SNP (Single Nucleotide Polymorphism) molecular marker related to Ningxiang pig body length character and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a Ningxiang pig body length character and an application of the SNP molecular marker. The molecular marker comprises an SNP1 and an SNP2; the SNP1 molecular marker takes a Sus Scrofa Build11.1 genome as a reference genome and is located at the 15776004 site of a No.1 chromosome, the polymorphism of the SNP1 molecular marker is T / C, and the serial number of the SNP1 molecular marker is rs334683505; the SNP2 molecular marker takes a Sus Scrofa Build11.1 genome as a reference genome and is located at the 15832958th site of a No.1 chromosome, the polymorphism of the SNP2 molecular marker is A / C, and the serial number of the SNP2 molecular marker is rs321288338. The molecular marker obviously related to the Ningxiang pig body length character is obtained, and an amplicon sequencing primer group capable of being used for identification analysis and large-scale detection is designed. The Ningxiang pig individuals with the BL sizes meeting the requirements can be efficiently and accurately identified and screened out.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a SNP molecular marker related to the body length trait of Ningxiang pigs and its application. BACKGROUND

[0002] Body length (BL) is one of the core indicators of pig body structure traits, referring to the horizontal straight-line distance from the occipital crest (the back of the skull) to the tail root. It is an objectively measurable body size trait, which, together with body height and chest circumference, reflects the physical structure and bone development level of pigs. Body length is a typical polygenic trait controlled by multiple genes, and its phenotype is influenced by both genetic and environmental factors. The heritability of body length is medium to high (0.34-0.41), higher than that of reproductive traits (such as litter size), and can be effectively improved through individual selection.

[0003] Its value in pig breeding lies in several aspects: first, in improving meat production efficiency: body length is significantly positively correlated with carcass length and dressing percentage, and each 1 cm increase in body length can increase lean meat yield by about 0.3-0.5 kg, directly increasing economic benefits; second, in reducing the rate of elimination: the proportion of sow elimination due to poor limb and hoof structure is 11% (second only to reproductive problems), and selecting pigs with moderate body length and uniform structure can extend the service life by 1-2 litters.

[0004] Single nucleotide polymorphism (SNP) has the characteristics of high density and stable inheritance, and has become the core tool of molecular marker-assisted selection (MAS). Early selection using SNP markers (such as eliminating individuals with unfavorable genotypes) can shorten the generation interval and improve the genetic progress of body length traits.

[0005] In summary, body length, as a key body size trait in pig breeding, has high heritability, economic value in meat production, and an indication of robustness. By incorporating it into the comprehensive breeding goal and combining it with SNP molecular marker breeding selection technology, the lifetime production efficiency of breeding pigs can be significantly improved, and scientific basis for balancing meat quality, reproduction, and other traits can be provided. In the future, standardized determination and cross-trait genetic mechanism research need to be promoted to achieve more precise genetic improvement. However, although there are related patents that combine SNP technology for non-Ningxiang pigs in the existing technology, the development of molecular markers for the key body structure trait of body length is still lacking. In particular, there have been no reports on SNP markers related to body length for Chinese local pig breeds (such as Ningxiang pigs). The molecular markers used in the existing technology are mostly based on simplified genome sequencing or low-density chip typing, which have the problems of low marker density and limited positioning accuracy, making it difficult to achieve precise genotype-phenotype correlation. In addition, existing patents and technologies are mostly focused on foreign commercial pig breeds (such as Landrace and Large White), which have significant differences in genetic background from Chinese local pig breeds, and direct application may result in insufficient or ineffective marker effects. SUMMARY

[0006] In order to solve the above problems in the prior art, the present application aims to provide a SNP molecular marker related to the body length trait of Ningxiang pigs and an application thereof.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The SNP molecular marker related to the body length trait of Ningxiang pigs comprises SNP1 and SNP2; the molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker is located at the 15776004th position of chromosome 1 with Sus Scrofa Build11.1 genome as a reference genome, and the polymorphism thereof is T / C; the SNP1 molecular marker is numbered as rs334683505; the SNP2 molecular marker is located at the 15832958th position of chromosome 1 with Sus Scrofa Build11.1 genome as a reference genome, and the polymorphism thereof is A / C; the SNP2 molecular marker is numbered as rs321288338.

[0008] The present application also provides a gene comprising the above-mentioned SNP1 molecular marker or SNP2 molecular marker related to the body length trait of Ningxiang pigs, wherein the gene comprises the nucleotide sequence of 1000 bp before and after the SNP1 molecular marker site or the SNP2 molecular marker site, specifically, the nucleotide sequence of 1000 bp before and after the SNP1 molecular marker site is shown in SEQ ID No. 1, and the nucleotide sequence of 1000 bp before and after the SNP2 molecular marker site is shown in SEQ ID No. 2.

[0009] The present application also provides a kit for detecting the SNP molecular marker related to the body length trait of Ningxiang pigs, wherein the kit comprises a sequencing primer pair for detecting the SNP1 molecular marker, and a sequencing primer pair for detecting the SNP2 molecular marker; the sequence of the sequencing primer pair for detecting the SNP1 molecular marker is shown in SEQ ID No. 3 and SEQ ID No. 4; and the sequence of the sequencing primer pair for detecting the SNP2 molecular marker is shown in SEQ ID No. 5 and SEQ ID No. 6.

[0010] The present application also provides an application of the SNP molecular marker in any one of the following aspects: Detecting body length trait size of Ningxiang pig: by extracting genomic DNA of the Ningxiang pig individual to be detected, the target region is amplified and sequenced by using the specific amplification primer or kit provided by the application to obtain the genotype information of SNP1 and SNP2. Specifically, if the genotype at the rs334683505 site is TC type, or the genotype at the rs321288338 site is AC type, then the individual has a larger body length trait (average BL ≈ 102.74 cm); if it is CC type or AA type homozygous, then the body length is relatively short (average BL ≈ 99.52 cm), and the difference is extremely significant (p<0.01). This method can realize early, accurate and non-destructive genotyping of body length traits without relying on phenotype measurement.

[0011] Early screening of Ningxiang pig growth and development performance: the above molecular marker genotyping results are associated with body length and other growth traits, which can predict the body length development potential of pigs at a young age (such as weaning period or nursery period). In actual production management, genotype detection can be performed on the reserve sow or commercial pig group to identify individuals with excellent body length genetic potential in advance, and provide molecular basis for realizing early mating selection, reasonable grouping, differential nutrition regulation and improving population uniformity and production efficiency.

[0012] Ningxiang pig molecular marker assisted breeding: the marker of the application can be included in the existing comprehensive breeding evaluation system of Ningxiang pig to guide the selection and matching of core groups. In the selection process, individuals with TC or AC genotypes can be preferentially selected as parents, thereby significantly increasing the frequency of obtaining large body length genotypes in offspring, accelerating genetic progress, shortening generation interval, and realizing precise and efficient genetic improvement of body length traits.

[0013] Ningxiang pig breed screening: by detecting these sites, Ningxiang pigs can be effectively distinguished from other pig breeds, and applied to breed purity identification, local genetic resource protection, and establishment of characteristic high body length lines.

[0014] The two sites of SNP1 and SNP2 have a linkage effect.

[0015] The application also provides a kit for detecting the SNP molecular marker related to the body length trait of Ningxiang pig, and the kit is applied to any one of the following: detecting the body length trait size of Ningxiang pig; early screening of Ningxiang pig growth and development performance; Ningxiang pig molecular marker assisted breeding; Ningxiang pig breed screening. The two sites of SNP1 and SNP2 have a linkage effect. The application mode is consistent with the application of the molecular marker, but the application of the kit has an advantage in high-throughput and standardized detection, and is especially suitable for large-scale population screening and commercial breeding application.

[0016] The application will be further described below: The application locates the linkage SNP marker sites (chr1:15776004 and chr1:15832958) which are significantly related to the body length of Ningxiang pigs by whole genome association analysis of 118 Ningxiang pigs resequencing detection data and BL phenotype, and designs an amplicon sequencing primer set which can be used for identification analysis and large-scale detection. The Ningxiang pig individuals with the required body length can be efficiently and accurately identified and screened.

[0017] Compared with the prior art, the application has the following beneficial effects: (1) The application obtains a molecular marker which is significantly related to the body length of Ningxiang pigs, and provides guidance for Ningxiang pig breeding by using the molecular marker, so that the body length can be accurately and efficiently predicted and typed, and Ningxiang pigs with excellent body length can be identified and screened, thereby improving the health level, production efficiency and breeding efficiency of Ningxiang pig population.

[0018] (2) The application provides an amplicon sequencing primer set for detecting the SNP molecular marker related to the body length of Ningxiang pigs, and the primer set has high specificity and accuracy, can accurately obtain the sequence containing the SNP molecular marker site, and can be applied to the breeding of Ningxiang pigs with excellent traits, and can efficiently identify the size of the body length.

[0019] (3) The molecular marker has heterozygote advantage, and the designed primer pair can be applied to a high-throughput SNP typing platform and can be applied to large-scale population breeding and analysis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a Manhattan plot for whole genome association analysis of BL phenotype of Ningxiang pigs. Figure 2 It is a molecular marker developed in the 500 kb region upstream and downstream of SNP rs321341167 (red for gene name, black for molecular marker site). Figure 3 It is an association analysis of different allelic genotypes of SNP chr1:15776004 and chr1:15832958 with BL. DETAILED DESCRIPTION

[0021] The specific embodiments of the application are described below to facilitate those skilled in the art to understand the application, but it should be clear that the application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the application defined and determined by the appended claims, and all applications utilizing the concept of the application are within the scope of protection.

[0022] 1. Material screening and phenotype recording At the Ningxiang Pig National Conservation Farm and the Fusi Breeding Farm of Hunan Liushahe Flower Pig Ecological Animal Husbandry Co., Ltd., the production performance of the Ningxiang pig herd was measured using 10 sets of automatic feeding systems from Shenzhen Runnong Company over a period of 90 days. At the time of departure, the horizontal straight-line distance from the occipital crest (posterior part of the skull) to the tail root was measured, obtaining the BL phenotypic data for these 118 Ningxiang pigs.

[0023] 2. Whole genome resequencing 2.1 Sample preparation and DNA extraction quality control Freshly collected pig ear tissue was lysed and digested. High-purity, high-quality DNA was extracted using the CTAB method. The concentration was determined using Qubit 3.0, the purity of the sample was detected using Nanodrop, and the integrity of the DNA was detected by agarose gel electrophoresis to ensure that the sample met the requirements for library construction.

[0024] 2.2 DNA Fragmentation and End Repair The DNA was broken down into 350bp fragments using Covaris sonication, followed by end repair and 3' A-tailing to facilitate adapter ligation.

[0025] 2.3 Connector Connection and Library Construction Sequencing adapters are ligated to both ends of the DNA fragment to form a library that can be used for amplification; target length fragments are screened using magnetic beads and then enriched by PCR amplification.

[0026] 2.4 Sequencing: The sequencing was technically supported by Beijing Novogene Technology Co., Ltd., and paired-end 150bp (PE150) sequencing was completed on the Illumina sequencing platform, with an average sequencing depth of 10X. The raw sequencing data were processed through a bioinformatics workflow, and the resequencing data were visualized to obtain high-quality genotyping data.

[0027] 3. Genome-wide association analysis to locate target SNP loci. 3.1 Genome-wide association analysis (GWAS) Based on 118 resequencing data from Ningxiang pigs, genome-wide association analysis was performed using the rMVP software package (Yin L, Zhang H, Tang Z et al. 2021). The analysis workflow is as follows: (1) Model construction: The mixed linear model (MLM) was selected as the core statistical method. The results of the first three principal component analyses (PCA) were included in the model as fixed effects covariates to correct for the group stratification effect. (2) Phenotype association: Construct association model for target BL traits, and evaluate the significant association between each SNP site and phenotype by likelihood ratio test; (3) Significance determination: Set threshold value by Bonferroni multiple correction method, and calculate P = 0.05 / N (N is the number of effective SNPs), take the value of -log10(P) to draw Manhattan plot (Fig. 2), and the region above the threshold line is determined as the significantly associated site, and finally the candidate SNP site significantly associated with BL traits is identified. Figure 1

[0028] 3.2 Mining significant marker sites In the whole genome range, the significant marker sites in BL phenotype traits are obtained, and the GWAS locates the significant SNP site rs321341167, and 5 molecular markers are designed in the upstream and downstream 500 kb regions ( Figure 2 ). Ningxiang pig is used as experimental material, and it is found that the molecular markers of chr1: 15776004 (rs334683505) and chr1: 15832958 (rs321288338) sites have significant phenotype effect.

[0029] 4. SNP site sequencing primer design 4.1 Primer design After obtaining the significant SNP marker of whole genome association analysis, the sequence of 1000 bp before and after the SNP is extracted (Table 1), the reference genome is Sus Scrofa Build11.1, and the BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) is used to design the amplicon primer (Table 2) for later material verification. The primer is entrusted to Invitrogen Company for synthesis.

[0030] 4.2 Primer information The amplicon sequencing primer is composed of two primers, including forward amplification primer Primer_F and reverse amplification primer Primer_R. The primer sequence needs to be added with specific universal adapter sequence before and after the sequencing platform to constitute the complete amplicon primer sequence.

[0031] Table 1. Site information table

[0032] Table 2. Amplicon sequencing primer sequence information table for detection of BL markers of Ningxiang pig

[0033] ​5. Material verification (second-generation amplicon sequencing verification) 5.1 DNA sample collection and quality control process 1) Sample pretreatment One week before inbound, 118 purebred Ningxiang pig right ear tissue samples were collected using sterile ear forceps. The ear tissue was placed in a 1.5 mL sterile enzyme-free centrifuge tube containing 75% ethanol and stored at -20°C for subsequent amplicon library construction.

[0034] DNA extraction and quality control Tissue lysis and genomic DNA extraction were performed by CTAB method. The DNA concentration was accurately determined by Qubit fluorescence quantifier, and the sample concentration was required to be greater than 10 ng / μL. The 260 / 280 absorbance ratio was detected by Nanodrop spectrophotometer, and the sample purity was between 1.8-2.2. The DNA integrity was detected by agarose gel electrophoresis, and the sample main band was required to be obvious without obvious degradation.

[0035] 5.2 Amplicon library construction process 1) Target region PCR amplification The designed complete amplicon primer (including sequencing platform specific adapter sequence) was used to amplify the target region of the 118 Ningxiang pig genomic DNA. The PCR system is shown in Table 3, and the PCR reaction program is shown in Table 4.

[0036] Table 3. Target region PCR reaction system

[0037] Table 4. Target region PCR reaction program

[0038] 2) PCR product quality control Agarose gel electrophoresis was used for quality inspection to check whether the band was clear and the band size was correct.

[0039] 3) PCR product magnetic bead purification The PCR1 product was purified using 1.2X purification magnetic beads, and the supernatant was aspirated after purification.

[0040] 4) Second PCR amplification The purified product was subjected to second PCR amplification using sequencing adapters to obtain a complete structure library. The PCR system is shown in Table 5, and the PCR reaction program is shown in Table 6.

[0041] Table 5. Second PCR reaction system

[0042] Table 6. Second PCR reaction procedure

[0043] 5) PCR product quality control Agarose gel electrophoresis quality inspection, check if the band is clear and the band size is correct.

[0044] 6) PCR product magnetic bead purification Use 1X purification magnetic beads to purify and recover PCR1 product, and use Qubit to measure the concentration.

[0045] 7) Sequencing Qubit quantification accurately detects library concentration, and Agilent 2100 bioanalyzer detects insert size. According to the sequencing throughput and data volume requirement, prepare the sequencing library pool, and perform PE150 sequencing on the illumina sequencing platform.

[0046] 5.3 SNP site genotyping After high-throughput sequencing, the raw data is processed by quality control and filtering to remove sequences containing adapter contamination and low-quality sequences. Use BWA to align with the reference genome (Sus Scrofa Build11.1), and use Freebayes to detect and filter SNPs. Then, the number of SNPs detected in all samples is counted, and the number and proportion of each genotype are analyzed. Finally, a VCF file containing specific site genotype information is generated. The SNP site genotyping data is shown in Table 7.

[0047] According to the genotyping results, the sample numbers corresponding to CC genotype of chr1:15776004 and AA genotype of chr1:15832958 are consistent; the sample numbers corresponding to TC genotype of chr1:15776004 and AC genotype of chr1:15832958 are consistent.

[0048] Table 7. SNP site genotyping

[0049] 5.4 Genotype-Phenotype Association Analysis 245 BL phenotypic data of Ningxiang pigs were collected, and SNP genotyping and phenotypic data were analyzed. Results showed that the chr1:15776004 locus had two genotypes (TC and CC) in the Ningxiang pig population. The average BL value for TC-type Ningxiang pigs was 102.74, while the average BL value for CC-type Ningxiang pigs was 99.52. The average BL value for TC-type Ningxiang pigs was significantly different from that for CC-type Ningxiang pigs. p < 0.01). The chr1:15832958 locus indicates two genotypes (AA and AC) in the Ningxiang pig population. The average BL value for AA-type Ningxiang pigs is 99.52; the average BL value for AC-type Ningxiang pigs is 102.74. The average BL value for AC-type Ningxiang pigs is significantly different from that for AA-type Ningxiang pigs. p < 0.01). Due to the strong association between this genotype and its significant correlation with the phenotype, it is speculated that chr1:15776004 and chr1:15832958 have a linkage effect.

Claims

1. A SNP molecular marker related to body length trait of Ningxiang pigs, characterized in that, The molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker is located at position 15776004 of chromosome 1 with reference to the Sus Scrofa Build11.1 genome, the polymorphism of which is T / C, and the SNP1 molecular marker is numbered as rs334683505; the SNP2 molecular marker is located at position 15832958 of chromosome 1 with reference to the Sus Scrofa Build11.1 genome, the polymorphism of which is A / C, and the SNP2 molecular marker is numbered as rs321288338.

2. The gene comprising the SNP molecular marker associated with the body length trait of Nanzhong pigs according to claim 1, characterized in that, The gene comprises the nucleotide sequence 1000 bp before and after the SNP1 molecular marker site or the SNP2 molecular marker site, the nucleotide sequence 1000 bp before and after the SNP1 molecular marker site is as shown in SEQ ID No. 1, and the nucleotide sequence 1000 bp before and after the SNP2 molecular marker site is as shown in SEQ ID No.

2.

3. A kit for detecting a SNP molecular marker associated with the body length trait of Ningxiang pigs, characterized in that, The kit comprises a sequencing primer pair for detecting the SNP1 molecular marker and a sequencing primer pair for detecting the SNP2 molecular marker; the sequence of the sequencing primer pair for detecting the SNP1 molecular marker is as shown in SEQ ID No. 3 and SEQ ID No. 4; and the sequence of the sequencing primer pair for detecting the SNP2 molecular marker is as shown in SEQ ID No. 5 and SEQ ID No.

6.

4. The SNP molecular marker of claim 1 is applied to any of the following: detecting the size of the body length trait of Ningxiang pigs; early screening of the growth and development performance of Ningxiang pigs; Ningxiang pig molecular marker assisted breeding; Ningxiang pig breed screening.

5. The use according to claim 4, wherein the compound is ###0002### The SNP1 and SNP2 sites have a linkage effect.

6. The kit of claim 3 is applied to any of the following: detecting the size of the body length trait of Ningxiang pigs; early screening of the growth and development performance of Ningxiang pigs; Ningxiang pig molecular marker assisted breeding; Ningxiang pig breed screening.

7. Use according to claim 6, wherein The SNP1 and SNP2 sites have a linkage effect.

Images