A snp molecular marker related to feed reward traits of ningxiang pigs and application thereof

Abstract

The application discloses a SNP molecular marker related to a feed reward character of Ningxiang pigs and application thereof. The SNP molecular marker related to the feed reward character of the Ningxiang pigs comprises SNP1 and SNP2. The SNP1 molecular marker is located at the 14729188th position of chromosome 9 with a Sus Scrofa Build11.1 genome as a reference genome, the polymorphism of the SNP1 molecular marker is T / A, and the SNP1 molecular marker is numbered as rs3470966449. The SNP2 molecular marker is located at the 15248361th position of chromosome 9 with the Sus Scrofa Build11.1 genome as the reference genome, the polymorphism of the SNP2 molecular marker is G / A, and the SNP2 molecular marker is numbered as rs343638482. The application obtains a molecular marker significantly related to the feed reward of the Ningxiang pigs, provides guidance for Ningxiang pig breeding by using the molecular marker, can accurately and efficiently predict the feed reward and typing, identifies and screens pig breeds with high feed reward, improves the breeding efficiency, and provides a reliable technical tool for genetic improvement of pig breeds and accurate selection of characters.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a SNP molecular marker related to feed efficiency traits in Ningxiang pigs and its application. Background Technology

[0002] Feed efficiency (FE) is an indicator that measures the efficiency with which livestock and poultry convert feed into products. Its core meaning is the ratio of feed input to product output. It is calculated as: FE = Daily weight gain (kg) / Daily feed intake (kg). Feed costs directly affect meat production costs, and FE directly determines the "cost-to-meat ratio" (feed cost per kilogram of weight gain), which is crucial for livestock profitability. Furthermore, pig breeds with high FE can reduce their reliance on concentrated feed (e.g., replacing 20-30 kg / head with forage), alleviating feed resource shortages. Given these characteristics, FE has the highest weighting coefficient in modern pig breeding and is a core standard for evaluating the quality of breeding pigs.

[0003] FE is a quantitative trait, regulated by multiple genes with minor effects and easily influenced by the environment. Currently, traditional breeding relies on phenotypic selection, but it has three major limitations: low heritability: the heritability of reproductive traits (such as semen quality) is only 0.1-0.2, and conventional selection is slow; phenotypic lag: feed efficiency testing takes a long time and is conducted at different stages, making early selection impossible; and the complexity of multiple gene interactions: it involves multiple pathways such as energy metabolism and fat deposition.

[0004] Molecular marker-assisted selection (MAS) can overcome the above bottlenecks and achieve early precision breeding by linking genotype and phenotype. Early RAPD and SSR markers are limited due to low polymorphism and poor stability, while single nucleotide polymorphisms (SNPs) have become the mainstream due to their wide distribution (one per 100-300 bp in the genome) and high stability (mutation rate lower than microsatellites).

[0005] Chinese invention patent CN 108419756 A discloses a breeding method for the Bahuai pig, but it mainly uses population successive selection and molecular marker-assisted selection, not specifically for Ningxiang pig breeding, nor does it apply SNP molecular markers related to feed conversion patterns. This indicates that although SNP-based molecular markers are widely used in breeding, research specifically on FE (feed conversion pattern) in pigs, especially Ningxiang pigs, is scarce. Ningxiang pigs, as a superior local pig breed in my country, are characterized by good meat quality and tolerance to roughage, but their slow growth and low feed conversion efficiency limit their industrial development. Therefore, developing FE-related molecular markers specific to Ningxiang pigs to achieve early and precise breeding has significant theoretical and practical value. Summary of the Invention

[0006] To address the aforementioned shortcomings in the prior art, the present invention aims to provide an SNP molecular marker related to feed efficiency traits in 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 feed efficiency traits in Ningxiang pigs include SNP1 and SNP2. SNP1, with the Sus Scrofa Build11.1 genome as a reference, is located at position 14729188 on chromosome 9, with a polymorphism of T / A, and is designated rs3470966449. SNP2, with the Sus Scrofa Build11.1 genome as a reference, is located at position 15248361 on chromosome 9, with a polymorphism of G / A, and is designated rs343638482.

[0008] The present invention also provides a gene containing the above-mentioned SNP1 or SNP2 molecular markers related to the feed efficiency traits 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 feed efficiency 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 or auxiliary detection of feed efficiency in Ningxiang pigs: Genomic DNA is extracted from individual Ningxiang pigs to be tested. Target regions are amplified and sequenced using the specific primer pairs or corresponding kits provided in this invention to obtain the genotypes at rs3470966449 and rs343638482 loci. Based on association analysis results: if the genotype at rs3470966449 is TT and the genotype at rs343638482 is GG, the individual is considered to have a high feed efficiency phenotype (i.e., excellent FE trait, average FE≈4.39); if the genotype at rs3470966449 is AA or the genotype at rs343638482 is GA, the individual has a lower feed efficiency (average FE≈5.07) and lower conversion efficiency. This molecular detection method can achieve rapid and objective evaluation of feed efficiency traits without relying on traditional time-consuming phenotyping.

[0011] Early screening for feed efficiency in pigs in Ningxiang: DNA extraction and genotyping were completed. Based on the genotyping results: individuals carrying the "TT / GG" genotype were selected as high-feed efficiency candidate breeding pigs; individuals with poor genotypes (such as "AA / GA") were considered for early culling or commercial use to significantly reduce ineffective feeding costs.

[0012] Molecular marker-assisted breeding of Ningxiang pigs: In the selection of the core population, individuals with the "TT / GG" genotype are given priority as parents to increase the frequency of superior alleles in the population; by monitoring the genotypes of offspring, the selection effect is evaluated, and feed efficiency traits are selected in a targeted manner to accelerate genetic progress; combined with molecular markers of other economic traits (such as meat quality and fertility), multi-trait synergistic selection is carried out to establish a new Ningxiang pig breed with better overall performance.

[0013] Ningxiang pig breed screening: By utilizing the genotype frequency characteristics of these two loci, Ningxiang pigs can be distinguished from other pig breeds.

[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 feed efficiency traits in Ningxiang pigs, applicable to any of the following: detection or auxiliary detection of feed efficiency in Ningxiang pigs; early screening for feed efficiency 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 utilizes whole-genome sequencing data from 118 purebred Ningxiang pigs and conducts genome-wide association analysis (GWAS) on the fetal phenotypic (FE) trait in pigs to locate two SNP linkage loci (chr9:14729188 and chr9:15248361) that have a significant phenotypic effect on Ningxiang pig FE. A matching set of amplicon sequencing primers was designed for these linkage loci, enabling precise genotyping and detection of the target loci, providing a reliable technical tool for pig genetic improvement and precise trait selection.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention has obtained a molecular marker that is significantly related to feed efficiency of Ningxiang pigs. Using this molecular marker to provide guidance for Ningxiang pig breeding can accurately and efficiently predict feed efficiency and typing, identify and screen pig breeds with high feed efficiency, and improve breeding efficiency.

[0018] (2) The present invention provides an amplicon sequencing primer set for detecting SNP molecular markers related to feed efficiency 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 Ningxiang pig assisted breeding and efficiently identify the level of feed efficiency.

[0019] (3) The molecular marker has codominant inheritance, 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 the Ningxiang Spotted Pig population; Figure 2 Molecular markers developed for the 500 kb region upstream and downstream of SNP rs343638482 (red indicates gene name, black indicates molecular marker site). Figure 3 Association analysis of different allelic genotypes of SNPs chr9:14729188 and chr9:15248361 with FE. 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 Breeding Farm and the Fusi Breeding Farm of Hunan Liushahe Flower Pig Ecological Animal Husbandry Co., Ltd., one week before entering the testing station, 118 right ear tissue samples of Ningxiang Flower Pigs were collected and placed in 1.5 mL sterile enzyme-free centrifuge tubes containing 75% alcohol, stored at -20℃, and subjected to whole-genome resequencing. The growth performance of the 118 Ningxiang pigs was measured using 10 sets of automated feeding systems from Shenzhen Runong Company over a period of 90 days. After 90 days at the testing station, total feed intake and total weight gain were measured. The FE phenotypic data of these 118 Ningxiang pigs were collected using the formula FE = total feed intake / total weight gain.

[0023] 2. Whole genome resequencing 2.1 DNA Sample Collection and Quality Control Process 1) Sample preprocessing One week prior to arrival at 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 whole-genome resequencing analysis.

[0024] DNA extraction and quality control Tissue lysis and genomic DNA extraction were performed using the CTAB method. DNA quality was assessed by precisely measuring DNA concentration using a Qubit fluorescence quantitative PCR instrument. The 260 / 280 and 260 / 230 absorbance ratios were measured using a Nanodrop spectrophotometer, and the integrity of DNA bands was observed using agarose gel electrophoresis, thereby ensuring that the DNA concentration, purity, and integrity met the requirements for library preparation.

[0025] 2.2 Library Construction and High-Throughput Sequencing 1) Library Construction DNA samples that passed quality control were processed using a Covaris fragmentation instrument, randomly breaking them into 350bp fragments. The fragmented DNA was then used in a standardized process involving end repair, A-tailing, adapter ligation, purification, and PCR amplification using an Illumina TruSeq library preparation kit.

[0026] Document Quality Control After the library was constructed, preliminary quantification was performed using Qubit 3.0, and the library was diluted to a working concentration of 1 ng / μL. Subsequently, the insert size was detected using an Agilent 2100 bioanalyzer. The effective concentration was accurately determined using qPCR to ensure that the effective concentration of the library was >2 nM.

[0027] 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.

[0028] 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: Model building The mixed linear model (MLM) was selected as the core statistical method, and the results of the first three principal component analyses (PCA) were included in the model as fixed-effect covariates to correct for the population stratification effect. Phenotypic Association Association models were constructed for the target FE trait, and the significance of the association between each SNP locus and the phenotype was evaluated by the likelihood ratio test. Significance determination The Bonferroni multiple correction method was used to set the threshold, calculated as P = 0.05 / N (where N is the number of valid SNPs). The Manhattan plot was then plotted using 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 FE trait were identified.

[0029] 3.2 Discovering significant marker sites Significant marker sites for the porcine febrile trait were identified across the entire genome, and the significant SNP site rs343638482 was located using GWAS. Two molecular markers were designed within a 500 kb region upstream and downstream of this site. Figure 2 In the Ningxiang pig population, molecular markers at the chr9:14729188 (rs3470966449) and chr9:15248361 (rs343638482) sites were found to have significant phenotypic effects.

[0030] 4. Primer design for SNP site sequencing 4.1 Primer Design After obtaining the target SNP markers (chr9:14729188 and chr9:15248361), 1000 bp sequences before and after the SNPs were extracted (Table 1). The reference genome was Sus Scrofa Build 11.1. Amplicon primers were designed using the BatchPrimer3 online tool (http: / / probes.pw.usda.gov / batchprimer3 / ) (Table 2) for later material validation. Primers were synthesized by Invitrogen.

[0031] 4.2 Primer Information An amplicon primer consists of two primers: a forward amplification primer (Primer_F) and a reverse amplification primer (Primer_R). Sequencing platform-specific universal adapter sequences are added before and after the primer sequences to form a complete amplicon primer sequence.

[0032] Table 1. Site Information Table

[0033] Table 2. Amplicon sequencing primer sequence information for FE marker detection in Ningxiang pigs

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Table 3. PCR reaction system for the target region

[0038] Table 4. PCR reaction procedure for the target region

[0039] 2) PCR product quality control Agarose gel electrophoresis quality inspection checks whether the bands are clear and whether the band size is correct.

[0040] 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.

[0041] 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.

[0042] Table 5. Second PCR reaction system

[0043] Table 6. Second PCR reaction procedure

[0044] 5) PCR product quality control Agarose gel electrophoresis quality inspection checks whether the bands are clear and whether the band size is correct.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] According to the genotyping results, the sample numbers corresponding to the AA genotype of chr9:14729188 and the GA genotype of chr9:15248361 are the same; the sample numbers corresponding to the TA genotype of chr9:14729188 and the AA genotype of chr9:15248361 are the same; and the sample numbers corresponding to the TT genotype of chr9:14729188 and the GG genotype of chr9:15248361 are the same.

[0049] Table 7. SNP locus genotyping

[0050] 5.4 Genotype-Phenotype Association Analysis Ninety days after entering the station, total feed intake and total weight gain were measured. FE phenotypic data of these 118 Ningxiang pigs were collected using the formula FE = total feed intake / total weight gain. Analysis of SNP genotyping and phenotypic data revealed three genotypes (AA, TA, and TT) at the chr9:14729188 locus in the Ningxiang pig population. The average FE for AA-type Ningxiang pigs was 5.07; for TA-type Ningxiang pigs, it was 4.69; and for TT-type Ningxiang pigs, it was 4.39. The FE of AA-type Ningxiang pigs was significantly different from that of TT-type Ningxiang pigs. p <0.01).

[0051] The chr9:15248361 locus indicates the presence of three genotypes (AA, GA, and GG) in the Ningxiang pig population. The average femoral epithelial response (FE) of GG-type Ningxiang pigs was 4.39; that of GA-type Ningxiang pigs was 5.07; and that of AA-type Ningxiang pigs was 4.69. The FE of GG-type Ningxiang pigs was significantly different from that of GA-type Ningxiang pigs. p <0.01) Figure 3 ).

[0052] Due to the strong association between this genotype and its significant correlation with the phenotype, it is speculated that chr9:14729188 and chr9:15248361 have a linkage effect, and the TT / GG genotype combination is a favorable genotype.

Claims

1. The application of a SNP molecular marker associated with feed efficiency traits in Ningxiang pigs in the detection or auxiliary detection of feed efficiency in Ningxiang pigs; the molecular marker includes SNP1 and SNP2; the SNP1 molecular marker is located at position 14729188 on chromosome 9, with a polymorphism of T / A, and is numbered rs3470966449; the SNP2 molecular marker is located at position 15248361 on chromosome 9, with a polymorphism of G / A, and is numbered rs343638482; if the genotype at rs3470966449 is TT and the genotype at rs343638482 is GG, then the individual is determined to have a high feed efficiency phenotype.

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

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