TSHR gene molecular marker related to egg laying traits and application of TSHR gene molecular marker

Abstract

The invention discloses a TSHR gene molecular marker related to egg laying traits and application of the TSHR gene molecular marker, and belongs to the technical field of molecular genetics. According to the invention, four molecular markers which are obviously associated with the egg laying traits such as the first laying day age (AFE), the 43-week-age total egg laying number (E43), the longest continuous laying day number (LCS) and the like of the chicken are obtained in a region of a chicken TSHR gene, which is located in a chromosome 5 and at the position of Chromosome 5, NC006092.5: (40967852-41029739), and the molecular markers can be used for breeding practice of high-yield laying hens.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetics, and particularly relates to a TSHR gene molecular marker related to egg production traits and its application. Background Art

[0003] The TSHR gene (thyroid stimulating hormone receptor gene) encodes a receptor protein mainly 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). Research shows that THs and its receptor are widely present in ovarian tissue, participating in the regulation of ovarian steroidogenesis and follicular development, and thus affecting female reproductive function. As a core component of the thyroid hormone signaling pathway, TSHR plays an important role in thyroid function regulation. It is known that as a "domesticated gene", the G558A mutation in its exon can directly affect the photoperiod response and seasonal reproductive characteristics of animals, but there are few reports on molecular markers in the intron region of the TSHR gene related to the egg production number trait in chickens. Summary of the Invention

[0004] Aiming at the above-mentioned existing technology, the purpose of the present invention is to provide a TSHR gene molecular marker related to egg production traits and its application. The present invention obtains 4 molecular markers significantly associated with egg production traits such as age at first egg (AFE), total number of eggs produced at 43 weeks of age (E43), and longest consecutive laying days (LCS) in the region of the chicken TSHR gene located on chromosome 5, position Chromosome 5, NC_006092.5: (40967852 - 41029739), which can be used in the breeding practice of high-egg-yielding chickens.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a combination of TSHR gene molecular markers related to egg production traits is provided, including a first molecular marker, a second molecular marker, and a third molecular marker;

[0007] The nucleotide sequence of the first molecular marker is as shown in SEQ ID NO.1, which contains SNP1 locus and SNP2 locus. The 286th base from the 5' end of the sequence shown in SEQ ID NO.1 is the SNP1 locus, 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 locus, and its base is A or T.

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

[0009] The nucleotide sequence of the third molecular marker is as 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 nucleotides of the first molecular marker are specifically as follows:

[0011]

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

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

[0014]

[0015]

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

[0017] The nucleotides of the third molecular marker are specifically as follows:

[0018]

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

[0020] In the second aspect of the present invention, there is provided the use of the above-mentioned TSHR gene molecular marker combination in chicken genetic breeding.

[0021] In the above-mentioned use, the chicken genetic breeding is: selection of laying hens with early age at first egg, large total number of eggs laid at 43 weeks of age, and long longest consecutive laying days.

[0022] Furthermore, in the TSHR gene molecular marker combination, individuals with the AG genotype at the SNP1 site, the TA genotype at the SNP2 site, the AG genotype at the SNP3 site, and the GC genotype at the SNP4 site exhibit laying traits of early age at first egg, large total number of eggs laid at 43 weeks of age, and long longest consecutive laying days.

[0023] In a third aspect of the present invention, there is provided a primer pair for detecting the above-mentioned TSHR gene molecular marker combination, 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;

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

[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 as follows:

[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 as follows:

[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 of the present invention, there is provided a kit for detecting the above-mentioned TSHR gene molecular marker combination, and the kit contains 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 the fifth aspect of the present invention, there is provided the use of the above-mentioned primer pair and / or kit in the assisted breeding of laying hen breeds; the laying hen breeds have laying traits of early age at first egg, large total number of eggs laid at 43 weeks of age, and long longest consecutive laying days.

[0035] In the sixth aspect of the present invention, there is provided a method for identifying the laying traits of laying hens, comprising the following steps:

[0036] Using the genomic DNA of the laying hen to be tested as a template, performing PCR amplification with primer pair A shown in SEQ ID NO.4 and SEQ ID NO.5 to obtain amplification product A; performing PCR amplification with primer pair B shown in SEQ ID NO.6 and SEQ ID NO.7 to obtain amplification product B; performing PCR amplification with primer pair C shown in SEQ ID NO.8 and SEQ ID NO.9 to obtain amplification product C; sequencing amplification product A, amplification product B and amplification product C, and identifying the laying traits of the laying hens according to the sequencing results.

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

[0038] Advantages of the present invention:

[0039] (1) In the present invention, 4 SNP loci significantly associated with laying traits such as age at first egg (AFE), total number of eggs laid at 43 weeks of age (E43), and longest consecutive laying days (LCS) of chickens are detected in the 8th intron and 3′ UTR region of the TSHR gene on chicken chromosome 5, namely 5_41012839, 5_41013711, 5_41016884 and 5_41023899. Based on the above 4 SNP loci, a molecular marker combination related to laying traits can be developed. Detecting the molecular marker combination is helpful for breeding high-yield laying hen breeds and provides favorable assistance for breeding work.

[0040] (2) Based on the four SNP loci 5_41012839, 5_41013711, 5_41016884, and 5_41023899, the present invention further obtained haplotypes significantly associated with the age at first egg, total number of eggs laid at 43 weeks of age, and longest consecutive laying days in chickens, improving the accuracy of result judgment and can be used in the breeding practice of chicken sexual precocity and high egg production traits. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0042] Figure 2 : LD linkage disequilibrium map of genome-wide association analysis under the MLM model for egg production traits. DETAILED DESCRIPTION OF THE INVENTION

[0043] It should be noted that the following detailed description is illustrative and is 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 of ordinary skill in the technical field to which the present application belongs.

[0044] As mentioned above, understanding the genetic mechanism of chicken egg production traits has important practical and economic significance, and it is necessary to study functional genes or molecular markers related to chicken egg production traits for laying hen breeding. However, due to the vast number of SNP variant sites in the chicken genome, it is difficult to screen SNP loci significantly associated with chicken egg production traits.

[0045] The present invention takes the chicken TSHR gene (Gene ID: 428900) as a reference and detects four SNP loci related to chicken egg production traits in the 8th intron region of the chicken TSHR gene, namely:

[0046] SNP1 locus: Physical position 5_41012839, corresponding to a mutation of A>G at the 286th base from the 5′ end of the sequence shown in SEQ ID NO.1.

[0047] SNP2 locus: Physical position 5_41013711, corresponding to a mutation of A>T at the 1158th base from the 5′ end of the sequence shown in SEQ ID NO.1.

[0048] SNP3 locus: Physical position 5_41016884, corresponding to a mutation of A>G at the 155th base from the 5′ end of the sequence shown in SEQ ID NO.2.

[0049] SNP4 locus: Physical position 5_41023899, corresponding to a mutation of G>C at the 147th base from the 5′ end of the sequence shown in SEQ ID NO.3.

[0050] The present invention has found through research that the above 4 SNP loci are significantly associated with laying traits such as the age at first egg, the total number of eggs laid at 43 weeks of age, and the longest consecutive laying days in chickens, and can be used in the breeding practice of early sexual maturity and high egg production traits in chickens.

[0051] 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 in conjunction with specific embodiments.

[0052] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specific conditions are carried out according to conventional test methods or the operation manuals recommended by the suppliers. Among them:

[0053] The Langya chickens used in the embodiments of the present invention are from Shandong Jihua Poultry Breeding Co., Ltd.

[0054] Example 1: Screening and identification of molecular markers in the intron region of the TSHR gene related to laying traits

[0055] Taking Langya chickens with production performance records as the research object, GWAS analysis was performed on their laying traits. As a result, signals related to the total number of eggs laid at 43 weeks of age were screened on chromosome 5 ( Figure 1 ). Further analysis found that the significant locus related to E43 (total number of eggs laid at 43 weeks of age) was located at 40.989 Mb - 41.024 Mb at the GGA5 position. A total of 59 trait - significant loci were extracted, and linkage disequilibrium analysis was performed in the Langya chicken population using Haploview software. The D′ values were all greater than 99%, indicating a linkage relationship among the loci. The results are as Figure 2 shown. According to the P - value, 4 of the most significant loci were selected, all of which were located on the TSHR gene, namely:

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

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

[0058] 1. Test method:

[0059] Another 1200 Langya chicken hens were taken as the experimental subjects. The chickens were all hatched in the same batch and were raised in the same pen and the same environment during the brooding and growing stages, with a unified lighting system and feeding management method.

[0060] The hens were individually caged. The age at first egg (AFE) of the chickens was measured and recorded, the total number of eggs produced at 43 weeks of age (E43) was counted, and the longest consecutive laying days (LCS) of each hen were statistically analyzed. After the recording was completed, blood samples of all hens were collected from the wing vein, anticoagulated with EDTA, and stored in a -20°C refrigerator for subsequent extraction of genomic DNA.

[0061] Taking the chicken TSHR gene (Gene ID: 428900) as a reference, three pairs of primers were designed respectively for the detection of 4 SNP loci screened in Example 1. The specific primers are as follows:

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

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

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

[0065] Gene sequencing was used to determine the genotypes of the 4 SNP loci screened in Langya chickens in Example 1, and the genotype data of the corresponding SNP loci were obtained; the genotype data of Langya chickens were correlated with the egg production data of Langya chickens.

[0066] 2. Test results:

[0067] The results are shown in Table 1.

[0068] Table 1: Association analysis between 4 significant SNP loci and egg production traits in Langya chicken population

[0069]

[0070]

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

[0072] Example 3: Haplotype construction and combined analysis of four SNPs

[0073] 1. Analysis of haplotype and diplotype frequency distribution:

[0074] According to the sequencing results of Langya chicken in Example 2, for each individual, the alleles at each locus were split and combined in the order of the 4 SNP loci: 5_41012839, 5_41013711, 5_41016884, and 5_41023899, and 7 haplotypes were constructed. After removing the haplotypes with a frequency less than 0.001, four haplotypes, namely ATAG, GAGC, GAAG, and AAGG, were obtained. Among them, ATAG and GAGC were the dominant haplotypes; the genotype frequency distribution characteristics of the dominant haplotypes and their haplotype combinations (diplotypes) were analyzed, and the results are shown in Tables 2 - 3.

[0075] Table 2: Analysis of dominant haplotype frequencies in Langya chicken population

[0076]

[0077] Table 3: Analysis of diplotype frequencies in Langya chicken population

[0078]

[0079]

[0080] 2. Association analysis of four SNPs haplotypes with egg production traits

[0081] The haplotypes of SNPs in the Langya chicken population were analyzed for their association with the egg production traits of age at first egg (AFE), egg production at 43 weeks (E43), and maximum consecutive laying series (LCS). The results are shown in Table 4.

[0082] Table 4: Association analysis of four SNPs haplotypes with egg production traits in the Langya chicken population

[0083]

[0084] Note: The values in the table are the least squares means ± standard errors of AFE (age at first egg), E43 (egg production at 43 weeks), and LCS (maximum consecutive laying series). A significant difference was observed at P < 0.05.

[0085] The results showed that the haplotypes constructed from the four SNPs were significantly associated with the traits of age at first egg, egg production at 43 weeks, and maximum consecutive laying series (P = 6.25E - 08; P = 9.47E - 11; P = 1.91E - 09). In Langya chickens, individuals with the haplotype H1H2 (ATAG / GAGC) had a relatively younger age at first egg and a higher egg production. Increasing the ratio of the H2 haplotype in the breeding population would help to advance the sexual maturity time of the chicken flock and increase egg production.

[0086] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A TSHR gene molecular marker combination related to egg-laying traits, characterized in that: comprising a first molecular marker, a second molecular marker and a third molecular marker; 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, and the 155th base from the 5′ end of the sequence shown in SEQ ID NO.2 is the 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. Application of the TSHR gene molecular marker combination described in claim 1 in chicken genetic breeding.

3. The use according to claim 2, characterized in that: The chicken genetic breeding is to select and breed laying hens with early laying age, high total egg production at 43 weeks of age, and longest continuous laying days.

4. The use according to claim 2, characterized in that: In the TSHR gene molecular marker combination, individuals with AG genotype at SNP1, TA genotype at SNP2, AG genotype at SNP3, and GC genotype at SNP4 exhibit egg-laying traits such as early onset of egg laying, high total number of eggs laid at 43 weeks of age, and longest continuous laying days.

5. A primer pair for detecting the TSHR gene molecular marker combination according to claim 1, characterized in that: include: A primer pair A for detecting a first molecular marker, a primer pair B for detecting a second molecular marker, and a 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.

6. A kit for detecting the TSHR gene molecular marker combination according to claim 1, characterized in that: The kit comprises the primer pair according to claim 5.

7. Use of the primer pair according to claim 5 and / or the kit according to claim 6 in assisting the selection of laying hen varieties, characterized in that: The laying hen breed has the egg-laying characteristics of an early start of laying age, a large number of eggs laid at 43 weeks of age, and a long maximum number of consecutive laying days.

8. A method for identifying egg-laying traits of laying hens, characterized in that: The following steps are involved: Taking the genomic DNA of the laying hen to be tested as a template, PCR amplification is performed using the primer pair A shown by SEQ ID NO.4 and SEQ ID NO.5 to obtain an amplified product A; PCR amplification is performed using the primer pair B shown by SEQ ID NO.6 and SEQ ID NO.7 to obtain an amplified product B; PCR amplification is performed using the primer pair C shown by SEQ ID NO.8 and SEQ ID NO.9 to obtain an amplified product C; the amplified products A, B and C are sequenced, and the egg-laying traits of the laying hen are identified according to the sequencing results.

9. The method according to claim 8, characterized in that If the sequencing result of amplified product A corresponds to the AG genotype at the 286th base from the 5′ end of the sequence shown in SEQ ID NO.1, and the 1158th base is the TA genotype; the sequencing result of amplified product B corresponds to the AG genotype at the 155th base from the 5′ end of the sequence shown in SEQ ID NO.2; the sequencing result of amplified product C corresponds to the GC genotype at the 147th base from the 5′ end of the sequence shown in SEQ ID NO.3; then it is identified as having egg-laying traits of early age of first laying, large number of eggs laid at 43 weeks of age, and longest number of consecutive days of laying.

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