LALBA gene single nucleotide polymorphism genetic marker related to sheep lactation character and application of LALBA gene single nucleotide polymorphism genetic marker

Abstract

The invention discloses a sheep lactation character related LALBA gene single nucleotide polymorphism genetic marker and application thereof. The genetic marker is one or any combination of three single nucleotide polymorphism sites of c.-89 G / A, c.106 C / T and c.160-13 G / A of a sheep LALBA gene at a 5 'UTR, a first exon and a first intron respectively. Wherein c.-89 G / A and c.106 C / T influence the milk fat rate and the lactose rate, and c.160-13 G / A influences the conductivity and the pH value; the two single nucleotide polymorphic sites c.89 G / A and c.106 C / T provided by the invention are in a linkage imbalance state, and in a haplotype combination formed by reconstruction of the two single nucleotide polymorphic sites, the cream fat rate of a CCGG combination is relatively high, and the lactose rate of a TTAA combination is relatively high; the method can be applied to molecular breeding for improving the lactation character of sheep.

Description

Technical Field

[0001] This invention belongs to the field of genetic marker breeding technology related to sheep lactation, specifically relating to a genetic marker related to sheep lactation traits. LALBA Genetic markers of single nucleotide polymorphisms and their applications. Background Technology

[0002] Lactation performance is an important economic trait of ewes, mainly including indicators such as milk yield and milk composition. It directly determines the growth rate of lambs during the lactation period and the quantity and quality of sheep milk. Sheep milk is known as "golden milk," with milk protein, milk fat, and lactose content of 5.7%, 7.4%, and 4.8%, respectively, significantly higher than that of goat milk and cow milk.

[0003] Alpha-lactalbumin (LALBA) is the main component of whey protein, accounting for approximately 22% of milk protein. It is an important energy source for growth and development. As a bioactive protein, LALBA has diverse biological functions, providing the body with a rich supply of essential amino acids. LALBA protein also contains a high amount of tryptophan, which can be used as a nutritional supplement to improve nerve function and sleep. Furthermore, it acts as a regulatory component of the lactose synthase system in lactating mammary glands. LALBA The transcription of genes increases significantly during lactation and is associated with increased lactose production. Molecular genetic markers, which are specific mutation sites in DNA sequences, are used to track the transmission of genetic information and play an important role in modern molecular breeding. If key molecular genetic markers regulating lactation traits can be identified, marker-assisted selection technology can be used to achieve precise selection of traits such as milk yield and milk composition in ewes.

[0004] Studies have shown that, LALBA Single nucleotide polymorphisms (SNPs) have a significant impact on lactation traits in goats, cows, and horses, but different species or breeds exhibit distinctly different polymorphic characteristics. For example, current techniques in Sarda goats... LALBA Two SNP sites were detected in the gene promoter region, revealing higher lactose content in the TT and TC genotypes at c.-358 T>C and the GA and GG genotypes at c.-163 G>A (P<0.05); in Barki ewes, this was also observed. LALBA Ewes with the BB genotype have significantly higher fat content than those with the AA and CC genotypes, and their protein and lactose content is also higher than other genotypes. Therefore, it is difficult to determine the exact composition of ewes based solely on the BB genotype or breed. LALBASingle nucleotide polymorphisms (SNPs) of genes are used to infer or conduct breeding work on other species or breeds. Therefore, it is necessary to conduct research on SNPs related to lactation traits in different species or breeds to provide supporting data for molecular marker-assisted selection breeding of sheep, which will help improve the efficiency and accuracy of dairy sheep breeding. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method related to sheep lactation traits. LALBA Genetic markers of single nucleotide polymorphisms and their application in sheep lactation breeding.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A trait associated with sheep lactation LALBA A single nucleotide polymorphism (SNP) genetic marker, wherein the genetic marker is from sheep. LALBA The gene is located at one or any combination of the three single nucleotide polymorphism sites at c.-89 G / A, c.106 C / T and c.160-13 G / A in the 5' UTR, exon 1 and intron 1, respectively.

[0007] Furthermore, the sheep are East Florida sheep, Hu sheep, or their first or second generation hybrid sheep.

[0008] This invention analyzes LALBA The correlation between gene polymorphism and sheep lactation traits was investigated. Three single nucleotide polymorphism sites (SNPs) were found at the 5' UTR, exon 1, and intron 1 in sheep: c.-89 G / A, c.106 C / T, and c.160-13 G / A. These sites significantly affected multiple sheep lactation traits, including milk fat percentage, lactose percentage, milk protein percentage, nonfat milk solids content, conductivity, and pH, but had no significant effect on milk yield and dry matter content.

[0009] Therefore, this invention claims protection for the following applications: This invention provides the application of the above-mentioned single nucleotide polymorphism genetic markers in the selection and breeding of sheep to improve the lactation trait.

[0010] This invention provides a trait related to sheep lactation. LALBA The application of haplotype combinations of single nucleotide polymorphism (SNP) genetic markers in improving sheep lactation traits, wherein the haplotype combinations are haplotype combinations of the aforementioned SNP genetic markers associated with sheep lactation traits, and the haplotype combination sequences are CCGG, CTGA, or TTAA, and the SNP genetic markers are sheep... LALBA The gene sequence was found at single nucleotide polymorphism sites c.-89 G / A and c.106 C / T in 5' UTR and exon 1, respectively.

[0011] The present invention provides primers for amplifying the above-mentioned single nucleotide polymorphism genetic markers, the primers comprising a forward primer and a reverse primer, the forward primer and the reverse primer being located upstream and downstream of the above three single nucleotide polymorphism sites, respectively.

[0012] Furthermore, the sequences of the primers are shown in SEQ ID No: 1~4 in the sequence listing.

[0013] The present invention provides a kit for detecting the above-mentioned single nucleotide polymorphism genetic markers, the kit comprising the above-mentioned primers.

[0014] This invention provides the application of the above-mentioned kit in the breeding of sheep to improve the lactation trait.

[0015] This invention provides a trait related to sheep lactation. LALBA The detection method for single nucleotide polymorphism (SNP) genetic markers includes the following steps: S1. Extract sheep genomic DNA; S2. Amplify sheep genomic DNA using primers to obtain locus information for single nucleotide polymorphism genetic markers; The primers are as described above. LALBA The gene was designed at one or any combination of three single nucleotide polymorphism sites: c.-89 G / A, c.106 C / T, and c.160-13 G / A, in the 5' UTR, exon 1, and intron 1.

[0016] Furthermore, the primers are those described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a single nucleotide polymorphism genetic marker associated with the lactation trait in sheep, the genetic marker being located at LALBA Three single nucleotide polymorphism (SNP) sites were detected in the gene 5' UTR, exon 1, and intron 1: c.-89 G / A, c.106 C / T, and c.160-13 G / A. These three SNP sites are significantly associated with sheep lactation traits and can be applied to the selection of sheep lactation traits. The detection method is also provided.

[0018] (2) The present invention provides primers for amplifying the above-mentioned single nucleotide polymorphism genetic markers and kits containing the primers, which can be applied to the breeding of sheep lactation traits.

[0019] (3) This invention provides a haplotype combination sequence of single nucleotide polymorphism genetic markers related to sheep lactation traits. The linkage disequilibrium analysis of the two single nucleotide polymorphism sites provided by this invention is complete linkage. The haplotype combination formed by the reconstruction of the two single nucleotide polymorphism sites can be applied to improve the breeding of sheep lactation traits. Attached Figure Description

[0020] Figure 1 sheep LALBA SNP site screening sequencing map and genotyping results of genes; Figure 1 In the diagram, a is the sequencing data for the c.-89 G / A screening, b is its genotyping result; c is the sequencing data for the c.106 C / T screening, d is its genotyping result; e is the sequencing data for the c.160-13 G / A screening, f is its genotyping result.

[0021] Figure 2 sheep LALBA Gene linkage disequilibrium analysis. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0023] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0024] Example 1: Related to sheep lactation traits LALBA Gene sequencing and analysis of gene SNPs 1. Experimental Methods (1) Experimental sheep and sample collection Gansu Yuansheng Agricultural and Animal Husbandry Technology Co., Ltd. selected 437 sheep that were in the lactation period and were being raised uniformly, including 31 East Fuli sheep. East Friensian sheep ,EF), 52 lake sheep ( Hu sheep 193 F1 hybrid sheep (male) and 161 F2 hybrid sheep (male) were included in the study. Data on age, parity, number of lambs, and daily milk production were collected. During lactation (approximately 30 days after lambing), 1 mL of blood was collected from each sheep via the jugular vein method, dripped onto an FTA card (Whatman, Middlesex, UK), air-dried, and stored for subsequent genomic DNA extraction. Simultaneously, 50 mL of milk samples were collected, and seven milk component indicators, including milk fat percentage, milk protein percentage, lactose percentage, and conductivity, were measured using a UL40BC milk component analyzer (U-Creative, Hangzhou, China).

[0025] (2) Blood DNA extraction Blood DNA was extracted using a two-step method involving alkali treatment and TE solution treatment. The specific procedure was as follows: A 1.2 mm diameter disc was removed from an FTA card using a sampler. The sample was then transferred to a 1.5 mL centrifuge tube, and 200 μL of 20 mmol / L NaOH solution was added. The tube was incubated at 50°C for 25 min. After incubation, the NaOH solution was removed, and 200 μL of TE solution was added to the centrifuge tube. The tube was allowed to stand at room temperature for 2 min, followed by washing three times. Finally, the TE solution was aspirated, and the tube was left exposed to air dry for later use.

[0026] (3) Primer design According to the Ensemble database LALBA The gene sequence (accession number: ENSOARG00025051024) was used to design 5 pairs of primers using Primer 5.0 software for amplification. LALBA The 5' UTR region, part of the first intron, and the four exons of the gene. See Table 1 for specific primer information.

[0027] Table 1 Sheep LALBA Primer information for genes

[0028] (4) PCR amplification and mixed-pool sequencing Five samples were randomly selected from each of the four populations for PCR amplification to screen for SNPs. A 20 μL PCR reaction system was used, including one 1.2 mm DNA card, 0.8 μL each of 10 μmol / L forward and reverse primers, 10.4 μL of Taq DNA polymerase, and 8.0 μL of DEPC water (ddH2O). After PCR amplification, the amplified products were detected by 1.5% agarose gel electrophoresis. The qualified PCR products were then mixed and subjected to Sanger sequencing, and SNPs were screened using Chromas software.

[0029] (5) PARMS typing and haplotype construction PARMS technology was used to genotype the selected SNP loci. Popgene 32.0 software was used to calculate the genotype frequency, allele frequency, heterozygosity (He), and effective number of alleles (Ne) for each variant locus. PIC software was used to calculate the polymorphism information content (PIC). Haploview v4.2 software was used to construct haplotype combinations for the SNP loci and to statistically analyze haplotype frequencies.

[0030] 2. Experimental Results (1) SNP site screening and genotyping In sheep LALBA Three SNP sites were found in the gene ( Figure 1 The three variants are c.-89 G / A, c.106 C / T, and c.160-13 G / A, located in the 5' UTR, exon 1, and intron 1, respectively. No variant sites were found in other regions. The variants are present at both c.-89 G / A and c.160-13 G / A sites. A and G Two alleles, of which c.-89 G / A has AA , GA and GG There are 3 genotypes, while c.160-13 G / A only has 3. GA and GG Two genotypes. Present at the c.106 C / T locus. C and T Two alleles constitute CC , CT and TT Three genotypes.

[0031] Based on the three discovered SNPs, primers for amplifying these SNPs can be designed, including forward and reverse primers, located upstream and downstream of the three single nucleotide polymorphism sites, respectively. Kits for detecting these SNPs can also be designed using existing techniques based on these primers.

[0032] (2) Analysis of genetic polymorphism At the c.-89 G / A and c.106 C / T loci, the dominant genotypes and dominant alleles differed among the four populations (Table 2). Among the c.-89 G / A groups, the Hu sheep were... GG and G East Florida sheep are AA and A; The F1 population is GA and G ; The F2 population is GA and A . In c.106 C / T, for Hu sheep, it is CC and C ; for East Friesian sheep, it is TT and T ; The F1 population is CT and C ; The F2 population is CT and T . Chi-square test found that only the F2 population deviated from the Hardy-Weinberg equilibrium at 2 loci ( P <0.05), and other populations were in Hardy-Weinberg equilibrium ( P >0.05).

[0033] At the c.160-13 G / A locus, the dominant genotypes and dominant alleles of the 4 populations were both <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​LALBA The effects of genotype, allele, and haplotype combinations on the lactation trait in sheep were investigated using the model: Y = μ + Genotype (Allele or Haplotype) + Population + Age + e, where Y represents the lactation trait; μ is the population mean; Genotype represents the genotype; Allele represents the allele; Haplotype represents the haplotype; Population is the experimental population; Age represents age; and e is the random effect. Results are expressed as mean ± standard error.

[0038] 2. Experimental Results (1) LALBA Association analysis between genotype and sheep lactation traits As shown in Table 4, in c.-89 G / A, GG Individuals with this type of body have a significantly higher milk fat percentage than AA Type of individual ( P <0.05). AA Individuals with this condition had significantly higher lactose levels than those with the following condition. GA and GG Type of individual ( P <0.05), GA and GG There was no significant difference in lactose content among individuals of the three genotypes. None of the three genotypes had a significant effect on other lactation traits. In c.106 C / T, CC Individuals with this type of body have a significantly higher milk fat percentage than TT Type of individual ( P <0.05). TT Individuals with this condition had significantly higher lactose levels than those with the following condition. CT and CC Type of individual ( P <0.05). The three genotypes had no significant effect on other lactation traits. In c.160-13 G / A, GG The conductivity and pH of the individual were significantly higher than those of the previous type. GA Type of individual ( P <0.05); the two genotypes had no significant effect on the remaining lactation traits.

[0039] Table 4 LALBA Association analysis between genotype and sheep lactation traits

[0040] Note: Different lowercase letters in the same column indicate significant differences. P <0.05).

[0041] (2) LALBA Correlation analysis between alleles and sheep lactation traits c.-89 G / A alleleG The presence of [a specific substance] was positively correlated with milk fat percentage (present: 5.163±0.595; absent: 4.456±0.646). P =0.043), while the lactose rate (present: 5.360±0.020; missing: 5.391±0.022) ( P =0.010) and nonfat milk solids content (present: 11.734±0.220; missing: 11.998±0.239) P =0.049) showed a negative correlation. c.106 C / T allele C The presence of [a substance] significantly reduced the milk protein percentage (present: 5.419 ± 0.182; absent: 5.638 ± 0.198). P =0.043), lactose percentage (present: 5.360±0.020; missing: 5.391±0.022) P =0.011) and nonfat milk solids content (present: 11.731±0.220; missing: 11.998±0.239) P =0.041). c.160-13 G / A allele A The presence of this substance is associated with relatively low electrical conductivity (present: 2.414 ± 0.116; missing: 2.574 ± 0.110). P =0.016) and pH value (present: 6.759±0.031; missing: 6.803±0.029) P =0.011) Related (Table 5) Table 5 LALBA Correlation between alleles and sheep lactation traits

[0042] (3) LALBA Association analysis between haplotypes and sheep lactation traits LALBA The linkage disequilibrium analysis results of the three SNP sites in the gene are shown below. Figure 2 The results showed that c.-89 G / A and c.106 C / T were in a state of chain disequilibrium (r 2 =0.99), which can form a single haplotype module. c.160-13 G / A is not in a chain-linked unbalanced state with c.-89 G / A and c.106 C / T.

[0043] There were four haplotype combinations between c.-89 G / A and c.106 C / T. Since the frequency of the TTGA combination was less than 5%, no correlation analysis was performed. The milk fat percentage of the CCGG haplotype combination was significantly higher than that of the TTAA combination. P<0.05%. The lactose content of the TTAA haplotype combination was significantly higher than that of the CCGG and CTGA combinations ( P <0.05), there was no significant difference in lactose rate between the CCGG and CTGA haplotype combinations. The three haplotype combinations had no significant effect on other lactation traits (Table 6).

[0044] Table 6 LALBA Correlation analysis between genotype and sheep lactation traits

[0045] This study focuses on sheep. LALBA One SNP was found in each of the 5' UTR region, exon 1, and intron 1 of the gene. Among these, c.-89 G / A and c.106 C / T affected milk fat percentage and lactose percentage, while c.160-13 G / A was correlated with conductivity and pH. Haplotype analysis showed that the CCGG combination had a higher milk fat percentage, while the TTAA combination had a higher lactose percentage.

[0046] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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. A genetic single nucleotide polymorphism marker associated with a sheep lactation trait, wherein the marker is located within the BTAO 1 gene. LALBA 1. A genetic single nucleotide polymorphism marker associated with a sheep lactation trait, wherein the marker is located within the BTAO 1 gene. The genetic marker is a sheep LALBA The genes are at one or any combination of three single nucleotide polymorphism sites in the 5' UTR, exon 1 and intron 1 at c.-89 G / A, c.106 C / T and c.160-13 G / A, respectively.

2. A genetic single nucleotide polymorphism marker associated with a sheep lactation trait according to claim 1. LALBA A genetic single nucleotide polymorphism marker associated with a sheep lactation trait according to claim 1. The sheep is East Fleece sheep, Hu sheep or a crossbreed or second-generation sheep thereof.

3. Use of the single nucleotide polymorphism genetic marker in claim 1 in improving selection of a sheep lactation trait.

4. A haplotype combination sequence of genetic single nucleotide polymorphism markers associated with lactation traits in sheep. LALBA The application of haplotype combination sequence of genetic single nucleotide polymorphism markers associated with lactation traits in sheep for improving the selection of lactation traits in sheep, characterized in that, The haplotype combination is the haplotype combination of the single nucleotide polymorphism genetic markers related to the lactation traits of sheep in claim 1, the haplotype combination sequence is CCGG, CTGA or TTAA, and the single nucleotide polymorphism genetic markers are respectively the single nucleotide polymorphism genetic markers of sheep LALBA The gene sequence is the single nucleotide polymorphism site of c.-89 G / A and c.106 C / T in the 5' UTR and the 1st exon, respectively.

5. A primer for amplifying the single nucleotide polymorphism genetic marker of claim 1, wherein, The primers comprise a forward primer and a reverse primer, which are located upstream and downstream of the three single nucleotide polymorphism sites in claim 1 respectively.

6. The primer according to claim 5, characterized in that, The primers have sequences shown in SEQ ID No: 1-4 in the sequence listing.

7. A kit for detecting the single nucleotide polymorphism genetic marker of claim 1, wherein, The primers comprise a forward primer and a reverse primer, which are located upstream and downstream of the three single nucleotide polymorphism sites in claim 1 respectively.

8. Use of the primers in any one of claims 5 or 6 or the kit in claim 7 in improving selection of a sheep lactation trait.

9. A genetic single nucleotide polymorphism marker associated with a sheep lactation trait LALBA A method of detecting a genetic single nucleotide polymorphism marker associated with a sheep lactation trait, the method comprising, The method comprises the following steps: S1. extracting sheep genomic DNA; S2. amplifying the sheep genomic DNA by using the primers to obtain site information of the single nucleotide polymorphism genetic marker; The primers are designed for the gene of claim 1 LALBA The primers are designed for one or any combination of three single nucleotide polymorphism sites of the gene at 5' UTR, c.-89 G / A, c.106 C / T and c.160-13 G / A of the 1st exon and the 1st intron, respectively.

10. The detection method according to claim 9, characterized in that, The primers are the primers in any one of claims 5 or 6.

Images