A SNP molecular marker related to average daily gain trait of ningxiang pigs and application thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a SNP molecular marker related to the average daily weight gain trait of Ningxiang pigs and application thereof. The molecular marker comprises SNP1 and SNP2; the SNP1 molecular marker corresponds to the 12624189th site from the 5' end on the chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or A; the SNP2 molecular marker corresponds to the 13057273th site from the 5' end on the chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or A. The present application obtains a molecular marker significantly related to the average daily weight gain of Ningxiang pigs, and provides guidance for the breeding of Ningxiang pigs by using the molecular marker, so that the average daily weight gain value and typing can be accurately and efficiently predicted, and the pig species with high average daily weight gain value can be identified and screened, and the economic benefit can be improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a SNP molecular marker associated with the average daily weight gain trait of Ningxiang pigs and its application. Background Technology

[0002] my country's pig farming industry has developed rapidly, making it a major pork producer and consumer in the world. However, my country is not yet a leading pig farming nation, and the overall level of its pig farming industry still lags far behind that of developed countries. Therefore, improving pig productivity and overall economic efficiency has always been the goal of pig farming enterprises and livestock workers.

[0003] Average Daily Gain (ADG) is a crucial indicator for measuring the growth rate of pigs, directly impacting feed utilization efficiency. In breeding, it is often used alongside traits such as lean meat percentage and backfat thickness as important breeding indicators. Studies have shown that increasing ADG can not only reduce feeding time but also lower feeding costs. Pig breeds with high ADG typically have higher lean meat growth potential and feed conversion efficiency, giving them a significant advantage in commercial farming. Currently, identifying candidate genes associated with ADG and developing molecular markers have become important strategies for rationally improving the economic benefits of pig farms.

[0004] SNP markers are widely used molecular markers. Compared with SSR molecular marker technology, ISSR molecular marker technology and other polymorphic markers have higher genetic stability. At the same time, due to the diversity of detection methods applicable to them, including TaqMan method, mass spectrometry, microarray method, sequencing method, etc., they can be used for rapid and large-scale screening. With the advancement of sequencing technology and the continuous reduction of sequencing costs, more and more SNP markers of species are being explored and applied.

[0005] Chinese invention patent CN 119614716 A discloses a SNP molecular marker related to the daily weight gain trait of pigs and its application. However, it is aimed at Duroc pigs. The prior art has not disclosed SNP molecular markers related to the ADG 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 ADG traits between Duroc pigs (lean type) and Ningxiang pigs (fat type). Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, the present invention aims to provide a SNP molecular marker associated with the average daily weight gain (ADG) trait of Ningxiang pigs and its application. This marker can assist in identifying or screening Ningxiang pigs with high ADG for breeding, gradually improving the growth rate and feed utilization efficiency of the entire pig herd and increasing economic benefits.

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

[0008] Firstly, a SNP molecular marker associated with the average daily weight gain 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 12624189th position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is either C or A. 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 13057273rd position from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is either G or A.

[0009] Secondly, this paper provides the application of the aforementioned SNP molecular markers in detecting or assisting in the detection of the average daily weight gain of Ningxiang pigs.

[0010] Thirdly, the application of the above-mentioned SNP molecular markers in early screening of average daily weight gain in Ningxiang pigs 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 CA / GA genotype combination is a favorable genotype.

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

[0015] (1) This invention has obtained a molecular marker that is significantly associated with the average daily weight gain of Ningxiang pigs. Using this molecular marker, we can provide guidance for the breeding of Ningxiang pigs, accurately and efficiently predict the average daily weight gain and genotyping, identify and screen pig breeds with high average daily weight gain, and improve economic benefits.

[0016] (2) The present invention provides sequencing primer pairs and KASP primer pairs for detecting SNP molecular markers related to the average daily weight gain of Ningxiang pigs. These primer pairs are highly specific and accurate, and can accurately obtain sequences containing the SNP molecular marker sites of the present invention. They can be applied to the breeding of Ningxiang pork quality and efficiently identify the level of average daily weight gain.

[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 genome-wide association analysis of ADG phenotype in Ningxiang pig population;

[0019] Figure 2 Molecular markers developed for the 500kb region upstream and downstream of SNP chr1:12624189 (gene names are marked in red, and molecular marker sites are marked in black);

[0020] Figure 3 Association analysis of different allelic genotypes of SNP chr1:12624189 and SNP chr1:13057273 with ADG;

[0021] Figure 4 KASP test charts for 244 materials labeled Chr1:12624189;

[0022] Figure 5 KASP test charts for 244 materials labeled Chr1:13057273. 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., 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. Data such as initial weight upon entry and final weight upon exit were collected, and ADG phenotypic data for 118 Ningxiang pigs were obtained using the formula: ADG = (final weight – initial weight) / number of days.

[0027] 2. Resequencing and Genotyping

[0028] 2.1 DNA Sample Extraction and Quality Control

[0029] One week before Ningxiang pigs entered the testing station, right ear tissue was collected from 118 purebred Ningxiang pigs using ear clippers. The tissue was placed in 1.5 mL sterile, enzyme-free centrifuge tubes containing 75% ethanol and stored at -20°C for whole-genome resequencing. DNA was extracted from the tissue samples using the CTAB method. The concentration of DNA samples was detected using a Qubit quantitative PCR instrument; purity was determined using a Nanodrop spectrophotometer based on absorbance ratios of 260 / 280 and 260 / 230. Samples that passed quality control were used for library preparation.

[0030] 2.2 Resequencing

[0031] DNA samples that passed inspection were randomly fragmented into 350bp fragments using a Covaris shredder. Library construction was performed using the TruSeq Library Construction Kit. The DNA fragments underwent end repair, polyA tailing, sequencing adapter addition, purification, and PCR amplification to complete the entire library preparation process. After library construction, preliminary quantification was performed using Qubit 3.0, diluting the library to 1ng / μL. Subsequently, the insert size of the library was detected using an Agilent 2100. Once the insert size met expectations, the effective concentration of the library was accurately quantified using q-PCR (effective concentration >2nM). After passing the library inspection, sequencing was performed on the Illumina platform using PE150 sequencing, based on the effective concentration and data output requirements. The sample genome resequencing depth was 10x. Library preparation and sequencing were professionally supported by Beijing Novogene Technology Co., Ltd. Bioinformatics analysis software was used to visualize the resequencing data, ultimately obtaining high-quality genotyping data.

[0032] 3. Genome-wide association analysis to locate target SNP loci.

[0033] 3.1 Genome-wide association analysis

[0034] Genome-wide association analysis (GWA) was performed on 118 resequencing data files of Ningxiang pigs using rMVP (Yin L, Zhang H, Tang Z et al. 2021). A mixed linear model (MLM) was used to calculate the model, and principal components were added as covariates to the model for correction. GWA analysis was then performed on the collected pig ADG phenotypic traits. The Manhattan plot is shown below. Figure 1 The significance threshold is the logarithm of the Bonferroni-corrected P-value (p = 0.05 / N, where N is the number of SNPs) minus log10, which is used to calculate the significantly associated SNP sites.

[0035] 3.2 Discovering significant marker sites

[0036] Significant marker sites for the ADG phenotypic trait were identified across the entire genome, including the significant SNP rs325055064 located by GWAS. Nine molecular markers were designed within a 500kb region upstream and downstream of the site. Figure 2 Using Ningxiang pigs as experimental material, molecular markers at the rs325055064 (chr1:12624189) and rs324871066 (chr1:13057273) sites were found to have significant phenotypic effects.

[0037] 4. Design of SNP site sequencing primers and KASP primers

[0038] After obtaining significant SNP markers from genome-wide association analysis, 1000 bp sequences before and after the SNP 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 BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) for later material validation. The primers were synthesized by Invitrogen.

[0039] The sequencing primers consist of two primers: the forward amplification primer Primer_F and the reverse amplification primer Primer_R.

[0040] The KASP marker consists of three primers: two allele-specific primers, X (Primer_X) and Y (Primer_Y), and one universal primer, C (Primer_C). The 5' ends of the allele-specific primers are connected to the LGC KASP reaction-specific fluorescent groups FAM and HEX, respectively. 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 (carrying both alleles X and Y).

[0041] Table 1: Site Information Table

[0042]

[0043]

[0044] Table 2: Sequencing primer sequence information for ADG marker detection in Ningxiang pigs

[0045]

[0046]

[0047] Table 3. Alleles (Allele_X, Allele_Y) and primer sequences of KASP markers detected by ADG markers in Ningxiang pigs

[0048]

[0049] 5. Material validation (next-generation sequencing validation, KASP validation)

[0050] 5.1 Sequencing Validation

[0051] The target SNP markers were detected using 224 Ningxiang pig samples, and ADG phenotype data of these 224 Ningxiang pig samples were collected. The association between marker genotype and phenotype was tested, and the T-test was used to compare and analyze different genotypes.

[0052] turn out: Figure 3 At the Chr1:12624189 locus, two genotypes (AA and CA) were observed in the Ningxiang pig population. The mean ADG value for AA-type Ningxiang pigs was 0.37, and the mean ADG value for CA-type Ningxiang pigs was 0.41. The ADG value of CA-type Ningxiang pigs was significantly different from that of GG-type Ningxiang pigs (p<0.01). At the Chr1:13057273 locus, two genotypes (GG and GA) were observed in the Ningxiang pig population. The mean ADG value for GG-type Ningxiang pigs was 0.37, and the mean ADG value for GA-type Ningxiang pigs was 0.41. The ADG value of GA-type Ningxiang pigs was significantly different from that of GG-type Ningxiang pigs (p<0.01).

[0053] The AA type Ningxiang pig at Chr1:12624189 and the GG type Ningxiang pig at Chr1:13057273 have the same genotype number, and the CA type Ningxiang pig at Chr1:12624189 and the GA type Ningxiang pig at Chr1:13057273 have the same genotype number. Due to the strong association between these genotypes and their significant correlation with the phenotype, it is speculated that Chr1:12624189 and Chr1:13057273 have a linkage effect, and the CA / GA genotype combination is a favorable genotype, showing obvious heterosis.

[0054] 5.2KASP tag verification

[0055] 5.2.1 KASP Reaction Procedure

[0056] 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.

[0057] PCR reaction system: The PCR amplification system was automatically assembled using NEXAR, and the PCR reaction system is shown in Table 4 below.

[0058] Table 4. PCR reaction system for KASP marker genotyping

[0059] 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

[0060] 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.

[0061] 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.

[0062] 5.2.1 KASP Test Results

[0063] To test the specificity and practicality of the markers in this invention, the target SNP markers were detected using 244 samples of Ningxiang pigs.

[0064] Validation showed that the KASP markers divided the cells into two distinct and compact clusters. The KASP marker genotyping diagram for chr1:12624189 is shown below. Figure 4 As shown in the figure. The lower right cluster of circles in the figure indicates that the sample contains homozygous A:A alleles at this KASP marker locus, and the middle cluster of circles indicates that the sample contains heterozygous C and A alleles at this KASP marker locus. The results showed that 166 samples contained homozygous A:A alleles, and 78 samples contained heterozygous C and A alleles, consistent with the genotyping results in the sequencing reaction. The KASP marker genotyping diagram for chr1:13057273 is shown below. Figure 5As shown in the figure, the lower right circle cluster indicates that the sample contains a homozygous G:G allele at this KASP marker site, and the upper left circle cluster indicates that the sample contains a heterozygous G and A allele at this KASP marker site. The results showed that 166 samples contained a homozygous G:G allele, and 78 samples contained a heterozygous G and A allele, consistent with the genotyping results in the sequencing reaction. This indicates a linkage effect between Chr1:12624189 and Chr1:13057273.

[0065] In summary, this invention, through genome-wide association analysis of resequencing data from 118 purebred Ningxiang pigs and the ADG phenotype, located two linked SNP marker sites (chr1:12624189 and chr1:13057273) significantly associated with the ADG phenotype in Ningxiang pigs. Sequencing primer pairs and KASP primer pairs were designed for identification analysis and large-scale detection. This allows for efficient and accurate identification and screening of ADG levels.

[0066] 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.

[0067] 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 reagent for detecting SNP molecular markers associated with the average daily weight gain trait of Ningxiang pigs in the detection or auxiliary detection of the average daily weight gain of Ningxiang pigs, characterized in that, The molecular markers are SNP1 and SNP2; the SNP1 molecular marker corresponds to the 12624189th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or A; the SNP2 molecular marker corresponds to the 13057273rd site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or A.

2. The application of a reagent for detecting SNP molecular markers associated with the average daily weight gain trait of Ningxiang pigs in early screening of average daily weight gain in Ningxiang pigs, characterized in that, The molecular markers are SNP1 and SNP2; the SNP1 molecular marker corresponds to the 12624189th site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is C or A; the SNP2 molecular marker corresponds to the 13057273rd site from the 5' end on chromosome 1 of the reference genome Sus Scrofa Build11.1, and is G or A.

3. The application according to claim 1 or 2, characterized in that, The reagents include a KASP labeling system for detecting the SNP molecular markers, wherein the primer sequences of the KASP labeling system for detecting the SNP1 molecular marker are shown in SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9; and the primer sequences of the KASP labeling system for detecting the SNP2 molecular marker are shown in SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.

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