A molecular marker related to early body weight gain of hu sheep, primer thereof and application

Abstract

The application discloses a FGF7 gene molecular marker related to early body weight gain of Hu sheep, primers and application thereof. The molecular marker comprises two completely linked single nucleotide polymorphism sites of Chr7:57740946G>A and Chr7:57741376T>G in a 3'UTR region of a FGF7 gene of Hu sheep. The application further discloses a primer pair for amplifying the above sites and a detection method. By detecting polymorphism of the above SNP sites, screening of the GG type and the GA type of the Chr7:57740946G>A site, the TT type and the TG type of the Chr7:57741376T>G site, and individuals with the double type H1H1 (GGTT) and H1H2 (GATG), the early body weight gain of Hu sheep can be screened and molecular marker assisted selection breeding is carried out. The application provides an effective molecular marker for genetic improvement of early body weight gain of Hu sheep, and can significantly improve breeding efficiency, accelerate breeding process and reduce breeding cost.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and relates to the application of the complete linkage sites of the FGF7 gene molecular markers Chr7:57740946G>A and Chr7:57741376T>G in molecular marker-assisted breeding of Hu sheep. Background Technology

[0002] Fibroblast growth factors (FGFs) were originally named for the mitotic capacity of fibroblasts. FGFs and their receptors are highly conserved in the animal kingdom. The mammalian FGF family includes 23 polypeptides that regulate various cell migration, proliferation, differentiation, survival, metabolic activities, and neural functions.

[0003] The sheep FGF7 gene is located on chromosome 7 and has four exons and three introns. Studies have shown that FGF7 plays a regulatory role in lambing number, hair follicle formation, and organ growth. Some intronic regions of the FGF7 gene exhibit rich polymorphism, and this polymorphism is related to lambing number in Hu sheep. FGF7 gene polymorphism is also associated with the risk of chronic obstructive pulmonary disease and ovarian cancer in humans, the adaptability of Hu sheep to high altitudes, and carcass and growth traits in chickens.

[0004] This invention utilizes PCR pooling technology to screen for two SNP sites in the FGF7 3'UTR region: Chr7:57740946G>A and Chr7:57741376T>G. The correlation between these sites and early body weight gain in Hu sheep was analyzed, and molecular markers related to early body weight gain were screened out, providing a scientific basis for the application of molecular marker-assisted selection technology to breed new Hu sheep breeds. Summary of the Invention

[0005] The purpose of this invention is to provide an SNP molecular marker that affects early body weight gain in Hu sheep breeding in order to improve the accuracy of early body weight gain selection and accelerate genetic progress.

[0006] Another object of the present invention is to provide primer pairs for the above-mentioned SNP molecular markers.

[0007] Another object of the present invention is to provide applications of molecular markers and primer pairs.

[0008] Molecular markers associated with early body weight gain in lambs were selected from the following two groups, with complete linkage between the two loci:

[0009] (1) The SNP site in the 3'UTR region of FGF7 in the Oar_v4.0 genome of Hu sheep, the SNP site being Chr7:57740946G>A site, which corresponds to the G>A variant at position 57740946 in Genbank accession number NC_019461.2; individuals with the GG genotype and GA genotype at this site have significantly higher weaning weight and birth-to-weaning weight gain than those with the AA genotype;

[0010] (2) The SNP site in the 3'UTR region of FGF7 in the Oar_v4.0 genome of Hu sheep, the SNP site being Chr7:57741376T>G site, which corresponds to the T>G variant at position 57741376 in Genbank accession number NC_019461.2; individuals with the TT genotype and TG genotype at this site have significantly higher weaning weight and birth-to-weaning weight gain than those with the GG genotype.

[0011] The molecular marker is preferably the 788bp amplification product obtained by PCR amplification of Hu sheep genomic DNA using the primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2. It contains two completely linked sites in the 3'UTR region of the Hu sheep FGF7 gene: Chr7:57740946G>A and Chr7:57741376T>G. It has three genotypes: GGTT, GATG, and AAGG. Among them, the weaning weight and birth-to-weaning weight gain of Hu sheep of the GGTT and GATG types are significantly higher than those of the AAGG type.

[0012] A kit for detecting the molecular markers or combinations of the molecular markers associated with weaning weight and birth-to-weaning weight gain in Hu sheep, comprising the primer pairs described herein.

[0013] A method for detecting the molecular markers associated with weaning weight and birth-to-weaning weight gain in Hu sheep includes the following steps:

[0014] (1) Extracting genomic DNA from Hu sheep;

[0015] (2) Using the primer pair described above, the genomic DNA is amplified by PCR to obtain the amplification product;

[0016] (3) Detect the polymorphism of the amplification product at the SNP site to determine the genotype of the SNP site in claim 1.

[0017] The detection described in step (3) is selected from flight mass spectrometry.

[0018] A method for screening early-stage rapid weight gain Hu sheep breeds includes:

[0019] (1) Using the primer pair described in this invention, amplify the gene fragment in the genomic DNA of the Hu sheep to be tested that includes the two SNP sites;

[0020] (2) Detect the genotype of the SNP loci;

[0021] (3) Select individuals with Chr7:57740946G>A locus of GG and GA type, Chr7:57741376T>G locus of TT and TG type, or diploid type H1H1 (GGTT) or diploid type H1H2 (GATG) as breeding parents.

[0022] Preferably, the method for detecting the genotype of the SNP site in step (2) is selected from MassARRAY mass spectrometry.

[0023] The application of the molecular markers, the molecular marker combinations, or the primer pairs in the preparation of a kit for detecting early body weight gain in Hu sheep.

[0024] The application of the molecular markers, the molecular marker combinations, or the primer pairs in marker-assisted selection breeding of Hu sheep is preferably any one of the following:

[0025] (1) Used for the early body weight gain of Hu sheep;

[0026] (2) Used for early prediction of individuals with high weaning weight and high birth-to-weaning weight gain traits;

[0027] (3) Used for genetic improvement of Hu sheep breeds.

[0028] The application of the primer pairs described in this invention in the breeding of Hu sheep preferably utilizes the primer pairs to amplify the molecular markers described in this invention in the Hu sheep genome by PCR. The amplified products are then sequenced to determine that the Hu sheep FGF7 gene Chr7:57740946G>A has G / A polymorphism and Chr7:57741376T>G has T / G polymorphism. Individuals with Chr7:57740946G>A of GG and GA type, Chr7:57741376T>G of TT and TG type, and diploids of H1H1 (GGTT) or H1H2 (GATG) are selected as breeding parents.

[0029] A method for detecting the molecular markers described in this invention includes using the primer pairs to amplify the molecular markers in the genome of a Hu sheep by PCR, sequencing the amplified products, and determining that the FGF7 gene Chr7:57740946G>A has G / A polymorphism and Chr7:57741376T>G has T / G polymorphism.

[0030] A method for screening Hu sheep for early rapid body weight gain was developed. PCR amplification sequencing and MassARRAY mass spectrometry were used to detect the G / A polymorphism of the FGF7 gene Chr7:57740946G>A and the T / G polymorphism of Chr7:57741376T>G in Hu sheep, followed by population genetic analysis. Haplotype and diploid statistics and their association with early body weight gain were performed using the SHEsis online website. Complete linkage was found between the two loci. Individuals with diploid types H1H1 (GGTT) and H1H2 (GATG) were selected for breeding.

[0031] The method described in this invention includes the following steps:

[0032] (1) Extracting genomic DNA from Hu sheep;

[0033] (2) Using the primer pair described above, amplify a partial fragment of the 3'UTR region of the FGF7 gene;

[0034] (3) Genotyping was performed using Sanger sequencing. Chr7:57740946G>A has three genotypes: GG, GA, and AA; Chr7:57741376T>G has three genotypes: TT, TG, and GG.

[0035] (4) Population genotyping of the above loci was performed using MassARRAY technology;

[0036] (5) Haplotype and diploid analysis were performed using the SHEsis online website;

[0037] (6) Individuals of the GG and GA types at the Chr7:57740946G>A site, the TT and TG types at the Chr7:57741376T>G site, and the doublet H1H1 (GGTT) and H1H2 (GATG) types were selected for breeding.

[0038] Beneficial effects

[0039] Growth traits in livestock are important economic traits, belonging to quantitative traits. Traditional phenotypic selection has low accuracy, long cycles, and slow genetic progress. This invention uses a combination of PCR amplification and sequencing to detect the G / A polymorphism at the Chr7:57740946G>A site and the T / G polymorphism at the Chr7:57741376T>G site in the FGF7 gene of Hu sheep. Association analysis between genotype and early body weight gain in Hu sheep revealed complete linkage between the Chr7:57740946G>A site and the Chr7:57741376T>G site in the FGF7 gene of Hu sheep. Among the individuals with Chr7:57740946G>A, those with the GG and GA types had significantly higher weaning weight and birth-to-weaning weight gain than those with the AA type. Similarly, those with Chr7:57741376T>G had significantly higher weaning weight and birth-to-weaning weight gain than those with the TT and TG types. Two haplotypes and three diplotypes were detected in the experimental population for both SNPs. The dominant haplotype was H1 (GT), and the dominant diplotype was H1H1 (GGTT). Association analysis showed that among the three diplotypes, individuals with H1H1 (GGTT) and H1H2 (GATG) outperformed individuals with H2H2 (AAGG) in weaning weight and birth-to-weaning weight gain. This invention reveals that two SNP sites of the FGF7 gene are significantly associated with weaning weight and birth-to-weaning weight gain traits in Hu sheep. These sites can be used as molecular auxiliary markers for Hu sheep, improving the accuracy of selection. By detecting genotypes at birth, selection can be made, accelerating the breeding process and reducing breeding costs. This has important practical significance for further improving the early body weight gain of Hu sheep and has important practical value for the breeding of Hu sheep breeds. Attached Figure Description

[0041] Figure 1 The positions of the Chr7:57740946G>A and Chr7:57741376T>G sites in the amplification product of the FGF7 gene in Hu sheep;

[0042] Figure 2 The mass spectrometry scatter plot of the FGF7 gene Chr7:57740946G>A locus in Hu sheep shows three genotypes: GG, GA and AA.

[0043] Figure 3 The mass spectrometry scatter plot of the Chr7:57741376T>G site of the FGF7 gene in Hu sheep shows three genotypes: TT, TG, and GG. Detailed Implementation

[0045] Unless otherwise specified, the experimental methods mentioned in the following examples are conventional methods. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd., and Sanger sequencing was outsourced to Nanjing Qingke Biotechnology Co., Ltd.

[0046] Example 1: Selection method of molecular markers Chr7:57740946G>A and Chr7:57741376T>G for the FGF7 gene in Hu sheep.

[0047] (a) Sheep

[0048] The 384 Hu sheep samples were fed and managed uniformly by Jiangsu Qianbao Livestock Co., Ltd., with consistent nutrition and management standards. Their weight data were provided by Jiangsu Qianbao Livestock Co., Ltd.

[0049] (II) Methods

[0050] 1. Sample collection

[0051] Ear tissue samples were collected from each individual, placed in dry ice, and brought back to the laboratory for storage at -80°C.

[0052] 2. DNA extraction

[0053] DNA was extracted using the conventional phenol and chloroform method, and electrophoresis and OD were performed. 260 / 280 Detect DNA quality.

[0054] 3. Amplification of FGF7 gene loci Chr7:57740946G>A and Chr7:57741376T>G sequences

[0055] Based on the sequence NC_019461.2, a pair of specific primers were designed using Primer Premier5 (Table 1) to amplify the sequences of the Hu sheep FGF7 gene containing the sites Chr7:57740946G>A and Chr7:57741376T>G. The positions of these two SNP sites in each amplification product are shown below. Figure 1 As shown.

[0056] Table 1 Primers for SNP sites

[0057]

[0058] The total reaction volume for PCR amplification was 25 μL, including 15-200 ng template DNA, 12.5 μL TaqMix, 1 μL (10 μmol) each of forward and reverse primers, and ddH2O to 25 μL. The PCR amplification reaction program was as follows: 97℃ pre-denaturation for 5 min; 97℃ denaturation for 15 s, 60℃ annealing for 20 s, 72℃ extension for 30 s, for 35 cycles; and 72℃ extension for 10 min.

[0059] 4. Sequencing and typing of PCR amplification products

[0060] Sanger sequencing was performed on the 788bp amplified product using primers F1 and R1. The results showed that the Chr7:57740946G>A site had three genotypes: GG, GA, and AA; and the Chr7:57741376T>G site had three genotypes: TT, TG, and GG.

[0061] 5. Sequenom MassARRAY Flight Mass Spectrometry Genotyping

[0062] Table 2 Primers for Flight Mass Spectrometry

[0063]

[0064] DNA samples were extracted from the ear tissue of 384 Hu sheep and sent to Beijing Compson Biotechnology Co., Ltd. for testing. PCR amplification primers and single-base extension primers were designed using Compson's Assay Design 3.1 software (specific sequences are shown in Table 2). The core experimental steps are as follows: PCR amplification: The reaction system was 5 μL / well, containing 10 ng / μL DNA template, 0.5 μM Primer Mix, and accompanying buffer. PCR cycling parameters: 94°C for 120s pre-denaturation; 94°C for 20s, 56°C for 30s, 72°C for 60s, for a total of 45 cycles; final extension at 72°C for 180s. SAP digestion: 2 μL of digestion system (containing 1.7 U / μL SAP enzyme) was added to remove free dNTPs. The program was 37°C for 40 min, followed by 85°C for 5 min inactivation. Single-base extension (iPLEX reaction): Add 2 μL of reaction solution containing iPLEX buffer, terminator, enzyme, and extension primers. The reaction program is 94°C for 30 s; followed by 40 large cycles (94°C for 5 s, nested with 5 microcycles: 52°C for 5 s and 80°C for 5 s); finally, extend at 72°C for 180 s. Purification and mass spectrometry: After diluting the extension product 3-fold, desalt it using resin. The sample is then spotted onto a sample target for natural crystallization, and time-of-flight mass spectrometry is performed to collect data.

[0065] 6. Use the SHEsis online website to perform haplotype and diploid analysis.

[0066] 7. The significance of differences in early body weight among different genotypes of Hu sheep was analyzed using the one-way ANOVA method in SPSS.

[0067] (III) Results and Analysis

[0068] 1. FGF7 gene locus Chr7:57740946G>A and Chr7:57741376T>G polymorphism

[0069] The DNA products from mixed-breed sheep samples were amplified by PCR and sequenced using Sanger sequencing with primers (Table 1). Mutations G / A and T / G were found at two sites. Subsequently, 384 DNA samples were analyzed by flight-of-flight mass spectrometry, and the results are as follows: Figures 2-3 One sample from each of the two loci did not detect a genotype.

[0070] 2. Significant difference in early body weight among different genotypes of Hu sheep at the FGF7 gene locus Chr7:57740946G>A and Chr7:57741376T>G locus

[0071] One sheep at each of the two loci had no genotype detected, and two other sheep in the population had missing weight data. One-way ANOVA was used to compare the early body weight differences among sheep with different genotypes in the 381 sheep with both genotype and phenotype. The results showed that for Chr7:57740946G>A, weaning weight and birth-to-weaning weight gain were significantly higher in GG and GA individuals than in AA individuals (Table 3); for Chr7:57741376T>G, weaning weight and birth-to-weaning weight gain were significantly higher in TT and TG individuals than in GG individuals (Table 4).

[0072] Two SNPs of the FGF7 gene were detected in the experimental population, resulting in two haplotypes and three diploids (Table 5), with complete linkage between the two loci. The dominant haplotype was H1 (GT), and the dominant diploid was H1H2 (GATG). Association analysis between diploid type and early body weight gain was performed on 380 sheep possessing both the diploid and phenotype (Table 6). The results showed that the H1H1 (GGTT) and H1H2 (GATG) genotypes outperformed the H2H2 (AAGG) genotype in weaning weight and birth-to-weaning weight gain. These results indicate that the Chr7:57740946G>A and Chr7:57741376T>G loci of the FGF7 gene are completely linked. Individuals with the GGTT and GATG genotypes showed significantly higher weaning weight and birth-to-weaning weight gain than those with the AAGG genotype, making them suitable as preferred genotypes for molecular-assisted breeding.

[0073] Table 3. Effects of different genotypes at the FGF7 gene Chr7:57740946G>A locus on early body weight in Hu sheep.

[0074]

[0075] Note: Different letters in the same row's shoulder mark indicate significant differences. p <0.05), with the same letter or no letter indicating no significant difference, and the number of individuals in parentheses. The same applies below.

[0076] Table 4. Effects of different genotypes at the FGF7 gene Chr7:57741376T>G locus on early body weight in Hu sheep.

[0077]

[0078] Table 5. Haplotype and diploid analysis of SNPs in the FGF7 gene of Hu sheep.

[0079]

[0080] Note: The values ​​in parentheses are frequencies.

[0081] Table 6. Effects of different diploids of the FGF7 gene on early body weight in Hu sheep.

[0082]

[0083] A comprehensive analysis of the above results shows that the Chr7:57740946G>A and Chr7:57741376T>G sites of the FGF7 gene are completely linked. Individuals with the GG and GA morphs at the Chr7:57740946G>A site, the TT and TG morphs at the Chr7:57741376T>G site, and the diploid types H1H1 (GGTT) and H1H2 (GATG) exhibit advantages in early body weight gain and can be considered for breeding.

Claims

1. Molecular markers associated with early body weight gain in Hu sheep, characterized by: Selected from at least one of the following: (1) The SNP site in the 3'UTR region of the FGF7 gene in the Hu sheep genome, the SNP site being Chr7:57740946G>A site, which corresponds to the G>A variant at position 57740946 of the Genbank accession number NC_019461.2; at this site, the weaning weight and birth-to-weaning weight gain of individuals with the GG genotype and GA genotype were significantly higher than those of individuals with the AA genotype. (2) The SNP site in the 3'UTR region of the FGF7 gene in the Hu sheep genome, the SNP site being Chr7:57741376T>G site, corresponding to the T>G variant at position 57741376 of the Genbank accession number NC_019461.2; at this site, the weaning weight and birth-to-weaning weight gain of individuals with the TT genotype and TG genotype were significantly higher than those of individuals with the GG genotype.

2. A set of molecular markers associated with early body weight gain in Hu sheep, characterized in that, The SNP sites Chr7:57740946G>A and Chr7:57741376T>G in the 3'UTR region of the FGF7 gene as described in claim 1 are fully linked, forming three diploid types: GGTT, GATG, and AAGG. Individuals with the GGTT and GATG diploid types have significantly higher weaning weight and birth-to-weaning weight gain than individuals with the AAGG diploid type.

3. A primer pair for amplifying the molecular marker of claim 1, characterized in that, The primer pair simultaneously amplifies the Chr7:57740946G>A site and the Chr7:57741376T>G site, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. A kit for detecting the molecular marker of claim 1 or the combination of molecular markers of claim 2, characterized in that, It includes the primer pair as described in claim 3.

5. A method for detecting the molecular marker of claim 1 or the combination of molecular markers of claim 2, characterized in that, Includes the following steps: (1) Extracting genomic DNA from Hu sheep; (2) Using the primer pair described in claim 3, the genomic DNA is amplified by PCR to obtain the amplification product; (3) Detect the polymorphism of the amplification product at the SNP site to determine the genotype of the SNP site according to claim 1.

6. The method according to claim 5, characterized in that, The detection in step (3) is selected from sequencing, mass spectrometry, TaqMan probes, PCR-RFLP or high-resolution melting curve analysis.

7. A method for screening Hu sheep with rapid early body weight gain, characterized in that, include: (1) Using the primer pair described in claim 3, amplify the gene fragment containing the SNP site described in claim 1 in the genomic DNA of the sheep to be tested; (2) Detect the genotype of the SNP site as described in claim 1; (3) Select individuals with the GG or GA genotype at the Chr7:57740946G>A site, the TT or TG genotype at the Chr7:57741376T>G site, or the double genotype GGTT or GATG as breeding parents.

8. The method according to claim 7, characterized in that, The method for detecting the genotype of the SNP site described in claim 1 in step (2) is MassARRAY mass spectrometry.

9. The application of the molecular marker of claim 1, the combination of molecular markers of claim 2, or the primer pair of claim 3 in the preparation of a kit for detecting early body weight gain traits in Hu sheep.

10. The application of the molecular marker of claim 1, the molecular marker combination of claim 2, or the primer pair of claim 3 in marker-assisted selection breeding of Hu sheep, characterized in that, The applications include: (1) Used for the early body weight gain of Hu sheep; (2) Used for early prediction of Hu sheep individuals with excellent early body weight gain, wherein excellent early body weight gain includes high weaning weight and high birth to weaning weight gain; (3) Used for genetic improvement of Hu sheep breeds.

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