A molecular marker for SNPs in Xiang pigs and its application

Abstract

This invention discloses a Hunan pig SNP molecular marker and its application. The Hunan pig SNP molecular marker of this invention includes at least one of 64,055 SNP molecular markers. The genomic positions of these 64,055 SNP molecular markers were determined by sequence alignment based on the pig reference genome Sscrofa11.1. The Hunan pig SNP molecular markers of this invention are derived from the diverse local pig breeds of Hunan, exhibiting broad coverage, good site specificity, strong universality, and high accuracy. They can effectively distinguish local pig breeds from different origins and provide important technical support for the molecular breeding application of Hunan pigs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a combination of SNP molecular markers from Xiang pigs and their applications. Background Technology

[0002] Hunan boasts abundant local pig genetic resources, including breeds such as Ningxiang pig, Shaziling pig, Daweizi pig, and Qianshao spotted pig. These breeds are highly popular among farmers and consumers due to their strong adaptability, tolerance to roughage, and unique meat flavor. Currently, the protection of local pig germplasm resources and the selection of superior breeds lag behind, leading to the degradation of desirable traits such as reproduction, meat quality, and disease resistance. Furthermore, the slow growth rate of local pigs severely impacts economic benefits. Utilizing efficient modern molecular breeding technologies and tools to develop and utilize the superior germplasm resources of local pigs in a multi-dimensional and multi-level manner is a crucial measure for revitalizing the Hunan pig (Xiang pig) industry.

[0003] Currently, widely used chips in pig molecular breeding, such as Neocate 50K and SMIC-1, are mainly designed for imported pig breeds. Due to the significant genetic differences between these breeds and Hunan pigs, these chips have poor applicability to Hunan pig populations and cannot meet the needs of Hunan pig molecular breeding. Therefore, there is an urgent need to develop a Hunan pig liquid-phase chip to effectively improve the accuracy of analyzing the genetic basis of important traits and mining superior gene resources in Hunan pigs, enhance the efficiency of independent breeding of core pig breeds, and lead the development of modern, distinctive, and high-quality pig breeding industry. This is of great strategic significance for promoting the high-quality development of the pig industry. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a SNP molecular marker for Xiang pigs.

[0005] The present invention also proposes a primer set and / or probe for detecting the above-mentioned SNP molecular markers.

[0006] This invention also proposes a liquid phase chip for Hunan pigs.

[0007] This invention also proposes a reagent kit for detecting Xiang pigs.

[0008] This invention also proposes the application of the above-mentioned Xiang pig SNP molecular markers, primer sets and / or probes, liquid phase chips or kits for detecting Xiang pigs.

[0009] This invention also proposes a breeding method for Xiang pigs.

[0010] In a first aspect, the present invention provides a Xiang pig SNP molecular marker, comprising at least one of 64,055 SNP molecular markers, wherein the genomic positions of the 64,055 SNP molecular markers are determined by sequence alignment based on the pig reference genome Sscrofa11.1, and the specific site information is shown in Table 1 below:

[0011] Table 1

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[0084] In some embodiments of the present invention, the Xiang pig includes at least one of Ningxiang pig, Daweizi pig, Xiaoer pig, Shaziling pig, Tiegu pig, Debao pig, Hechuan pig, and Qianshao flower pig.

[0085] In a second aspect of the invention, a primer set and / or liquid-phase probe for detecting the above-mentioned Xiang pig SNP molecular markers is proposed.

[0086] In a third aspect of the present invention, a Hunan pig liquid phase chip is provided, the Hunan pig liquid phase chip comprising the above-mentioned primer set and / or liquid phase probe.

[0087] In a third aspect of the invention, a kit is provided comprising the above-described primer set and / or liquid phase probe.

[0088] In some embodiments of the present invention, the kit further includes at least one of PCR reaction reagents and nucleic acid extraction reagents.

[0089] In a fourth aspect of the present invention, the application of the above-mentioned Xiang pig SNP molecular markers, kits or Xiang pig liquid phase chips is proposed, wherein the application is in the genotyping of Xiang pigs.

[0090] In some embodiments of the present invention, the application is applied to the cluster analysis of Hunan pigs.

[0091] In some embodiments of the present invention, the application is in the breeding or assisted breeding of Xiang pigs.

[0092] In some embodiments of the present invention, the breeding or assisted breeding of Xiang pigs includes at least one of the following: assessment of genetic diversity of Xiang pigs, genome-wide association analysis of Xiang pigs, identification of kinship of Xiang pigs, construction and gene localization of genetic maps of Xiang pigs, identification of germplasm resources of Xiang pigs, mining, identification and functional analysis of genes for important traits of Xiang pigs, molecular marker-assisted selection breeding of Xiang pigs, and genome-wide selection breeding of Xiang pigs.

[0093] In a fourth aspect of the present invention, a method for breeding Xiang pigs is proposed, comprising the following steps: using the above-mentioned Xiang pig SNP molecular marker, reagent kit or Xiang pig liquid phase chip to detect the pig DNA of the sample to be tested, and selecting Xiang pigs for subsequent breeding.

[0094] In some embodiments of the present invention, the detection is performed based on liquid-phase probe capture sequencing genotyping technology.

[0095] The present invention has at least the following beneficial effects:

[0096] The SNP molecular markers of this invention are derived from diverse local pig breeds in Hunan Province (Ningxiang pig, Daweizi pig, Xiaoer pig, Shaziling pig, Tiegu pig, Debao pig, Hechuan pig, and Qianshao spotted pig), offering broad coverage, good locus specificity, strong versatility, and high accuracy. They can effectively distinguish local pig breeds from different origins, providing crucial technical support for molecular breeding applications of Hunan pigs. The SNP molecular markers of this invention are used to prepare a 64K SNP liquid phase chip for Hunan pigs, capable of detecting 64K marker data simultaneously. Compared to traditional molecular markers such as SSR markers, this chip offers advantages such as high throughput and low cost per marker. Genotyping data is accurate and reliable, exhibiting good genetic stability and repeatability, facilitating automated detection, reducing labor costs, and enabling the integration of multiple genotyping data results. Utilizing the 64K SNPs of Hunan pigs of this invention... SNP liquid phase chips can be used for genotyping, genetic diversity assessment, cluster analysis, germplasm resource and kinship identification, gene map construction and gene localization, identification and functional analysis of important trait genes, molecular marker-assisted selection, genome-wide association analysis, and genome-wide selection of Xiang pig breed resources, and have broad application prospects.

[0097] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0098] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0099] Figure 1 This is a distribution map of SNP sites on chromosomes in Embodiment 2 of the present invention;

[0100] Figure 2 This is a statistical diagram showing the distribution of SNP sites on chromosomes in Embodiment 2 of the present invention;

[0101] Figure 3 This is a flowchart of the detection process based on liquid-phase probe capture sequencing genotyping technology in Embodiment 3 of the present invention;

[0102] Figure 4 This is a graph showing the results of sample detection rate and heterozygosity rate in Example 3 of the present invention;

[0103] Figure 5 This is a graph showing the genotypic consistency rate of duplicate samples in Example 3 of the present invention;

[0104] Figure 6 This is a clustering analysis diagram from Embodiment 4 of the present invention. Detailed Implementation

[0105] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0106] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0107] Example 1

[0108] This embodiment provides a combination of SNP molecular markers for Xiang pigs, including 64,055 SNP sites. The physical locations of the 64,055 SNP sites were determined based on sequence alignment with the pig reference genome (Sscrofa11.1).

[0109] The specific screening process is as follows:

[0110] 1. Acquisition of sequencing data for Xiang pig germplasm resources

[0111] Using second-generation DNA sequencing technology, whole-genome resequencing was performed on the diverse local pig breeds in Hunan and other regions (Ningxiang pig, Daweizi pig, Xiaoer pig, Shaziling pig, Tiegu pig, Debao pig, Hechuan pig, Qianshao spotted pig, Lantang pig, Guangdong black pig, Guangdong Xiaoer spotted pig, and Dahua white pig), obtaining whole-genome resequencing data of 131 representative local pig germplasm resources.

[0112] The obtained sequencing data were analyzed, and reads contaminated with adapters and low-quality reads (base quality value ≤ 5) were removed to obtain high-quality clean reads. The clean reads were aligned to the pig reference genome (Sscrofa11.1) using BWA software to obtain SAM files. The SAM files of each sample were merged using samtools, and an index was added to obtain BAM files. The BAM files were sorted using Picard software, removing unaligned reads and marking duplicate reads. Realignment was performed using the IndelRealigner command in the GATK package, and base quality value calibration was performed using BaseRecalibrator. The genotype of each sample was detected using GATK to obtain single-sample GVCF files. Finally, the CombineGVCFs command in the GATK package was used to merge all sample GVCF files. The merged files were filtered using the following criteria: QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0, resulting in a VCF file containing SNP variation information for 131 samples.

[0113] 2. Site screening

[0114] Based on the VCF files of all samples, the MAF value, detection rate, heterozygosity, and sequencing depth of each locus were calculated. Initial screening of loci was performed according to MAF ≥ 0.15, detection rate ≥ 0.9, heterozygosity ≤ 0.5, and sequencing depth ≥ 10X, resulting in 3,978,252 porcine SNP polymorphic loci.

[0115] Based on the physical location of porcine SNP polymorphic sites, probes were designed, and successfully designed sites were retained. Density adjustments were made based on the uniformity of sites across the entire genome to obtain 64,055 high-quality porcine SNP polymorphic sites, which were used to prepare a Xiang pig 64K SNP liquid-phase chip. The physical locations of the 64,055 SNP sites are shown in the table below.

[0116] Example 2

[0117] This embodiment provides a Xiangzhu 64K SNP liquid phase chip.

[0118] Based on the upstream and downstream sequences of 64,055 SNP polymorphic sites in Xiang pigs, a specific liquid-phase microarray probe was designed for each site, resulting in the preparation of a Xiang pig 64K SNP liquid-phase microarray. Using this microarray, multiple target sequences located at different genomic positions were captured and enriched through liquid-phase hybridization. Next-generation sequencing was then performed on the captured and enriched target regions to obtain the genotypes of all loci within the target regions.

[0119] The distribution of the selected 64K chips on pig chromosomes was statistically analyzed, and the results are as follows: Figure 1 As shown, 64K SNP sites were found to be evenly distributed across porcine chromosomes, with an average interchromosomal distance of 37.7 kb. The number of 64K chips screened on 20 chromosomes was statistically analyzed, and the results are as follows: Figure 2 As shown, since chromosome 1 is the longest, the 64K SNP sites are most numerous on chromosome 1, totaling 7392.

[0120] Example 3: Application of Xiangzhu 64K SNP liquid phase chip in porcine genotyping

[0121] This embodiment provides an application of the Xiangzhu 64K SNP liquid phase chip in pig genotyping.

[0122] Based on liquid-phase probe capture sequencing genotyping technology, the genotypes of 54 pig samples (including 6 duplicate samples) were identified using the Xiang pig 64K SNP liquid-phase chip prepared in Example 2. The specific method is as follows: Figure 3 As shown, the steps are as follows:

[0123] (1) After extracting the whole genome DNA of pigs, the DNA was digested into fragments of 100-500bp using enzyme digestion reagents, and then Taq enzyme was added for end repair.

[0124] (2) Using T4 ligase, the sequencing adapters were ligated to both ends of the fragmented DNA, and the ligation products were purified by magnetic beads and amplified to complete the library construction.

[0125] (3) Hybridize the probe with the library to be tested in solution. After the probe has hybridized with the target region, use streptavidin-coated magnetic beads to capture the probe-target region binding in liquid phase.

[0126] (4) After washing the captured library with cleaning solution, amplification and enrichment were performed, and the library was sequenced using a high-throughput sequencing platform.

[0127] (5) The raw data after high-throughput sequencing were processed by quality control filtering to remove reads containing adapter contamination and low quality. The BWA software was used to compare with the reference genome, and then the GATK software was used to analyze the mutation sites of the sequencing results to obtain genotype data for 64K sites.

[0128] Genotyping was performed on 48 samples, with 6 samples used for technical replication experiments, resulting in a total of 54 sample data. The results are as follows: Figures 4-5 As shown, from Figure 4 As can be seen, the average detection rate was 99.23%, and the average heterozygosity was 25.73%; from Figure 5 The results show that the genotype comparison of the six technically replicated samples had an average genotype consistency rate of 99.31%. The application results of the Xiangzhu 64K SNP liquid phase chip in pig genotyping show that the Xiangzhu 64K SNP liquid phase chip has a high genotype detection rate, high genotype accuracy, good stability, and excellent overall performance.

[0129] Example 4

[0130] This embodiment provides an application of the Xiangzhu 64K SNP liquid phase chip in cluster analysis.

[0131] Cluster analysis was performed on 105 local pig breeds (breeds known) from different sources using the 64K SNP liquid phase chip of Xiang pig prepared in Example 2. The experimental procedure was the same as in Example 3. After obtaining the 64K genotype data, cluster analysis was performed using Plink software.

[0132] The results are as follows Figure 6 As shown, the Xiangzhu 64K SNP liquid phase chip can effectively distinguish local pig breeds from different sources (Ningxiang pig, Xiaoer pig, Debao pig, Lantang pig, Dahua white pig, Hechuan pig, Yuedong black pig, Guangdong Xiaoerhua pig, Qianshao Hua pig, Tiegu pig, Daweizi pig and Shaziling pig), and the results are consistent with those of known breeds. Therefore, the Xiangzhu 64K SNP liquid phase chip of the present invention can be used for cluster analysis and identification of pigs.

[0133] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A liquid phase chip for Hunan pigs, characterized in that, The Xiang pig liquid phase chip includes a liquid phase probe for detecting Xiang pig SNP molecular markers; the Xiang pig SNP molecular markers consist of 64,055 SNP molecular markers, and the genomic positions of the 64,055 SNP molecular markers are determined by sequence alignment based on the pig reference genome Sscrofa11.1, and the specific site information is shown in Table 1 of the specification; the Xiang pig is at least one of Ningxiang pig, Daweizi pig, Shaziling pig, Tiegu pig and Qianshao flower pig.

2. A reagent kit, characterized in that, The kit includes a liquid-phase probe for detecting SNP molecular markers in Xiang pigs; the Xiang pig SNP molecular markers consist of 64,055 SNP molecular markers, the genomic locations of which are determined by sequence alignment based on the pig reference genome Sscrofa11.1, and the specific site information is shown in Table 1 of the instruction manual; the Xiang pig is at least one of Ningxiang pig, Daweizi pig, Shaziling pig, Tiegu pig, and Qianshao spotted pig.

3. The application of the Xiang pig liquid phase chip of claim 1 or the kit of claim 2 in the genotyping of Xiang pigs; wherein the Xiang pig is at least one of Ningxiang pig, Daweizi pig, Shaziling pig, Tiegu pig and Qianshao flower pig.

4. The application of the Xiang pig liquid phase chip of claim 1 or the kit of claim 2 in the cluster analysis of Xiang pigs; wherein the Xiang pig is at least one of Ningxiang pig, Daweizi pig, Shaziling pig, Tiegu pig and Qianshao flower pig.

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