Application of a MAP10 gene mutation in the selection of lambing number in Macheng black goats

Abstract

This invention discloses a SNP marker for the MAP10 gene that affects the number of lambs born in goats and its application. The marker is located in the MAP10 gene on chromosome 28 of goats, specifically at the C / A base mutation at 277 bp of SEQ ID NO:1 in the sequence listing. At this site, goats with the AA and CA genotypes have significantly higher lambing numbers than those with the CC genotype. Using Macheng Black Goats as the research subject, this invention utilizes PCR to amplify the DNA sequence of exon 1 of the goat MAP10 gene. This sequence contains a SNP mutation site. The resulting amino acid changes are analyzed, and the effect of the specific genotype at this site on the number of lambs born in Macheng Black Goats is analyzed. Based on this analysis, Macheng Black Goat individuals can be selected for breeding, which can be used to increase the number of lambs born in Macheng Black Goats and provides marker resources for marker-assisted selection breeding of goat lambing traits.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding technology and relates to the application of SNP markers related to the lambing number trait of Macheng Black Goats in the breeding of Macheng Black Goats. Background Technology

[0002] Reproductive traits are crucial economic indicators in goat farming, and litter size is a vital reproductive indicator that directly impacts the economic benefits of the industry. Therefore, increasing litter size is a key research direction in goat breeding. However, litter size is a low-heritability trait, and genetic progress achieved through traditional selection methods is slow and inconsistent. With the development of molecular biology techniques, high-throughput sequencing, genome selection, molecular markers, and genetic modification have become increasingly sophisticated. More and more candidate genes related to high litter size in goats are being discovered and identified, and applied to the improvement of local goat breeds and the development of new varieties. This invention uses whole-genome resequencing technology to screen for genes affecting litter size in Macheng Black Goats, discovering that a single base mutation in the MAP10 gene is significantly correlated with litter size.

[0003] MAPs (MT-associated proteins) are proteins that bind to microtubules and stabilize their structure, playing crucial roles in spindle assembly during mitosis and meiosis, neuronal development, and axon formation. In mammals, the MAP family includes MAP1, MAP2, MAP4, MAP5, MAP7, MAP8, MAP9, MAP10, MAP11, and MAPT, which are involved in nervous system development and cellular material transport. Overexpression and hyperphosphorylation of the MAPT gene inhibit the transport of synaptic vesicles and organelles in vivo; mice lacking MAPT protein exhibit severe neurodegenerative diseases at 12 months of age. Purkinje cells in mice that do not express the MAP1 gene undergo degeneration, and MAP2 or MAP5 gene knockout mice show defective neurodevelopment. In octopus melanocytes, MAP4 specifically inhibits dynein-driven transport and activates kinin 2-driven transport. MAP2 participates in material sorting in the pre-axonal region, and MAP7 regulates the transport of intracellular organelles and macromolecules by activating kinin.

[0004] The MAPs family is involved in the reproduction of individual animals. MAP9 is involved in the formation of the spindle during mitosis; inhibiting MAP9 gene expression blocks zebrafish development, leading to embryonic death or severe developmental defects. It is an essential protein in early zebrafish development; the MAP9 gene is also a gene affecting the reproductive traits of Beijing Black Pigs. MAP7 and its related proteins interact with DNA repair proteins, promoting the repair of DNA double-strand breaks during the G1 cell cycle. MAP1 is downregulated in epithelial ovarian cancer, affecting tumor tissue by influencing autophagy. MAP10 promotes microtubule stability and participates in cell division. This invention, through whole-genome resequencing and association analysis, discovered a novel mutation site in the MAP10 gene, which is significantly correlated with litter size in goats, providing marker resources for marker-assisted selection breeding of litter size in Macheng Black Goats. Summary of the Invention

[0005] This invention provides a solution to the technical problems existing in the current breeding process for goat litter size.

[0006] This invention provides an SNP marker that affects the number of lambs born in goats. The marker corresponds to the coding region of the goat MAP10 gene (sequence number: ENSCHIG00000005553) published in the Ensembl database. The difference in the C / A ratio of nucleotides at this marker site leads to different numbers of lambs born in goats.

[0007] This invention provides a method for selecting goats for breeding based on a marker genotype. According to an embodiment of the invention, the SNP marker is located at the 277th base of the nucleotide sequence shown in SEQ ID NO:1. Goats with the AA and CA genotypes at this SNP marker site have significantly higher lambing numbers than those with the CC genotype. By detecting the aforementioned SNP marker in Macheng Black Goats, their lambing number can be effectively predicted, thereby allowing for the assessment of lambing performance based on the genotype at this SNP marker site. Therefore, the SNP marker of this invention is closely related to the lambing number trait of Macheng Black Goats and can be effectively used for marker-assisted selection breeding of Macheng Black Goats. This allows for the selection of goat breeding stock according to actual breeding needs, enabling accurate and efficient selection of superior individuals with high lambing numbers, thus improving the efficiency and accuracy of breeding selection.

[0008] This invention provides a primer pair for detecting the SNP marker described in claim 1. According to an embodiment of the invention, the primers have the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3, and are used to detect the SNP marker. According to an embodiment of the invention, the primer pair can effectively amplify the fragment containing the SNP marker related to the litter size trait in the tested goat by PCR, and sequencing can effectively detect this SNP marker, determine the genotype of the tested goat at the SNP marker site, and thus effectively predict the litter size of the tested goat.

[0009] Specifically, individuals with the AA and CA genotypes at the SNP marker locus had significantly higher litter sizes than those with the CC genotype, indicating that the AA genotype at this SNP locus can serve as an important criterion for determining the number of lambs produced in goats. In goat breeding, the genome sequence of individual goats is amplified and sequenced using the primers for the SNP marker described in this invention to determine their genotype. Individuals with the AA genotype at the SNP marker locus can be retained for breeding, or individuals with the AA genotype can be mated with individuals with the CA genotype. In the hybrid offspring, individuals with the AA genotype are selected for breeding, while individuals with the CC genotype at the SNP marker locus are culled. This gradually increases the number of lambs produced in the goat population, enabling the low-cost and high-accuracy selection of goat individuals with high litter sizes, thus achieving molecular marker-assisted breeding based on the number of lambs produced in goats.

[0010] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly correlated with the number of lambs born in Macheng Black Goats. The number of lambs born in Macheng Black Goats with genotypes AA and CA is significantly higher than that of individuals with genotype CC. (2) The SNP marker can be used for auxiliary selection of the lambing trait in Macheng Black Goats, screening for Macheng Black Goats with high lambing numbers. It has important practical application value for further improving the reproductive capacity of Macheng Black Goats and for breeding (or strain) using specific Macheng Black Goats as material. Attached Figure Description

[0011] The description of the embodiments will be more readily understood from the above aspects of the invention in conjunction with the accompanying drawings. Figure 1 The sequencing peak diagrams of the CC, CA, and AA genotypes of the SNP marker sites of this invention are shown. Specific Implementation

[0012] The embodiments of the present invention are described in detail below. The present invention will be further described in detail with reference to the embodiments. The embodiments are only used to illustrate the present invention and should not be construed as limiting the present invention.

[0013] 1. Experimental Samples

[0014] The 147 adult Macheng black goat ewes of Hubei Jinyang (Macheng) Livestock Co., Ltd., which have records of one to five litters, are kept under the same feeding, management and environmental conditions.

[0015] 2. Genomic DNA extraction

[0016] Blood from the jugular vein of the above-mentioned sample (5 mL / animal) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulation). Genomic DNA was extracted from the blood sample of Macheng black goat using the blood genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0017] 3. Primer design

[0018] Based on the goat MAP10 gene sequence (Ensembl database gene sequence number: ENSCHIG00000005553), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using Primer 5.0 software. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. These specific primers were used to amplify a DNA sequence containing the mutation site in the first exon of the MAP10 gene. The amplification product was 913p, and the nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0019] 4. PCR amplification of the MAP10 gene target sequence in the sample DNA and determination of genotype.

[0020] (1) PCR amplification system (25 μL): DNA 1 μL, primers SEQ ID NO:2 and SEQ ID NO:3 (10 μM) 1 μL each, 2×RapidTaq Master Mix 12.5 μL (Nanjing Novizan Biotechnology Co., Ltd.), ddH2O 9.5 μL. Amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 54℃ annealing for 15 s, 72℃ extension for 14 s, 35 cycles; 72℃ extension for 5 min.

[0021] (2) The PCR amplification products were sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 277bp site of the nucleotide sequence shown in SEQ ID NO:1, as shown in the attached figure. Figure 1 As shown, the genotype of unimodal C is CC, the genotype of unimodal A is AA, and the genotype of bimodal is CA.

[0022] 5. Variations in the MAP10 gene sequence of Macheng black goats

[0023] In the sequence listing, the base C of the SNP marker site at 277bp of SEQ ID NO:1 is mutated to A, changing the codon encoding alanine (Ala) to the codon encoding aspartic acid (Asp).

[0024] 6. Association analysis of MAP10 gene SNP markers and lambing number trait in Macheng black goats

[0025] One-way ANOVA was used in SPSS software to conduct an association analysis between genotype and litter size. The specific linear analysis model was: Yij = μ + Gi + Eij

[0026] Where: Yij represents individual phenotypic records; μ represents the population mean; Gi represents the genotypic effect at each point; and Eij represents random error.

[0027] 7. Significant difference in litter size among different genotypes of Macheng black goats

[0028] The results of the litter size analysis of different genotypes in Macheng Black Goats are shown in Table 1. Table 1 shows that there are three genotypes at this locus. One-way ANOVA comparing the differences in litter size among different genotypes revealed that the litter size of Macheng Black Goats with genotypes AA and CA was significantly higher than that with genotype CC (p<0.05). This indicates that the AA and CA genotypes at this SNP marker locus can serve as important criteria for judging high litter size in Macheng Black Goats. In the breeding of Macheng Black Goats, individuals with the AA genotype at this SNP marker locus can be retained for breeding, while individuals with the CC genotype can be culled. Alternatively, individuals with the CA genotype can be mated with individuals with the AA genotype to obtain more AA-type offspring, thereby gradually increasing the litter size of the Macheng Black Goat population.

[0029] Table 1. Correlation between different genotypes of the MAP10 gene mutation site in Macheng black goats and the number of lambs born.

[0030]

[0031] Note: Different shoulder letters in the same column indicate significant differences (p<0.05).

Claims

1. A method for selecting high-producing goats based on molecular markers associated with the trait of goat litter size, wherein the molecular marker exhibits a C / A polymorphism at position 277 of the nucleotide sequence SEQ ID NO:1, wherein goat individuals with the AA and CA genotypes at position 277 of the sequence have significantly higher litter sizes than individuals with the CC genotype; The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 913bp amplification product. The two specific primer sequences are SEQ ID NO:2 and SEQ ID NO:

3. (3) Sequencing the PCR amplification products to obtain sequencing results; (4) Determine the genotype of the goat individual to be tested based on the sequencing results; (5) Select goat individuals with the AA genotype of the above molecular markers for breeding; The goat in question is the Macheng Black Goat.

2. The application of the molecular marker described in claim 1 in screening Macheng Black Goats with high lambing rates, wherein individuals with the AA genotype of the molecular marker are selected for breeding.

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