LHR gene, molecular marker and application for influencing goat lambing traits

Abstract

The present application relates to the field of modern livestock breeding technology, and more particularly to a LHR gene affecting goat lambing traits, a molecular marker and application. A molecular marker related to goat lambing traits exists at the 211bp of the nucleotide sequence of the LHR gene, and the polymorphism thereof is G / A. When the genotype of the polymorphic site contained in the molecular marker is AA, the corresponding relative advantage of the lambing traits of the goat to be tested is obtained; when the genotype of the polymorphic site contained in the molecular marker is GA or GG, the corresponding relative disadvantage of the lambing traits of the goat to be tested is obtained. The molecular marker provided by the present application can be applied to the identification or prediction of the goat lambing state, and can also be applied to the field of molecular breeding. Based on the analysis of the genotype of the goat, the genetic potential of the goat is judged, and the breeding is left. The present application has great significance for the modern livestock breeding of goats.

Description

Technical Field

[0001] This invention relates to the field of modern animal husbandry technology, and in particular to an LHR gene, molecular marker, and application that affects lambing traits in goats. Background Technology

[0002] The reproductive performance of goats has always been a crucial factor restricting the development of the goat industry. Increasing the number of lambs born to goats can not only increase mutton production but also improve breeding efficiency. Traditional breeding methods mainly rely on phenotypic selection, which is time-consuming, costly, inefficient, and lacks guaranteed selection accuracy. In recent years, with the rapid development of molecular biology techniques and the maturation of gene analysis methods, new ideas and methods have been provided for improving the reproductive performance of goats. Molecular marker-assisted selection (MAS) technology combines animal genetics with sequencing technology, thereby greatly shortening the breeding process. Therefore, MAS technology has been widely used in livestock and poultry breeding. By analyzing the association between SNP loci and traits such as goat weight and body size, genetic markers related to meat production performance can be quickly identified, thereby improving breeding efficiency.

[0003] The LHR gene, or luteinizing hormone receptor gene, encodes a G protein-coupled receptor (GPCR) primarily found in the ovaries and testes, but also in many extraglandular organs such as the uterus and mammary glands. The LHR gene consists of 674 amino acids, with a molecular weight of approximately 85-95 kDa depending on the degree of glycosylation. In goat reproduction, the specific functions of the LHR gene mainly include promoting estrus and ovulation, increasing conception rate, and maintaining pregnancy and parturition. Studies have shown that the LHR gene is expressed in the goat brain, hypothalamus, and ovaries, with high expression in the ovaries. Currently, there are few studies using the LHR gene as a candidate gene for screening molecular markers related to goat lambing performance.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an LHR gene, molecular marker, and application that influences lambing traits in goats.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of the LHR gene in detecting lambing traits in goats, the LHR gene comprising the nucleotide sequence shown in SEQ ID NO. 01.

[0008] Preferably, the lambing traits include at least one of the following: number of lambs born in a first litter, number of lambs born in a second litter, number of lambs born in a third litter, number of lambs born in a fourth litter, number of lambs born multiparously, and average number of lambs born; especially the performance in the number of lambs born in a third litter, number of lambs born multiparously, and average number of lambs born.

[0009] Secondly, the present invention provides a molecular marker associated with lambing traits in goats, the molecular marker being located at 211 bp of the nucleotide sequence shown in SEQ ID NO. 01, with a polymorphism of G / A.

[0010] Preferably, the genotype of the polymorphic site contained in the molecular marker is AA, which corresponds to the relative advantage of the lambing trait in the goat to be tested.

[0011] Preferably, the genotype of the polymorphic site contained in the molecular marker is GA or GG, corresponding to a relative disadvantage in lambing traits of the goat being tested.

[0012] Preferably, the primer sequences for amplifying the molecular marker are shown in SEQ ID NO.02 and SEQ ID NO.03.

[0013] Thirdly, the present invention provides specific primer pairs for amplifying the aforementioned molecular markers.

[0014] Preferably, the primer sequences of the specific primer pair are shown in SEQ ID NO.02 and SEQ ID NO.03.

[0015] Fourthly, the present invention provides a reagent or kit for detecting lambing traits in goats, which contains the aforementioned specific primer pairs.

[0016] Fifthly, the present invention provides the use of the molecular marker, the specific primer pair, or the reagent or kit in at least one of the following:

[0017] (1) Application in identifying lambing traits in goats;

[0018] (2) Application in predicting lambing traits in goats;

[0019] (3) Application in goat resource identification, improvement or molecular marker-assisted breeding.

[0020] Sixthly, the present invention provides a molecular breeding method for high-quality goats, comprising:

[0021] (1) Extract total DNA from the goats to be tested;

[0022] (2) Using DNA as a template, detect the genotype of the molecular marker described in any one of claims 2-4;

[0023] (3) Analyze the genotype to determine the genetic potential of goats for breeding and propagation.

[0024] Preferably, step (3) includes: performing allele detection on the amplification product and determining the genotype based on the genotyping results: the genotype of the polymorphic site contained in the molecular marker is AA, which corresponds to the relative advantage of the lambing trait of the goat to be tested; and / or, the genotype of the polymorphic site contained in the molecular marker is GA or GG, which corresponds to the relative disadvantage of the lambing trait of the goat to be tested.

[0025] Beneficial effects:

[0026] This invention provides the LHR gene, molecular marker, and applications that influence lambing traits in goats. A molecular marker associated with lambing traits in goats exists at position 211 bp of the LHR gene nucleotide sequence, with a polymorphism of G / A. When the genotype of the polymorphic site on the molecular marker is AA, it corresponds to a relative advantage in lambing traits in the goats being tested; when the genotype of the polymorphic site is GA or GG, it corresponds to a relative disadvantage in lambing traits in the goats being tested. The molecular marker provided by this invention can be applied to the identification or prediction of lambing status in goats, and can also be applied in the field of molecular breeding, based on the analysis of goat genotypes, to determine the genetic potential of goats for breeding and propagation. This invention has significant implications for modern goat husbandry. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0028] Figure 1 The results of gel recovery and purification of a partial nucleotide sequence of the goat LHR gene in Example 1 (i.e., the sequence shown in SEQ ID NO:1).

[0029] Figure 2 This is a SnapGene software reading result of the g.30580464G>A site in the goat LHR gene in Example 1; Figure 2 In the diagram, A represents the GG genotype; B represents the G genotype. A Genotype; Figure C: AA genotype. Detailed Implementation

[0030] This invention has discovered a molecular marker in the goat LHR gene that is associated with goat lambing traits, providing a new molecular marker for goat lambing trait detection or marker-assisted breeding.

[0031] Specifically, the technical solution of the present invention is as follows:

[0032] This invention provides the application of single nucleotide polymorphisms (SNPs) at goat SNP sites or substances for detecting SNPs at goat SNP sites in the detection or auxiliary detection of goat litter size traits, wherein the SNP site is a molecular marker of the g.30580464G>A site in the goat LHR gene.

[0033] The molecular marker of the g.30580464G>A site is that there is a G>A base mutation at position 211bp in the sequence shown in SEQ ID NO.1.

[0034] The single nucleotide polymorphisms at the above-mentioned SNP sites in this invention are related to reproductive traits such as the number of third-parity lambs, the number of multiparous lambs, and the average number of lambs per litter in goats.

[0035] This invention does not limit the breed of goat, but may include breeds such as Chubao Blackhead Goat, Macheng Black Goat, Boer Goat, Yichang White Goat, and Matou Goat.

[0036] The present invention also provides a method for detecting the lambing number trait in goats. The method detects the base type at the 211bp position in the sequence shown in SEQ ID NO:1 of goats. The AA genotype has significantly higher triple litter number, multiparous litter number, and average litter number than the GG genotype.

[0037] The present invention also provides substances for detecting single nucleotide polymorphisms at goat SNP sites, including PCR primers for amplifying genomic DNA fragments including the SNP sites or kits containing the primers.

[0038] The present invention also provides a molecular marker in the LHR gene associated with the number of lambs born in goats, the nucleotide sequence of which is shown in SEQ ID NO.1, wherein there is a G>A base mutation at position 211 bp in SEQ ID NO.1.

[0039] The substances for detecting single nucleotide polymorphisms at SNP sites in goats or the molecular markers associated with goat litter size traits described above in this invention can be used in goat genetic breeding to increase the litter size of offspring goats.

[0040] This invention provides a genetic breeding method to increase the number of lambs born in goats. The method involves determining the single nucleotide polymorphism (SNP) at the aforementioned SNP site in the core goat population and making corresponding selections based on the SNP at the aforementioned SNP site: in the successive generation selection of breeding goats, individuals with the AA type at the 211 bp of the sequence shown in SEQ ID NO.1 are selected, while individuals with the GG and GA types are eliminated, so as to increase the frequency of the AA gene at this site in each generation, thereby increasing the number of triple litters, the number of multiparous lambs, and the average number of lambs born in offspring.

[0041] Based on the above-mentioned scheme provided by this invention, this invention has discovered molecular markers in the goat LHR gene associated with litter size, wherein the molecular markers contain the SNP site g.30580464G>A; the haplotypes formed by the above SNP sites can serve as molecular markers for litter size in goats. This invention has verified the effects of the above SNP molecular markers on the number of third-parity lambs, multiparous lambs, and average litter size in goats, and can be applied to the genetic improvement of breeding sheep to increase litter size, thereby improving the reproductive performance of offspring and increasing the market competitiveness of breeding enterprises. This invention provides a new molecular marker for marker-assisted breeding of the litter size trait in goats, enabling early selection of the litter size trait in goats and shortening the breeding process; the detection method is rapid, accurate, and unaffected by environmental factors in the breeding environment.

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0046] The sequences involved in the following embodiments include:

[0047] SEQ ID NO.01:

[0048] TACACTGTTGTCTCTTGGGGGAGATGTAAATTTTACCTCCTCTGTATTCCAGGATCATTTGACCCTTAGTTGGACATGTGTACCCACGTACTTATTAATTCCTTATCTTCTCTAGATGATTGCTAATTGGACCATTCATAGGGAGGCCTTGTGGTACAGTGGAAAATGCACAAGCTTGGAGGACATGGGCTGGGGTGAATTCCAGCCCTG G CATGTCGAAGGTGCCACTTTGGCCAAGTTAACGACCCTTCTGAGCCTCCATTCCTTCCTGTGAACAAAAGGGATAATTACCCACTTTGCAGGGCAGCTGCAAGGATTAAATGACACAACCTAGAGGAAA TAAAGGCTGAGCACAAGTGCTCAAAATATATTCGTTGCTGATATCTTTTCTGCTTCTTAACTTTGGAAACTCCTCTGCTGATGCAGCTTGTAGTCGTTCAGATTTGGGACAAACTAGGCAGCAGTAAGAA.

[0049] The sequence listing SEQ ID NO:1 is a partial sequence of the LHR gene of Chubao black-headed sheep, which is the DNA sequence used as the molecular marker of this invention; at the 211th base of this sequence, there is an allele mutation, that is, the base "G" is mutated to the base "A".

[0050] Example 1

[0051] Obtaining SNP fragments of goat LHR gene and establishing a method for detecting polymorphic sites.

[0052] 1. Extraction of goat genomic DNA

[0053] The Chubao Blackhead Goat, a breed of meat goat improved from Macheng Black Goat, was selected as the experimental animal. Samples were obtained from the breeding farm of the Animal Husbandry and Veterinary Research Institute of the Hubei Academy of Agricultural Sciences. Whole-genome DNA was extracted from the goats using a blood genomic DNA extraction kit (manufactured by Beijing Tiangen Biotech Co., Ltd.), following the kit's instructions. The obtained genomic DNA was tested for concentration and quality, labeled, and stored at -80℃ for later use.

[0054] 2. Obtaining SNP genetic marker fragments:

[0055] (1) PCR amplification:

[0056] Primers were designed based on the goat LHR genome sequence. The primer sequence information is as follows:

[0057] Upstream primer (SEQ ID NO.02): GATCATTTGACCCTTAGTTGGA (5'→3');

[0058] Downstream primer (SEQ ID NO.03): TTACTGCTGCCTAGTTTGTCCC (5'→3').

[0059] The above primers were used to amplify the genomic DNA of Chubao black-headed sheep by PCR. The reaction system is shown in Table 1.

[0060] Table 1 PCR reaction system

[0061]

[0062] The PCR reaction program was set as follows: 98℃ pre-denaturation for 45s; 98℃ denaturation for 10s; annealing temperature set at 55℃ for 30s; extension at 72℃ for 25s; cycle number fixed at 34×; final extension at 72℃ for 5 min; 12℃ for 1 min.

[0063] Store PCR amplification products at 4°C.

[0064] (2) Purification of PCR products:

[0065] The PCR products were purified using a gel extraction kit (Shanghai Sangon Biotech Co., Ltd.). For specific steps, please refer to the instruction manual.

[0066] 3. SNaPshot method for detecting molecular markers:

[0067] Design SNaPshot extension primers for the G211A site based on the goat LHR gene genome sequence:

[0068] Add 5U SAP and 2U ExoI to 15μL of purified PCR product, vortex to mix, incubate at 37℃ for 1h, then incubate at 75℃ for 15min to inactivate SAP and ExoI enzymes; use the SNaPshot Multiplex Kit (Applied Biosystems) to aspirate 3μL of the treated 15μL PCR product for SNaPshot detection. The PCR reaction system is 10μL, containing 5μL Reaction Mix reagent, 3μL of PCR product treated with SAP and ExoI enzymes, 0.5μL each of extension primers, and 1μL of deionized water. The PCR amplification program is 96℃ denaturation for 10s, 50℃ annealing for 5s, 60℃ extension for 30s, 25 cycles, and storage at 4℃; dilute the SNaPshot product 20-fold in the following dilution system: Hi-Di Formamide 9.25μL, GS-120LIZ. 0.25 μL of SNaPshot product and 0.5 μL of sNaPshot product were added to the reaction system, which involved denaturation at 95 °C for 5 min followed by an ice bath for 4 min. A mixture containing 350 μL of Hi-Di formamide and 50 μL of Matrix standard was prepared, denatured at 95 °C for 5 min, and rapidly cooled for 5 min. The mixture was then divided into two equal tubes, aliquoted onto the instrument plate, and the 3730XL DNA Analyzer was spectrally calibrated. The prepared samples were then subjected to capillary electrophoresis using the 3730XL DNA Analyzer, and the signals were collected. Finally, the experimental results were analyzed using SnapGene software (e.g., ...). Figure 2 (As shown).

[0069] Example 2

[0070] The polymorphism distribution of the molecular markers prepared in this invention was detected in a goat population.

[0071] In this embodiment, the polymorphism of the g.30580464G>A site in the goat LHR gene was detected in a black-headed sheep population. The detection results are shown in Table 2.

[0072] Table 2. Genotype and gene frequencies of the g.30580464G>A locus in the goat LHR gene.

[0073]

[0074] Table 2 shows that the LHR gene g.30580464G>A locus in the Chubao black-headed sheep population exhibits three genotypes: GG, GA, and AA, with the homozygous AA genotype being the dominant genotype. The base frequency of allele G is 0.95. Chi-square test indicates that the genotype distribution at this locus does not conform to Hardy-Weinberg equilibrium.

[0075] Example 3

[0076] The present invention relates to the correlation analysis and application of molecular markers with lambing traits in goats.

[0077] To determine whether the LHR gene g.30580464G>A in goats is related to lambing performance, 500 Chubao black-headed goats were selected as experimental materials. Sample collection and relevant lambing information were obtained from the breeding farm of the Animal Husbandry and Veterinary Research Institute of the Hubei Academy of Agricultural Sciences. Direct sequencing was used to detect the genotypes of different individuals, and the correlation between individuals with different genotypes and their lambing traits was analyzed. The GLM program in SAS statistical analysis software was used to perform association analysis between molecular markers and traits of different genotypes. The model used was:

[0078] Model 1: Y = Population mean + Genotype + Sheep farm environmental effect + Residual;

[0079] Model 2: Y = Population mean + Additive effect + Dominant effect + Sheep farm environmental effect + Residual.

[0080] Where Y represents the phenotypic value. Additive effect = (homozygote 1 - homozygote 2) / 2, with 1, 0, and -1 representing homozygote 1, heterozygote 1, and homozygote 2, respectively; dominant effect = heterozygote - (homozygote 1 + homozygote 2) / 2, with 1, -1, and 1 representing homozygote 1, heterozygote 1, and homozygote 2, respectively. The statistical analysis results are shown in Table 3.

[0081] Table 3. Association analysis between the g.30580464G>A site in the LHR gene and lambing traits.

[0082]

[0083] The association analysis of the LHR gene g.30580464G>A locus and lambing traits in Chubao black-headed sheep using SAS 9.4 data analysis software is shown in Table 3. The LHR gene g.30580464G>A locus has three genotypes: GG, GA, and AA. The AA genotype showed significantly higher rates of third-parity lambing, multiparous lambing, and average lambing compared to the GG and GA genotypes (P<0.05). Therefore, screening for individuals with the AA genotype at the LHR gene g.30580464G>A locus in goats aims to obtain sheep with higher lambing rates.

[0084] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of specific primer pairs for amplifying molecular markers associated with goat lambing traits, or reagents or kits containing said specific primer pairs, in identifying or predicting goat lambing traits, wherein the lambing trait is at least one of triplets, multiparous lambs, or average number of lambs per litter; and the molecular marker is located at 211 bp of the nucleotide sequence shown in SEQ ID NO.1 and has a polymorphism of G / A; The genotype of the polymorphic site contained in the molecular marker is AA, which corresponds to the relative dominance of the lambing trait in the goat being tested; The genotype of the polymorphic site contained in the molecular marker is GA or GG, which corresponds to a relative disadvantage in lambing trait of the goat being tested; the goat is the Chubao Blackhead Goat.

2. A molecular breeding method for high-quality goats, characterized in that, include: (1) Extract total DNA from the goats to be tested; (2) Using DNA as a template, detect the genotypes of molecular markers related to lambing traits in goats; (3) Analyze the genotype to determine the genetic potential of goats for breeding and propagation; Step (3) includes: performing allele detection on the amplification products and determining the genotype based on the genotyping results: the genotype of the polymorphic site contained in the molecular marker is AA, which corresponds to the relative advantage of the lambing trait in the goat to be tested; and / or, the genotype of the polymorphic site contained in the molecular marker is GA or GG, which corresponds to the relative disadvantage of the lambing trait in the goat to be tested. The molecular marker is located at 211 bp of the nucleotide sequence shown in SEQ ID NO.1, and the polymorphism is G / A; the goat is the Chubao Blackhead Goat; the lambing trait is at least one of the following: number of lambs born in triplets, number of lambs born multiparously, and average number of lambs born per litter.

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