A snp molecular marker related to body length trait of ningxiang pigs and application thereof

Abstract

The application discloses a SNP molecular marker related to a body length character of Ningxiang pigs and application of the molecular marker. The molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker is located at the position of 15776004 of chromosome 1 with Sus Scrofa Build11.1 genome as a reference genome, the polymorphism of the SNP1 molecular marker is T / C, and the SNP1 molecular marker is numbered as rs334683505; the SNP2 molecular marker is located at the position of 15832958 of chromosome 1 with Sus Scrofa Build11.1 genome as a reference genome, the polymorphism of the SNP2 molecular marker is A / C, and the SNP2 molecular marker is numbered as rs321288338. The application obtains the molecular marker which is significantly related to the body length character of the Ningxiang pigs, and designs an amplicon sequencing primer group which can be used for identification analysis and large-scale detection. The Ningxiang pig individual with the required BL size can be efficiently and accurately identified and screened.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a SNP molecular marker related to the body length trait of Ningxiang pigs and its application. Background Technology

[0002] Body length (BL) is one of the core indicators of body structure traits in pigs, 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 type trait, and together with height and chest girth, it reflects the pig's physical structure and skeletal development. Body length is controlled by multiple genes and is a typical quantitative trait, its phenotype influenced by both genetic and environmental factors. The heritability of body length is moderate to high (0.34–0.41), higher than reproductive traits (such as litter size), and can be effectively improved through individual selection.

[0003] Its value in pig breeding is reflected in several aspects: First, in improving meat production efficiency: body length is significantly positively correlated with carcass length and dressing percentage. For every 1 cm increase in body length, lean meat production can be increased by about 0.3–0.5 kg, directly increasing economic benefits; Second, in reducing culling rate: poor limb and hoof structure leads to the culling of sows accounting for 11% (second only to reproductive problems). Selecting breeding pigs with moderate body length and symmetrical structure can extend their service life by 1–2 litters.

[0004] Single nucleotide polymorphisms (SNPs) are characterized by high density and stable inheritance, and have become a core tool of marker-assisted selection (MAS). Utilizing SNP markers for early selection (such as eliminating individuals with unfavorable genotypes) can shorten generation intervals and improve the genetic progression of body length traits.

[0005] In summary, body length, as a key conformation trait in pig breeding, possesses high heritability, economic value in meat production, and serves as an indicator of robustness. Incorporating it into comprehensive breeding goals, combined with SNP molecular marker-based breeding selection techniques, can significantly improve the lifetime productivity of breeding pigs and provide a scientific basis for balancing traits such as meat quality and reproduction. Future research should focus on standardized measurement and cross-trait genetic mechanisms to achieve more precise genetic improvement. However, while there are existing patents combining SNP technology for non-Ningxiang pigs, the development of molecular markers for body length, a key structural trait, remains relatively lacking. In particular, research on body length-related SNP markers for local Chinese pig breeds (such as Ningxiang pigs) has not been reported. Existing molecular markers are mostly based on simplified genome sequencing or low-density microarray genotyping, which suffer from low marker density and limited localization accuracy, making it difficult to achieve precise genotype-phenotype associations. Furthermore, existing patents and technologies are mostly concentrated on foreign commercial pig breeds (such as Landrace and Large White pigs), whose genetic backgrounds differ significantly from those of local Chinese pig breeds; direct application may result in insufficient or ineffective marker effects. Summary of the Invention

[0006] To address the aforementioned shortcomings in the existing technology, the present invention aims to provide an SNP molecular marker related to the body length trait of Ningxiang pigs and its application.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: The SNP molecular markers associated with the body length trait of Ningxiang pigs include SNP1 and SNP2; the molecular markers include SNP1 and SNP2; the SNP1 molecular marker is located at position 15776004 on chromosome 1, with the Sus Scrofa Build11.1 genome as the reference genome, and its polymorphism is T / C, and the SNP1 molecular marker number is rs334683505; the SNP2 molecular marker is located at position 15832958 on chromosome 1, with the Sus Scrofa Build11.1 genome as the reference genome, and its polymorphism is A / C, and the SNP2 molecular marker number is rs321288338.

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

[0009] The present invention also provides a kit for detecting SNP molecular markers related to body length traits in Ningxiang pigs. The kit includes sequencing primer pairs for detecting SNP1 molecular markers and sequencing primer pairs for detecting SNP2 molecular markers. The sequences of the sequencing primer pairs for detecting SNP1 molecular markers are shown in SEQ ID No. 3 and SEQ ID No. 4. The sequences of the sequencing primer pairs for detecting SNP2 molecular markers are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0010] This invention also provides the application of SNP molecular markers in any of the following: Detection of body length trait in Ningxiang pigs: Genomic DNA was extracted from individual Ningxiang pigs to be tested, and the target regions were amplified and sequenced using the specific amplification primers or kits provided in this invention to obtain the genotype information of SNP1 and SNP2. Specifically, if the genotype at rs334683505 is TC, or the genotype at rs321288338 is AC, the individual has a larger body length (average BL≈102.74 cm); if it is homozygous for CC or AA, the body length is relatively shorter (average BL≈99.52 cm), with highly significant differences (p<0.01). This method can achieve early, accurate, and non-destructive genotypic identification of body length traits without relying on phenotypic measurements.

[0011] Early screening of growth and development performance in Ningxiang pigs: By correlating the results of the above molecular marker genotyping with growth traits such as body length, the growth potential of adult body length can be predicted in pigs during their early stages (such as weaning or nursery periods). In actual production management, genotyping of replacement breeding pigs or commercial pig herds can be performed to identify individuals with excellent genetic potential for body length in advance, providing a molecular basis for early indirect selection, rational grouping, differentiated nutritional regulation, and improving herd evenness and production efficiency.

[0012] Molecular marker-assisted breeding of Ningxiang pigs: The markers of this invention can be incorporated into the existing comprehensive breeding evaluation system for Ningxiang pigs to guide the selection of core herds. During the selection process, individuals with TC or AC genotypes can be preferentially selected as parents, thereby significantly increasing the frequency of obtaining the large body length genotype in offspring, accelerating genetic progress, shortening generation intervals, and achieving precise and efficient genetic improvement of body length traits.

[0013] Ningxiang pig breed screening: By detecting these loci, Ningxiang pigs can be effectively distinguished from other pig breeds, and can be applied to breed purity identification, local genetic resource protection, and the establishment of distinctive tall-body-length strains.

[0014] The two sites, SNP1 and SNP2, exhibit a linkage effect.

[0015] This invention also provides a kit for detecting SNP molecular markers associated with the body length trait of Ningxiang pigs, applicable to any of the following: detection of body length in Ningxiang pigs; early screening of growth and development performance in Ningxiang pigs; marker-assisted breeding of Ningxiang pigs; and breed screening of Ningxiang pigs. The SNP1 and SNP2 sites exhibit a linkage effect. Its application is consistent with that of molecular markers, but the kit's advantages in high-throughput, standardized detection make it particularly suitable for large-scale population screening and commercial breeding applications.

[0016] The present invention will be further described below: This invention, through genome-wide association analysis of resequencing data from 118 purebred Ningxiang pigs and the BL phenotype, located linkage SNP markers (chr1:15776004 and chr1:15832958) significantly associated with BL size in Ningxiang pigs, and designed an amplicon sequencing primer set for identification analysis and large-scale detection. This allows for efficient and accurate identification and screening of Ningxiang pig individuals with BL sizes meeting the requirements.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention has obtained molecular markers that are significantly related to the body length trait of Ningxiang pigs. Using these molecular markers to guide the breeding of Ningxiang pigs can accurately and efficiently predict body length traits and genotypes, identify and screen pig breeds with excellent body length traits, and improve the health level, production efficiency and breeding efficiency of Ningxiang pig herds.

[0018] (2) The present invention provides an amplicon sequencing primer set for detecting SNP molecular markers related to body length traits of Ningxiang pigs. The primer set has high specificity and accuracy, and can accurately obtain sequences containing SNP molecular marker sites of the present invention. It can be applied to the selection of superior traits of Ningxiang pigs and efficiently identify the size of body length traits.

[0019] (3) This molecular marker has heterozygous advantage, and the designed primer pairs are applicable to high-throughput SNP genotyping platforms and can be applied to large-scale population selection and analysis. Attached Figure Description

[0020] Figure 1 Manhattan plot for genome-wide association analysis of BL phenotype in Ningxiang pigs; Figure 2 Molecular markers developed for the 500 kb region upstream and downstream of SNP rs321341167 (red indicates gene name, black indicates molecular marker site). Figure 3 Association analysis of different allelic genotypes of SNPs chr1:15776004 and chr1:15832958 with BL. Detailed Implementation

[0021] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0022] 1. Material screening and phenotypic 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) Phenotypic association: An association model was constructed for the target BL trait, and the significance of the association between each SNP site and the phenotype was evaluated by the likelihood ratio test; (3) Significance determination: The Bonferroni multiple correction method was used to set the threshold, and the calculation method was P=0.05 / N (N is the number of valid SNPs). The Manhattan plot was drawn by taking the -log10(P) value. Figure 1 The region above the threshold line was identified as a significantly associated site, and finally candidate SNP sites that were significantly associated with the BL trait were identified.

[0028] 3.2 Discovering significant marker sites Significant marker sites for the BL phenotypic trait were identified across the entire genome. The significant SNP site rs321341167 was located using GWAS, and five molecular markers were designed within a 500 kb region upstream and downstream of it. Figure 2 Using Ningxiang pigs as experimental material, it was found that molecular markers at the chr1: 15776004 (rs334683505) and chr1:15832958 (rs321288338) sites had significant phenotypic effects.

[0029] 4. Primer design for SNP site sequencing 4.1 Primer Design After obtaining significant SNP markers from genome-wide association analysis, 1000 bp sequences before and after each SNP were extracted (Table 1). Using Sus Scrofa Build 11.1 as the reference genome, amplicon primers were designed using BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) (Table 2) for subsequent material validation. Primers were synthesized by Invitrogen.

[0030] 4.2 Primer Information Amplicon sequencing primers consist of two primers: a forward amplification primer (Primer_F) and a reverse amplification primer (Primer_R). Platform-specific universal adapter sequences are added before and after the primer sequences to form a complete amplicon primer sequence.

[0031] Table 1. Site Information Table

[0032] Table 2. Amplicon sequencing primer sequence information for BL marker detection in Ningxiang pigs

[0033] 5. Material validation (second-generation amplicon sequencing validation) 5.1 DNA Sample Collection and Quality Control Process 1) Sample preprocessing One week prior to entry into the station, right ear tissue samples were collected from 118 purebred Ningxiang pigs using sterile ear clippers. The ear tissues were placed in 1.5 mL sterile enzyme-free centrifuge tubes 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 using the CTAB method. DNA quality was assessed by precisely determining DNA concentration using a Qubit quantitative PCR instrument, requiring a sample concentration greater than 10 ng / μL. The 260 / 280 absorbance ratio was measured using a Nanodrop spectrophotometer, with sample purity between 1.8 and 2.2 for the 260 / 280 ratio. DNA integrity was assessed using agarose gel electrophoresis, requiring a clear main band and no significant degradation.

[0035] 5.2 Amplicon Library Construction Process 1) PCR amplification of the target region Using the designed complete amplicon primers (containing sequencing platform-specific adapter sequences), the target regions of the 118 Ningxiang pig genomic DNA samples were amplified by PCR. The PCR system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0036] Table 3. PCR reaction system for the target region

[0037] Table 4. PCR reaction procedure for the target region

[0038] 2) PCR product quality control Agarose gel electrophoresis quality inspection checks whether the bands are clear and whether the band size is correct.

[0039] 3) Purification of PCR products using magnetic beads The PCR1 product was purified using 1.2X purification magnetic beads, and the supernatant was collected after purification.

[0040] 4) Second PCR amplification The purified product was amplified a second time using sequencing adapters to obtain a complete structural library. The PCR system is shown in Table 5, and the PCR reaction procedure 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 checks whether the bands are clear and whether the band size is correct.

[0044] 6) Purification of PCR products using magnetic beads The PCR1 product was purified and recovered using 1X purification magnetic beads, and the concentration was measured using Qubit.

[0045] 7) Sequencing on the machine Qubit quantification was used to accurately determine library concentration, and insert size was determined using an Agilent 2100 bioanalyzer. Sequencing libraries were prepared into pools based on sequencing throughput and data volume requirements, and PE150 sequencing was performed on the Illumina sequencing platform.

[0046] 5.3 SNP locus genotyping The raw data after high-throughput sequencing underwent quality control filtering to remove sequences containing adapter contamination and low-quality sequences. BWA was used to align with the reference genome (Sus Scrofa Build 11.1), and SNPs were detected and filtered using Freebayes. The number of SNPs detected in all samples was then counted, and the number and proportion of each genotype were analyzed. Finally, a VCF file containing genotype information at specific loci was generated. Locus genotyping data are shown in Table 7.

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

[0048] Table 7. SNP locus 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. Application of a detection reagent for SNP molecular markers related to body length in Ningxiang pigs in detecting body length, wherein the molecular markers include SNP1 and SNP2; the SNP1 molecular marker is located at position 15776004 on chromosome 1, with a polymorphism of T / C, and is numbered rs334683505; the SNP2 molecular marker is located at position 15832958 on chromosome 1, with a polymorphism of A / C, and is numbered rs321288338.

2. The application as described in claim 1, characterized in that, The two sites, SNP1 and SNP2, exhibit a linkage effect.

3. The application as described in claim 1, characterized in that, The detection reagents for SNP molecular markers related to body length traits in Ningxiang pigs include sequencing primer pairs for SNP1 molecular marker detection and sequencing primer pairs for SNP2 molecular marker detection; the sequences of the sequencing primer pairs for SNP1 molecular marker detection are shown in SEQ ID No. 3 and SEQ ID No. 4; the sequences of the sequencing primer pairs for SNP2 molecular marker detection are shown in SEQ ID No. 5 and SEQ ID No. 6.

Images