A molecular marker related to the meat ratio trait of ningxiang pig and application thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a molecular marker related to the feed-meat ratio trait of Ningxiang pigs and application thereof. The molecular marker comprises SNP1 and SNP2. The SNP1 molecular marker corresponds to the 97217694th site from the 5' end on the chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or T. The SNP2 molecular marker corresponds to the 97590304th site from the 5' end on the chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or T. The present application obtains a molecular marker significantly related to the feed-meat ratio trait of Ningxiang pigs. The present application can provide guidance for the breeding of Ningxiang pigs by using the molecular marker, and can accurately and efficiently predict the feed-meat ratio and typing, identify and screen pig breeds with low feed-meat ratio, and improve the breeding efficiency.

Description

Technical Field

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

[0002] The global pork industry is currently undergoing a transformation towards large-scale and intensive production, with feed costs becoming a core factor restricting industry efficiency. The feed-to-gain ratio (F:G), a key indicator measuring how well an animal converts feed into body weight gain, is a core parameter reflecting feed conversion efficiency (FCE) and directly determines the amount of feed consumed per unit of output. Reducing the F:G ratio can significantly lower production costs and is a key breakthrough for improving the economics and sustainability of pig farming.

[0003] Traditional F:G improvement methods mainly rely on phenotypic selection, which has two major bottlenecks: First, F:G is a complex trait, and the phenotypic data collection cycle is long and easily affected by environmental interference, resulting in insufficient accuracy; Second, traditional methods cannot accurately locate key genomic regions, which limits the efficiency of genetic improvement.

[0004] Single nucleotide polymorphisms (SNPs), as third-generation genetic markers, are characterized by high density, high stability, and wide genomic distribution. Genome-wide association studies (GWAS) can systematically elucidate the genetic basis of F:G pairs. This technology can directly link genotype and phenotypic variations, enabling precise analysis of complex traits and promoting the large-scale application of molecular-assisted breeding.

[0005] Chinese invention patent CN 120210383 A discloses a method for assisting in the identification of feed conversion ratio (FCR) in pigs and its molecular markers. However, it targets Large White pigs. Existing technologies have not yet disclosed SNP molecular markers related to the FCR trait of Ningxiang pigs. Large White pigs and Ningxiang pigs are two significantly different breeds; Large White pigs have a faster growth rate and higher feed conversion ratio, while Ningxiang pigs have a slower growth rate and lower feed conversion ratio. Although the traits are similar, they are influenced by many loci, leading to significant differences in SNP molecular markers related to the FCR trait between Large White (lean) and Ningxiang (fat) pigs. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, the present invention aims to provide a molecular marker related to the feed conversion ratio (FCR) trait of Ningxiang pigs and its application. This marker can assist in screening for the F:G trait in Ningxiang pigs, thereby improving the breeding of superior traits in Ningxiang pigs.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] In the first aspect, a molecular marker related to the feed conversion ratio trait of Ningxiang pigs is provided. The molecular marker includes SNP1 and SNP2. The sequence of the nucleic acid containing the SNP1 molecular marker is shown in SEQ ID No. 1. The SNP1 molecular marker corresponds to the 97217694th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or T. The sequence of the nucleic acid containing the SNP2 molecular marker is shown in SEQ ID No. 2. The SNP2 molecular marker corresponds to the 97590304th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or T.

[0009] Secondly, this paper provides the application of the aforementioned SNP molecular markers in the detection or auxiliary detection of the feed conversion ratio trait in Ningxiang pigs.

[0010] Thirdly, the application of the above-mentioned SNP molecular markers in the early screening of feed conversion ratio in pigs in Ningxiang is provided.

[0011] Fourthly, the application of the aforementioned SNP molecular markers in marker-assisted breeding of Ningxiang pigs is provided.

[0012] Fifthly, the application of the aforementioned SNP molecular markers in the screening of Ningxiang pig breeds is provided.

[0013] Furthermore, there is a linkage effect between the SNP1 and SNP2 loci, and the CC / GG genotype combination is a favorable genotype.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) This invention obtained a molecular marker that is significantly related to the feed conversion ratio of Ningxiang pigs. Using this molecular marker, we can provide guidance for breeding Ningxiang pigs, accurately and efficiently predict the feed conversion ratio and genotyping, identify and screen pig breeds with low feed conversion ratios, and improve breeding efficiency.

[0016] (2) This invention provides sequencing primer pairs and KASP primer pairs for detecting SNP molecular markers related to feed conversion ratio of Ningxiang pigs. These primer pairs are highly specific and accurate, and can accurately obtain sequences containing SNP molecular marker sites of this invention. They can be applied to Ningxiang pig assisted breeding to efficiently identify the feed conversion ratio.

[0017] (3) The molecular markers of this invention have codominant inheritance, and the designed primer pairs are applicable to multiple platforms such as high-throughput SNP genotyping platform and high-throughput KASP detection platform, and can be applied to large-scale population selection and analysis. Attached Figure Description

[0018] Figure 1 Manhattan plot for F:G genome-wide association analysis of Ningxiang pig population;

[0019] Figure 2 Molecular markers developed for the 500kb region upstream and downstream of SNP rs329321577 (gene names are marked in red, and molecular marker sites are marked in black);

[0020] Figure 3 Association analysis of different allelic genotypes of SNPs chr1:97217694 and chr1:97590304 with F:G;

[0021] Figure 4 301 KASP detection maps labeled chr1:97217694;

[0022] Figure 5 301 KASP detection maps labeled chr1:97590304. Detailed Implementation

[0023] 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.

[0024] Example

[0025] 1. Material screening and phenotypic recording

[0026] 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 F:G phenotypic data of these 118 Ningxiang pigs were collected using the formula F:G = Total Feed Intake / Total Weight Gain.

[0027] 2. Whole genome resequencing

[0028] Genomic DNA was extracted from collected pig ear tissues, and its purity was controlled by agarose gel electrophoresis. The quality-controlled genomic DNA was randomly fragmented into 350 bp segments, followed by end repair, addition of polyA tails, addition of sequencing adapters, purification, and PCR amplification to prepare a complete library. Paired-end PE150 sequencing was performed using the Illumina sequencing platform at a sequencing depth of 10×. Novogene provided professional technical services for both library preparation and sequencing.

[0029] 3. Genome-wide association analysis to locate target SNP loci.

[0030] 3.1 Genome-wide association analysis

[0031] After library construction and sequencing, raw reads were obtained. Low-quality bases, adapter sequences, and short reads were first filtered to obtain clean reads. Quality-controlled data were aligned to a reference genome using BWA. Genome-wide association analysis (GWA) was performed on the quality-controlled whole-genome resequencing data using rMVP (Yin L, Zhang H, Tang Ze et al. 2021). Trait association analysis was performed using a mixed linear model (MLM), with principal components added as covariates for model correction. GWA was performed on the collected pig F:G traits, and significantly associated genetic loci were identified using Manhattan plots. Figure 1 The significance threshold was calculated by taking the logarithm of the Bonferroni-corrected P-value (p = 0.05 / N, where N is the number of SNPs) and then subtracting log10. The significantly associated SNP sites were then obtained.

[0032] 3.2 Discovering significant marker sites

[0033] Significant marker sites for the porcine F:G trait were identified across the entire genome, including the significant SNP rs329321577 located by GWAS. Nine molecular markers were designed within a 500kb region upstream and downstream of this site. Figure 2 In the Ningxiang pig population, molecular markers at the rs326246446 (chr1: 97217694) and rs329321577 (chr1: 97590304) loci were found to have significant phenotypic effects.

[0034] 4. Design of SNP site sequencing primers and KASP primers

[0035] After obtaining the target SNP markers (chr1: 97217694 and chr1: 97590304), 1000 bp sequences before and after the SNPs were extracted (Table 1). The reference genome was Sus Scrofa Build 11.1. Sequencing primers (Table 2) and KASP primers (Table 3) were designed using the BatchPrimer3 online tool (http: / / probes.pw.usda.gov / batchprimer3 / ) for later material validation. The primers were synthesized by Invitrogen. The sequencing primers consist of two primers: a forward amplification primer Primer_F and a reverse amplification primer Primer_R.

[0036] The KASP marker system consists of three primers: two allele-specific primers, primer X (Primer_X) and primer Y (Primer_Y), and one universal primer C (Primer_C). The 5' ends of the two allele-specific primers are connected to the fluorophores FAM and HEX, respectively, specific to the KASP reaction from LGC. In genotyping, if only FAM fluorescence is detected in the sample, the genotype is homozygous allele X (Allele_X); if only HEX fluorescence is detected, the genotype is homozygous allele Y (Allele_Y); if both FAM and HEX fluorescence are detected, the genotype is heterozygous, meaning the sample carries both alleles X and Y.

[0037] Table 1. Site Information Table

[0038]

[0039]

[0040]

[0041] Table 2. Sequencing primer sequence information for F:G marker detection in Ningxiang pigs.

[0042]

[0043] Table 3. Alleles (Allele_X, Allele_Y) and primer sequences of KASP markers detected by F:G markers in Ningxiang pigs.

[0044]

[0045] 5. Material validation (next-generation sequencing validation, KASP validation)

[0046] 5.1 Sequencing Validation

[0047] Phenotypic data of 301 Ningxiang pigs (F:G) were collected. Target SNP markers were detected using these 301 Ningxiang pig samples. The association between marker genotype and phenotype was tested, and the T-test was used for comparative analysis between different genotypes.

[0048] The results showed that the chr1:97217694 locus represented three genotypes (CC, CT, and TT) in the Ningxiang pig population. The average F:G ratio was 4.43 for CC-type Ningxiang pigs, 4.61 for CT-type, and 4.83 for TT-type. The F:G ratio of CC-type Ningxiang pigs was significantly different from that of CT-type (p < 0.05), and highly significantly different from that of TT-type (p < 0.01).

[0049] Furthermore, at the chr1:97590304 locus, three genotypes (GG, GT, and TT) were observed in the Ningxiang pig population. The average F:G ratio in GG-type Ningxiang pigs was 4.43; in GT-type Ningxiang pigs, it was 4.61; and in TT-type Ningxiang pigs, it was 4.83. The F:G ratio in GG-type Ningxiang pigs was significantly different from that in GT-type Ningxiang pigs (p < 0.05), and the F:G ratio in GG-type Ningxiang pigs was extremely significantly different from that in TT-type Ningxiang pigs (p < 0.01). Figure 3 ).

[0050] Based on the data analysis, 181 Ningxiang pig samples had the genotype CC at chr1:97217694 and the genotype GG at chr1:97590304; 104 Ningxiang pig samples had the genotypes CT and GT at these two loci; and the remaining 16 Ningxiang pig samples had the genotype TT at both chr1:97217694 and chr1:97590304. Genotyping analysis of the Ningxiang pig samples revealed a clear genotype correspondence between chr1:97217694 and chr1:97590304: 181 samples were CC / GG, 104 were CT / GT, and 16 were TT / TT. Due to this strong association between genotypes and their significant correlation with phenotype, it is speculated that there is a linkage effect between these two loci, and the CC / GG genotype combination is a favorable genotype.

[0051] 5.2KASP tag verification

[0052] 5.2.1 KASP Reaction Procedure

[0053] KASP-tagged reactive sequencing was performed using the Douglas Scientific Array Tape system. The Array Tape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0054] PCR reaction system: The PCR amplification system was automatically assembled using NEXAR, and the PCR reaction system is shown in Table 4 below.

[0055] Table 4. PCR reaction system for KASP marker genotyping

[0056]

[0057]

[0058] PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: 94℃ for 15 minutes; 94℃ for 20 seconds, 65℃-57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 30 cycles.

[0059] Signal scanning and genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotyping and data analysis were performed using INTELLICS.

[0060] 5.2.2 KASP Test Results

[0061] To test the specificity and practicality of the markers in this invention, the target SNP markers were detected using 301 samples of Ningxiang pigs.

[0062] Verification showed that the chr1:97217694 locus marker divided the genotypes into three distinct and compact clusters. The KASP marker genotyping diagram is shown below. Figure 4 As shown in the figure, the upper left circle cluster indicates that the sample contains a homozygous C:C allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous T:T allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous C and T allele at this KASP marker site. The results showed that 181 samples contained a homozygous C:C allele; 16 samples contained a homozygous T:T allele; and 104 samples contained a heterozygous C and T allele, consistent with the genotyping results in the sequencing reaction.

[0063] The chr1:97590304 locus markers divided the genotypes into three distinct and compact clusters. The KASP marker genotyping diagram is shown below. Figure 5As shown in the figure, the upper left circle cluster indicates that the sample contains a homozygous G:G allele at this KASP marker locus, the lower right circle cluster indicates that the sample contains a homozygous T:T allele at this KASP marker locus, and the middle circle cluster indicates that the sample contains a heterozygous G and T allele at this KASP marker locus. The results showed that 181 samples contained a homozygous G:G allele; 16 samples contained a homozygous T:T allele; and 104 samples contained a heterozygous G and T allele, consistent with the genotyping results in the sequencing reaction. These results indicate a linkage effect between the chr1:97217694 and chr1:97590304 loci.

[0064] In summary, this invention, based on whole-genome sequencing data from 118 purebred Ningxiang pigs, combined with genome-wide association analysis of the F:G trait, identified two SNP linkage loci (chr1: 97217694 and chr1: 97590304) that have significant phenotypic effects on Ningxiang pig F:G. For these linkage loci, a matching sequencing primer set and KASP primer set were designed, enabling precise genotyping detection of the target loci, providing a reliable technical tool for pig genetic improvement and precise trait selection.

[0065] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0066] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of a molecular marker associated with the feed conversion ratio trait of Ningxiang pigs in the detection or auxiliary detection of the feed conversion ratio trait of Ningxiang pigs, characterized in that, The molecular markers are SNP1 and SNP2; the SNP1 molecular marker corresponds to the 97217694th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or T; the SNP2 molecular marker corresponds to the 97590304th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or T.

2. The application of a molecular marker associated with the feed conversion ratio trait in Ningxiang pigs in early screening of the feed conversion ratio, characterized in that, The molecular markers are SNP1 and SNP2; the SNP1 molecular marker corresponds to the 97217694th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or T; the SNP2 molecular marker corresponds to the 97590304th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or T.

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