPs diploid types with egg production in Langya chicken population

[0083]

[0084] Note: The values ​​in the table are the least squares mean ± standard error of AFE (age at first laying), E43 (number of eggs laid at 43 weeks), and LCS (longest consecutive laying). P < 0.05 indicates a significant difference.

[0085] The results showed that the diploid types constructed from the four SNP loci were significantly associated with age at first laying, egg production at 43 weeks of age, and maximum consecutive laying count (P = 6.25E-08; P = 9.47E-11; P = 1.91E-09). In Langya chickens, individuals with the diploid type H1H2 (ATAG / GAGC) were associated with a younger age at first laying and a higher egg production. Increasing the proportion of the H2 haplotype in the breeding population helps to advance the sexual maturity time of the flock and increase egg production.

[0086] 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 TSHR Gene molecular marker combination, 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 286th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP1 site, and its base is A or G; the 1158th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP2 site, and its base is A or T. The nucleotide sequence of the second molecular marker is shown in SEQ ID NO.

2. The 155th base from the 5′ end of the sequence shown in SEQ ID NO.2 is an SNP3 site, and its base is A or G. The nucleotide sequence of the third molecular marker is shown in SEQ ID NO.

3. The 147th base from the 5′ end of the sequence shown in SEQ ID NO.3 is the SNP4 site, and its base is G or C.

2. The claim 1 TSHR The application of gene molecular marker combinations in chicken genetic breeding is characterized by, The chicken genetic breeding refers to the selection and breeding of Langya chickens, which have an early onset of egg production, a high total egg production at 43 weeks of age, and the longest consecutive laying period.

3. The application according to claim 2, characterized in that, The TSHR Among the gene molecular marker combinations, individuals with the AG genotype at SNP1, the TA genotype at SNP2, the AG genotype at SNP3, and the GC genotype at SNP4 exhibited laying traits such as early onset of egg production, a large total number of eggs laid at 43 weeks of age, and the longest consecutive laying period.

4. The test according to claim 1 TSHR The application of primer pairs combining gene molecular markers in assisted breeding of laying hen breeds is characterized by, The egg-laying hen breed is Langya chicken, which has the traits of early onset of egg production, high total egg production at 43 weeks of age, and the longest 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; The TSHR Among the gene molecular marker combinations, individuals with the AG genotype at SNP1, the TA genotype at SNP2, the AG genotype at SNP3, and the GC genotype at SNP4 exhibited laying traits such as early onset of egg production, a large total number of eggs laid at 43 weeks of age, and the longest consecutive laying period.

Images