SNP (Single Nucleotide Polymorphism) molecular marker related to Ningxiang pig feed conversion efficiency character and application of SNP molecular marker

Abstract

The invention relates to the technical field of biology, in particular to an SNP molecular marker related to the Ningxiang pig feed conversion efficiency character and application of the SNP molecular marker. The molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker is A or G corresponding to the 97685244th site from the 5'end on a chromosome 1 of which the reference genome is Sus Scrofa Build11.1; the SNP2 molecular marker is A or C corresponding to the 97782417th site from the 5'end on the chromosome 1 of which the reference genome is Sus Scrofa Build11.1. The invention also discloses a method for detecting the Sus Scrofa Build11.1 in the invention, and the Sus Scrofa Build11.1 in the invention. The molecular marker remarkably related to the feed conversion efficiency of the Ningxiang pigs is obtained, guidance is provided for breeding of the Ningxiang pigs by utilizing the molecular marker, the feed conversion efficiency and typing can be accurately and efficiently predicted, pig breeds with high feed conversion efficiency and excellent meat quality can be identified and screened, and the breeding efficiency is improved.

Description

Technical Field

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

[0002] As a core source of protein for humans, improving pork production efficiency has become one of the core development directions of animal husbandry. Feed conversion efficiency (FCE), a key indicator for measuring how well an animal converts feed into weight gain, directly determines the amount of feed consumed per unit of output. Optimizing FCE can significantly reduce production costs and is a key breakthrough for improving the economics and sustainability of animal husbandry.

[0003] Traditional breeding methods rely on phenotypic selection, which suffers from drawbacks such as long research cycles and slow genetic progress, making it difficult to meet the needs of the modern pig industry. 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 free cervical ovarian hyperplasia (FCE). Therefore, utilizing novel molecular breeding techniques such as molecular markers to conduct genome-wide association studies on important traits like FCE, identify relevant candidate genes, and develop genomic information-based assisted breeding technologies is an inevitable choice to overcome industry bottlenecks.

[0004] Chinese invention patent CN 113699246 A discloses a SNP molecular marker that affects the feed conversion efficiency trait of pigs and its uses. However, it is aimed at Duroc pigs. The prior art has not disclosed SNP molecular markers related to the feed conversion efficiency trait of Ningxiang pigs. Duroc pigs and Ningxiang pigs are two significantly different pig breeds. Although they have the same traits, the traits are affected by many sites, resulting in significant differences in SNP molecular markers related to the FCE trait between Duroc pigs (lean type) and Ningxiang pigs (fat type). Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, the present invention aims to provide a SNP molecular marker related to feed conversion efficiency (FCE) traits in Ningxiang pigs and its application. This marker can assist in screening for feed conversion efficiency (FCE) in Ningxiang pigs, thereby improving the breeding of superior traits in Ningxiang pigs.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] Firstly, a SNP molecular marker related to the feed conversion efficiency 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 97685244th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or G. 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 97782417th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or C.

[0008] Secondly, this paper provides the application of the aforementioned SNP molecular markers in detecting or assisting in the detection of feed conversion efficiency in Ningxiang pigs.

[0009] Thirdly, the application of the above-mentioned SNP molecular markers in the early screening of feed conversion efficiency in Ningxiang pigs is provided.

[0010] Fourthly, the application of the aforementioned SNP molecular markers in marker-assisted breeding of Ningxiang pigs is provided.

[0011] Fifthly, the application of the aforementioned SNP molecular markers in the screening of Ningxiang pig breeds is provided.

[0012] Furthermore, there is a linkage effect between the SNP1 and SNP2 loci, and the AA / AA genotype combination is a favorable genotype.

[0013] The beneficial effects of this invention are as follows:

[0014] (1) This invention has obtained a molecular marker that is significantly related to the feed conversion efficiency of Ningxiang pigs. Using this molecular marker to provide guidance for the breeding of Ningxiang pigs, it can accurately and efficiently predict feed conversion efficiency and typing, identify and screen pig breeds with high feed conversion efficiency and excellent meat quality, and improve breeding efficiency.

[0015] (2) This invention provides sequencing primer pairs and KASP primer pairs for detecting SNP molecular markers related to feed conversion efficiency 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 the breeding of Ningxiang pork quality and efficiently identify the feed conversion efficiency.

[0016] (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

[0017] Figure 1Manhattan plot for genome-wide association analysis of FCE in Ningxiang pig population;

[0018] 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);

[0019] Figure 3 Association analysis of different allelic genotypes at SNP chr1:97685244 and chr1:97782417 with FCE;

[0020] Figure 4 338 KASP detection maps labeled chr1:97685244;

[0021] Figure 5 338 KASP detection maps labeled chr1:97782417. Detailed Implementation

[0022] 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.

[0023] Example

[0024] 1. Material screening and phenotypic recording

[0025] 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 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 FCE phenotypic data of these 118 Ningxiang pigs were collected using the formula FCE = Total Feed Intake / Total Weight Gain.

[0026] 2. Whole genome resequencing

[0027] 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.

[0028] 3. Genome-wide association analysis to locate target SNP loci.

[0029] 3.1 Genome-wide association analysis

[0030] 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 porcine FCE 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.

[0031] 3.2 Discovering significant marker sites

[0032] Significant marker sites for the porcine FCE 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 a population of 118 Ningxiang pigs, molecular markers at the loci rs338257182 (chr1: 97685244) and rs331204472 (chr1: 97782417) were found to have significant phenotypic effects. NCBI sequence alignment confirmed that both loci were annotated onto the ZBTB7C gene.

[0033] 4. Design of SNP site sequencing primers and KASP primers

[0034] After obtaining the target SNP markers (chr1: 97685244 and chr1: 97782417), 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.

[0035] 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 fluorescent groups FAM and HEX, specific to the KASP reaction from LGC, respectively. 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.

[0036] Table 1. Site Information Table

[0037]

[0038]

[0039]

[0040] Table 2. Sequencing primer information for FCE marker detection in Ningxiang pigs

[0041]

[0042] Table 3. Alleles (Allele_X, Allele_Y) and primer sequences of KASP markers detected by FCE markers in Ningxiang pigs

[0043]

[0044] 5. Material validation (next-generation sequencing validation, KASP validation)

[0045] 5.1 Sequencing Validation

[0046] Target SNP markers were detected in 338 Ningxiang pig samples. The association between marker genotype and phenotype was tested, and the T-test was used to compare and analyze different genotypes.

[0047] The results showed that the chr1:97685244 locus represented three genotypes (AA, AG, and GG) in the Ningxiang pig population. The average free calorie (FCE) of AA-type Ningxiang pigs was 4.43; that of AG-type Ningxiang pigs was 4.59; and that of GG-type Ningxiang pigs was 4.83. The FCE of AA-type Ningxiang pigs was significantly different from that of AG-type Ningxiang pigs (p < 0.05); and the FCE of AA-type Ningxiang pigs was extremely significantly different from that of GG-type Ningxiang pigs (p < 0.01).

[0048] The chr1:97782417 locus identified three genotypes (AA, AC, and CC) in the Ningxiang pig population. The mean free calorie (FCE) of AA-type Ningxiang pigs was 4.43; that of AC-type Ningxiang pigs was 4.59; and that of CC-type Ningxiang pigs was 4.83. The FCE of AA-type Ningxiang pigs was significantly different from that of AC-type Ningxiang pigs (p < 0.05); and the FCE of AA-type Ningxiang pigs was extremely significantly different from that of CC-type Ningxiang pigs (p < 0.01).

[0049] According to the data analysis, 204 Ningxiang pig samples showed genotypes of AA at both chr1:97685244 and chr1:97782417 loci; 118 samples showed genotypes of AG and AC at these two loci; and the remaining 16 samples showed genotypes of GG and CC at these two loci. Genotyping analysis of the Ningxiang pig samples revealed a clear genotype correspondence at chr1:97685244 and chr1:97782417 loci: 204 samples were AA / AA, 118 were AG / AC, and 16 were GG / CC. 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 AA / AA genotype combination is a favorable genotype.

[0050] 5.2KASP tag verification

[0051] 5.2.1 KASP Reaction Procedure

[0052] 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.

[0053] PCR reaction system: The PCR amplification system was automatically assembled using NEXAR, and the PCR reaction system is shown in Table 4 below.

[0054] Table 4. PCR reaction system for KASP marker genotyping

[0055] reagents Final concentration Actual usage 100μM Primer_C 0.42μM 0.0033μL 100μM Primer_X 0.17μM 0.0013μL 100μM Primer_Y 0.17μM 0.0013μL 2×KASP Master Mix 1× 0.3945μL Ultrapure water 0.3995μL DNA (DNA is added to a tape membrane and then dried) 20ng-50ng Total volume 0.8μL

[0056] 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.

[0057] 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.

[0058] 5.2.2 KASP Test Results

[0059] To test the specificity and practicality of the markers in this invention, the target SNP markers were tested using 338 samples of Ningxiang pigs.

[0060] Verification showed that the KASP marker at the chr1:97685244 locus was divided 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 A:A allele at this KASP marker site, the lower right circle cluster indicates that the sample contains a homozygous G:G allele at this KASP marker site, and the middle circle cluster indicates that the sample contains a heterozygous A and G allele at this KASP marker site. The results showed that 204 samples contained a homozygous A:A allele; 16 samples contained a homozygous G:G allele; and 118 samples contained a heterozygous A and G allele, consistent with the genotyping results in the sequencing reaction.

[0061] The KASP marker at chr1:97782417 locus was divided 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 A:A allele at this KASP marker locus, the lower right circle cluster indicates that the sample contains a homozygous C:C allele at this KASP marker locus, and the middle circle cluster indicates that the sample contains a heterozygous A and C allele at this KASP marker locus. The results showed that 204 samples contained a homozygous A:A allele; 16 samples contained a homozygous C:C allele; and 118 samples contained a heterozygous A and C allele, consistent with the genotyping results in the sequencing reaction. These results indicate a linkage effect between the chr1:97685244 and chr1:97782417 loci.

[0062] In summary, this invention, based on whole-genome sequencing data from 118 purebred Ningxiang pigs and combined with genome-wide association analysis of the pig FCE trait, located two SNP linkage loci (chr1: 97685244 and chr1: 97782417) significantly associated with Ningxiang pig FCE. For these two 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.

[0063] 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.

[0064] 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. A SNP molecular marker associated with feed conversion efficiency traits in Ningxiang pigs, characterized in that, The molecular markers include SNP1 and SNP2; the sequence of the nucleic acid containing the SNP1 molecular marker is shown in SEQ ID No. 1, and the SNP1 molecular marker corresponds to the 97685244th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or G; the sequence of the nucleic acid containing the SNP2 molecular marker is shown in SEQ ID No. 2, and the SNP2 molecular marker corresponds to the 97782417th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is A or C.

2. The sequencing primers for SNP molecular marker detection as described in claim 1, characterized in that, The primer sequences for the SNP1 molecular marker are shown in SEQ ID No. 3 and SEQ ID No. 4; the primer sequences for the SNP2 molecular marker are shown in SEQ ID No. 5 and SEQ ID No.

6.

3. The KASP labeling system for SNP molecular marker detection as described in claim 1, characterized in that, The KASP marker system consists of three primers: two allele-specific primers, namely primer X and primer Y, and one universal primer C. The primer sequences of the KASP marker system for SNP1 molecular marker detection are shown in SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9; the primer sequences of the KASP marker system for SNP2 molecular marker detection are shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No.

12.

4. The application of the SNP molecular marker as described in claim 1 in detecting or assisting in the detection of feed conversion efficiency in Ningxiang pigs.

5. The application of the SNP molecular marker as described in claim 1 in screening feed conversion efficiency for pigs in Ningxiang.

6. The application of the SNP molecular marker as described in claim 1 in marker-assisted breeding of Ningxiang pigs.

7. The application of the SNP molecular marker as described in claim 1 in the screening of Ningxiang pig breeds.

8. The application according to any one of claims 4-7, characterized in that, The two loci, SNP1 and SNP2, exhibit a linkage effect, and the AA / AA genotype combination is a favorable genotype.

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