SNP (Single Nucleotide Polymorphism) molecular marker related to reproductive traits of Songliao black pigs and application thereof

Abstract

The invention provides an SNP molecular marker related to reproductive traits of Songliao black pigs and application of the SNP molecular marker, and belongs to the technical field of biomolecular markers. The SNP molecular marker is located on the fifth intron and the eighth exon of an LHX8 gene, the nucleotide sequences of the SNP molecular marker are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and Tgt exists at the 28th basic group of the fifth intron; c mutation, which is divided into three genotypes of TT, TC and CC; ggt exists at the 231st basic group of the 8th exon; a mutation is carried out, and the gene is divided into three genotypes of GG, GA and AA. The polymorphism of the T28C site is related to birth weight and the number of weaned piglets, and the weaned number of piglets produced by GG individuals at the G231A site is higher than that of GA and AA individuals. The breeding traits of the Songliao black pigs can be accurately identified, and a new molecular marker is provided for molecular marker-assisted selective breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of biomolecular markers, and in particular to SNP molecular markers related to the reproductive traits of Songliao black pigs and applications thereof. Background Art

[0002] Molecular markers are genetic markers based on nucleotide sequence variation within genetic material between individuals, and are a direct reflection of genetic polymorphism at the DNA level. Molecular markers reveal variations from DNA; they affect the expression of target traits, are linked to biological traits, and have simple and rapid detection methods. With the development of molecular biology technology, there are dozens of DNA molecular marker technologies, which are widely used in genetic breeding, genome mapping, gene positioning, species kinship identification, gene bank construction, gene cloning, etc. Single nucleotide polymorphism (SNP) markers are the phenomenon that there are differences in single nucleotides in the allele sequence of genomic DNA of different individuals of the same species. It specifically refers to DNA sequence polymorphism caused by single nucleotide variation in the genome, including base conversion, transversion, single base insertion or deletion, etc., and is recognized as the latest third-generation DNA molecular marker.

[0003] The pig industry occupies an important position in my country's animal husbandry, and the price of pork is closely related to the consumer price index. At present, the main supplier of pork in the market is the imported white pig breed. Songliao black pig, as a lean black pig breed in the north, has excellent production performance, but its reproductive performance is no longer superior to that of white pig breeds. Therefore, it is of great significance to select Songliao black pigs with excellent reproductive performance. The invention patent with application number "201610543064.5" and invention name "SNP in the promoter region of pig NTF3 as a molecular marker and application of boar reproductive traits" discloses molecular markers related to boar reproductive traits, but there is no report on molecular markers for the reproductive performance of Songliao black pig sows.

[0004] The transcriptional regulator LIM homebox protein 8 (LHX8) gene is a member of the homeobox gene family, located on chromosome 6 of pigs, with two conserved domains, and is involved in physiological processes such as cell differentiation and organ development. Currently, there is a lack of reports on the correlation between the polymorphism of this gene and the reproductive performance of Songliao black pigs. Summary of the invention

[0005] The purpose of the present invention is to provide SNP molecular markers related to the reproductive traits of Songliao black pigs and their applications, to solve the problem of low reproductive performance of Songliao black pigs and to provide technical support for the breeding of Songliao black pigs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a SNP molecular marker, and the molecular marker is shown in the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. There is a T>C mutation at the 28th base of the nucleotide sequence shown in SEQ ID NO.1, which is divided into three genotypes: TT, TC and CC; there is a G>A mutation at the 231st base of the nucleotide sequence shown in SEQ ID NO.2, which is divided into three genotypes: GG, GA and AA.

[0008] The present invention also provides the use of the molecular marker in any of the following:

[0009] (1) Application in the selection of birth weight and number of weaned piglets produced by Songliao black pig sows;

[0010] (2) Application in Songliao black pig breeding.

[0011] The present invention also provides a primer combination for detecting the molecular marker according to claim 1, comprising a primer pair LHX8-45F / R and a primer pair LHX8-8F / R, and the nucleotide sequences thereof are shown in SEQ ID NOs. 3 to 6, respectively.

[0012] The present invention also provides a reagent or a kit containing the primer combination.

[0013] The present invention also provides a method for identifying the reproductive traits of Songliao black pig sows, comprising the following steps:

[0014] S1. Extracting genomic DNA from the Songliao black pig sow to be identified, and performing PCR amplification using primer pair LHX8-45F / R and primer pair LHX8-8F / R respectively;

[0015] S2. Sequence the amplified products, detect the genotype of the SNP locus, and identify the reproductive traits of Songliao black pig sows.

[0016] Preferably, the reaction system for PCR amplification is: 0.5 μL of upstream and downstream primers, 0.5 μL of DNA template, 10 μL of 2×Premix buffer, and water is added to make up to 20 μL.

[0017] Preferably,

[0018] Preferably, the reaction program of the PCR amplification is: 95°C for 5 min, 95°C for 30 s, 58°C for 30 s, 72°C for 120 s, 30 cycles, 72°C for 5 min.

[0019] Preferably, the concentrations of the upstream and downstream primers are 10 μM respectively, and the concentration of the DNA template is 50 ng / μL.

[0020] Preferably, the judging criteria for the reproductive traits of Songliao black pig sows are as follows: if the 28th base position of intron 5 is TC genotype, the piglets produced by Songliao black pig sows have a large birth weight; if the 231st base position of exon 8 is GG genotype, the number of weaned piglets of Songliao black pig sows is large.

[0021] By adopting the above technical scheme, the present invention has the following beneficial effects: the SNP molecular marker of the present invention is located on the 5th intron and the 8th exon of the LHX8 gene, and its nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. There is a T>C mutation at the 28th base of the 5th intron, which is divided into three genotypes: TT, TC and CC; there is a G>A mutation at the 231st base of the 8th exon, which is divided into three genotypes: GG, GA and AA. The genomic DNA of Songliao black pigs was detected using the molecular markers, and it was found that the above two SNP sites were significantly correlated with the reproductive traits of Songliao black pigs. The present invention can accurately identify the reproductive traits of Songliao black pigs and provide new molecular markers for molecular marker-assisted selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 1 is the target fragment amplified by primer pair LHX8-45 F / R and primer pair LHX8 F / R, M, Marker5000; 1, 2, 3 are the amplification products of primer LHX8-45; 4, 5, 6 are the PCR amplification products of primer LHX8-8.

[0023] Figure 2 This is the sequencing diagram of different genotypes of the LHX8 gene SNPs site. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1. Acquisition of Songliao Black Pig LHX8 Gene and SNPs Detection

[0026] The molecular markers related to the reproductive traits of Songliao black pigs of the present invention are shown in SEQ ID NO.1 and SEQ ID NO.2. SEQ ID NO.1 is located in the 5th intron of LHX8 gene, and SEQ ID NO.2 is located in the 8th exon of LHX8 gene.

[0027] 120 healthy Songliao black sows collected from the original pig farm of Jilin Academy of Agricultural Sciences were selected as the research subjects. Ear tissues the size of soybeans were collected using ear tag forceps, and the surface blood stains were cleaned with physiological saline and placed in tissue preservation solution and stored at 4°C. The ear tissues were first cut into pieces with sterile ophthalmic scissors, homogenized, and then genomic DNA was extracted using a DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP304). The concentration and purity of the DNA solution were determined according to the instructions, and the DNA concentration was adjusted to 50 ng / μL and stored at -20°C for subsequent PCR.

[0028] Primers were designed based on the sequence of porcine LHX8 in Ensemble (Ensembl number: ENSSSCT00000004181.5), and sequence synthesis was performed by Suzhou Genewise Biotechnology Co., Ltd. The sequence information is shown in Table 1.

[0029] Table 1 Primer information

[0030]

[0031] 60 sample DNAs were randomly selected, and 5 μL of each sample was selected to form a DNA pool sample. The sample was used as a template and PCR amplification was performed using the primers listed in Table 2. The reaction system was: 0.5 μL of each upstream and downstream primer (10 μM), 0.5 μL of DNA template (50 ng / μL), 10 μL of 2xPremixbuffer, and water was added to 20 μL; the reaction procedure was: pre-denaturation: 95°C for 5 min, denaturation: 95°C for 30 s, annealing: 58°C for 30 s, extension: 72°C for 120 s, 30 cycles, 72°C for 5 min, and cooling to 4 degrees Celsius for subsequent operations. After obtaining the target fragment, electrophoresis was performed using 1% agarose gel, and the pressure was stabilized at 100

[0032] V, electrophoresis for 20 min, obtain a single-band PCR product, such as Figure 1 Direct sequencing to screen SNP sites.

[0033] SeqMan in DNASTAR was used to compare the Ensemble database sequence with the sequenced nucleotide sequence to find the mutation site in the target fragment and record the mutation type of the corresponding site (T28C, G231A) in each individual. The SeqMan comparison results are as follows: Figure 2 shown. Figure 2 The results showed that there was one SNP site (T28C, G231A) in intron 5 and one SNP site (G231A) in exon 8, and there were three genotypes: natural homozygous, heterozygous and mutant homozygous.

[0034] The nucleotide sequences and mutation sites of the 5th intron and 8th exon of the LHX8 gene amplified by the primer pair LHX8-45F / R and the primer pair LHX8F / R are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. Lowercase letters represent intron regions, uppercase letters represent exon regions, underlined sequences are positions that bind to primers, and bordered sites are SNP sites.

[0035] SEQ ID NO.1:

[0036]

[0037]

[0038] SEQ ID NO.2:

[0039]

[0040] Then, PCR amplification of the polymorphic fragments was performed on 120 samples using LHX8-45F / R and primer pair LHX8F / R as templates. The amplification system and amplification procedure were the same as above. The gene and genotype frequencies of the two SNP sites of the LHX8 gene in the 120 Songliao black pigs tested are shown in Table 2.

[0041] Table 2 Analysis of genotype frequency and gene frequency

[0042]

[0043]

[0044] df = 2, χ 2 (0.05) = 5.99, χ 2 (0.01) = 9.21

[0045] The results of genotype frequency and gene frequency analysis showed that CC and GA were dominant alleles, and C and G were dominant alleles. The chi-square test showed that the T28C locus was in a Hartmann-Wenzhou equilibrium state (P>0.05), while the G231A locus deviated from this equilibrium state (P<0.05).

[0046] The population genetics indicators were statistically analyzed using Excel, and the results are shown in Table 3.

[0047] Table 3 Genetic diversity of SNPs

[0048]

[0049] The genetic diversity analysis of SNPs is used to analyze the proportion of homozygosity and heterozygosity at the site, and the state of polymorphism. The population diversity analysis results in Table 4 show that the homozygosity of the two SNP sites is high, and both show moderate polymorphism (0.25<PIC<0.5).

[0050] Example 2. Application of molecular markers in identifying reproductive traits of Songliao black pigs

[0051] In order to establish the relationship between the LHX8 gene and the reproductive traits of Songliao black pigs, 120 Songliao black pigs collected in Example 1 were used as experimental materials, and the experimental population was tested using the SNPs detection method mentioned in Example 1. The association analysis between the SNP loci and the reproductive trait indicators was analyzed using SPSS, and the results are shown in Table 4.

[0052] Table 4 Correlation between SNPs and reproductive traits

[0053]

[0054]

[0055] In terms of sow reproductive performance, quantity-related indicators include the number of piglets born alive, and quality-related indicators mainly include the pass rate and whether weaning is qualified. Weaning weight is an important measurement indicator.

[0056] As shown in Table 4, in the 120 Songliao black pig populations, the SNP site T28C was found in the 5th intron of the LHX8 gene. The polymorphism of the T28C site was associated with birth weight and the number of weaned piglets. The birth weight of piglets produced by TC genotype individuals was advantageous, but the number of weaned piglets was lower than that of TT and CC genotype individuals. The number of live piglets and weaned piglets produced by TT sows were higher than those of TT and CC genotype individuals, which increased the amount of meat provided by them annually. Considering all factors in a balanced way, although the birth weight of individuals produced by TT sows was smaller, their growth during lactation compensated and they were no longer at a disadvantage at weaning. Therefore, TT sows should be selected for the production of commercial pigs. The G231A site in the 8th exon was associated with reproductive traits. The number of weaned piglets produced by GG individuals was higher than that of GA and AA individuals. Therefore, GG individuals were given priority for commercial pig production.

[0057] It can be seen from the above embodiments that the present invention provides SNP molecular markers related to the reproductive traits of Songliao black pigs and applications thereof. The molecular markers of the present invention can be used to identify the reproductive traits of Songliao black pigs.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SNP molecular marker, characterized in that: The molecular marker is shown in the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.

2. There is a T>C mutation at the 28th base of the 5th intron, which is divided into three genotypes: TT, TC and CC; there is a G>A mutation at the 231st base of the 8th exon, which is divided into three genotypes: GG, GA and AA.

2. Use of the molecular marker according to claim 1 in any of the following: (1) Application in the selection of birth weight and number of weaned piglets produced by Songliao black pig sows; (2) Application in Songliao black pig breeding.

3. A primer combination for detecting the molecular marker according to claim 1, characterized in that: It comprises a primer pair LHX8-45F / R and a primer pair LHX8-8F / R, and the nucleotide sequences thereof are shown in SEQ ID NOs. 3 to 6 respectively.

4. A reagent or kit containing the primer combination described in claim 3.

5. A method for identifying the reproductive traits of Songliao black pig sows, characterized in that: The following steps are involved: S1. Extracting genomic DNA from the Songliao black pig sow to be identified, and performing PCR amplification using primer pair LHX8-45F / R and primer pair LHX8-8F / R respectively; S2. Sequence the amplified products, detect the genotype of the SNP locus, and identify the reproductive traits of Songliao black pig sows.

6. The method according to claim 5, characterized in that The reaction system of the PCR amplification is: 0.5 μL of upstream and downstream primers, 0.5 μL of DNA template, 10 μL of 2×Premix buffer, and water is added to make up to 20 μL.

7. The method according to claim 5, characterized in that The reaction program of the PCR amplification is: 95°C for 5 min, 95°C for 30 s, 58°C for 30 s, 72°C for 120 s, 30 cycles, 72°C for 5 min.

8. The method according to claim 6, characterized in that The concentrations of the upstream and downstream primers were 10 μM respectively, and the concentration of the DNA template was 50 ng / μL.

9. The method according to claim 5, characterized in that The criteria for judging the reproductive traits of Songliao black pig sows are as follows: if the 28th base of intron 5 is TC genotype, the piglets produced by Songliao black pig sows will have a large birth weight; if the 231st base of exon 8 is GG genotype, the number of weaned piglets of Songliao black pig sows will be large.

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