Landrace growth and reproduction SNP (Single Nucleotide Polymorphism) molecular marker and application thereof

Abstract

The invention relates to the field of gene molecular breeding, and discloses a Landrace pig growth and reproduction SNP molecular marker and application thereof, and is characterized in that the Landrace pig SNP molecular marker is composed of 306 SNP molecular markers, and the physical positions of the 306 SNP molecular markers are determined by sequence alignment based on a pig reference genome Sscrofa11.1; the site information of the SNP molecular marker is specifically shown in the specification table 1. The Landrace growth and reproduction SNP molecular marker has the beneficial effects that the Landrace growth and reproduction SNP molecular marker provided by the invention can be used for low-cost and large-scale genetic typing of the Landrace, the sites are flexible based on a liquid chip technology, and new functional marker sites can be added at any time in the later period.

Description

Technical Field

[0001] The invention relates to the field of gene molecular breeding, and in particular to a Landrace pig growth and reproduction SNP molecular marker and application thereof. Background Art

[0002] Landrace pigs have the characteristics of fast growth, high feed utilization rate, high lean meat rate, etc., and sows have more litters and more milk, and have very high breeding value. Landrace pigs are often used as maternal pigs. When used to cross with other pig breeds, they can effectively improve the growth and reproductive performance of offspring. In modern pig production, Landrace pigs are an indispensable pig breed in any hybrid combination, and any supporting system cannot do without Landrace pigs. Therefore, the breeding and improvement of Landrace pigs is of great significance to the development of the pig industry.

[0003] With the development of molecular biology technology, molecular markers have gradually been applied to pig breeding. Among them, single nucleotide polymorphism (SNP), as a third-generation genetic marker, is widely used for its high coverage density, wide distribution, abundant number, low mutation rate and high genetic stability. It is also considered to be the best molecular marker at present. At the same time, SNP chips capable of high-throughput genotyping have emerged and have been widely used in various fields. At present, the mainstream SNP chips in the market are mainly developed based on solid-phase technology, with high detection accuracy, but have disadvantages such as poor flexibility, strict sample size requirements and high customization costs. Limited by the high cost of resequencing methods and the relatively low site density and flexibility of traditional solid-phase chips, it is difficult to promote and apply them on a large scale in the production practice of Landrace pigs. Therefore, we developed an efficient, fast and low-cost large-scale genotyping tool based on genotyping by target sequencing (GBTS) technology for important traits of Landrace pigs. In the field of pig breeding, the development of liquid phase chips is still in its infancy. Compared with solid-phase chips, liquid-phase chips do not require solid-phase carriers, which greatly improves the application flexibility of liquid-phase chips. At the same time, liquid-phase chips have higher throughput detection capabilities, simple operation, and larger reaction space. In addition, while the liquid-phase chip detects the genotype of the target site, the polymorphic sites upstream and downstream of the target site can also be genotyped, which is called multiple single nucleotide polymorphism (mSNP) cluster. Based on the multiple single nucleotide polymorphism technology, liquid-phase chips can significantly increase the effective SNP markers without increasing the target site markers, thereby improving the information content of the chip. Therefore, liquid-phase chips have broader application prospects.

[0004] Site information is the data basis for the design of gene chip probes, and site selection is the core content and key link in the chip design process. Its effect directly determines the practical value of the chip. In pigs, a variety of SNP chips have been developed, among which chips containing about 50,000 markers (50K) are the mainstream.

[0005] Geneseek, Neogen, Jiangxi Agricultural University, etc. have successively developed Porcine SNP 50KBeadchip (Geneseek), KPS Porcine Breeding Chip v2 (SMIC One), and Porcine GGP 50Kchip. All three chips are solid-phase chips, and all contain about 50,000 SNP markers covering the entire pig genome. These chips are designed for the entire pig breed, covering a variety of traits of all breeds of pigs, and the SNP sites for specific traits of specific breeds of pigs are not precise enough. Considering the actual breeding work in the current pig farming industry, a large number of Landrace pigs need to be selected as maternal parents. For the breeding of Landrace pigs, we pay more attention to their growth and reproductive performance, so it is necessary to design and develop special breeding chips for the selection of these two traits for this specific breed. Summary of the invention

[0006] The present invention provides a SNP molecular marker for growth and reproduction of Landrace pigs and its application, aiming to solve one of the technical problems existing in the above-mentioned prior art. It can be used for early selection of growth and reproduction traits of Landrace pigs, and provides an important tool for molecular marker-assisted breeding and combined breeding of Landrace pigs, playing an important role in the research of Landrace pig breeding.

[0007] In order to achieve the above-mentioned purpose, an application of a Landrace pig SNP molecular marker in the breeding of Landrace pig growth and reproduction traits is provided, characterized in that the Landrace pig SNP molecular marker consists of 306 SNP molecular markers, and the physical positions of the 306 SNP molecular markers are determined by sequence alignment based on the pig reference genome Sscrofa11.1; the SNP molecular marker site information is specifically shown in Table 1 of the specification.

[0008] A Landrace pig SNP liquid phase gene chip, characterized in that the SNP liquid phase gene chip comprises a primer set and / or a liquid phase probe for detecting a Landrace pig SNP molecular marker as described in claim 1.

[0009] A kit, characterized in that the kit comprises a primer set and / or a liquid phase probe for detecting the Landrace pig SNP molecular marker described in claim 1.

[0010] Application of at least one of the Landrace pig SNP liquid phase gene chip and the kit in Landrace pig genome-wide association analysis.

[0011] The invention discloses an application of at least one of a Landrace pig SNP molecular marker, a Landrace pig SNP liquid phase gene chip and the above-mentioned kit in Landrace pig cluster analysis and kinship identification.

[0012] The invention discloses an application of at least one of a Landrace pig SNP molecular marker, a Landrace pig SNP liquid phase gene chip and the above-mentioned kit in the identification of genetic diversity of Landrace pigs.

[0013] A use of at least one of a Landrace pig SNP molecular marker, a Landrace pig SNP liquid-phase gene chip and the above-mentioned kit in Landrace pig breeding or assisted breeding.

[0014] Furthermore, the breeding or assisted breeding includes at least one of assisted major effect gene selection, molecular assisted breeding, whole genome selection breeding, genetic map construction, gene positioning, species evolution analysis and germplasm resource identification.

[0015] A method for breeding Landrace pigs, characterized in that it comprises the following steps:

[0016] A Landrace pig SNP molecular marker, a Landrace pig SNP liquid phase gene chip and at least one of the above-mentioned kits are used to detect the DNA of the pig to be tested, and Landrace pigs are selected for subsequent breeding.

[0017] The beneficial effects of the present invention are:

[0018] (1) The SNP molecular markers for growth and reproduction of Landrace pigs provided by the present invention can be used for low-cost and large-scale genotyping of Landrace pigs. Based on liquid phase chip technology, the sites are flexible, and new functional marker sites can be added at any time in the later stage.

[0019] (2) The present invention uses the multi-single nucleotide polymorphism technology to develop a 50kSNP liquid phase chip for the whole genome of Landrace pigs. Compared with solid phase chips, the SNP liquid phase chip designed by the present invention can produce multiple SNP markers at a single amplification site (target site), expanding the number of detectable SNPs to more than 1.5-2 times the number of sites, that is, effectively increasing the marker information without increasing the cost.

[0020] (3) The present invention mainly identifies related sites and designs chips for growth and reproduction traits of Landrace pigs through genome-wide association analysis. Compared with the same type of 50K chip, it not only has better breed representativeness, but also designs more accurate probe sequences specifically for Landrace pigs. In my country, Landrace pigs are often used as maternal parents, and their reproduction and growth traits are the two traits that breeders are most concerned about. The chip designed for specific traits based on this single breed is highly targeted, more accurate, and has a low chip cost. Whether from a breeding perspective or an economic perspective, the chip has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The statistical results of the number of all SNP markers on different chromosomes of the 50K liquid phase chip for the growth and reproduction of Landrace pigs: a. The horizontal axis is the chromosome number; b. The left vertical axis represents the number of sites and segments; c. The right vertical axis represents the chromosome length

[0022] Figure 2 This is the distribution of all SNP markers on chromosomes of the 50K liquid phase chip for growth and reproduction of Landrace pigs. The darker the color, the greater the density of marker distribution.

[0023] Figure 3 This is the distribution of all SNP markers in the gene structure of the 50K liquid phase chip for growth and reproduction of Landrace pigs. The horizontal axis is the gene structure type, and the vertical axis is the number of sites under this type.

[0024] Figure 4 In order to use the 50K liquid phase chip for growth and reproduction of Landrace pigs, the MAF values ​​of all sites were actually detected in 105 Landrace pigs. The larger the MAF value, the better the polymorphism of the site. The horizontal axis MAF is the different ranges of MAF values, and the vertical axis is the number of sites in each MAF range. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0026] Example 1

[0027] This embodiment designs and prepares a liquid phase chip for Landrace pig breeding, and the specific steps are as follows:

[0028] 1) Obtain the SNP candidate site set at the whole genome level:

[0029] Step 1: In order to obtain the whole genome loci, the actual production data and chip data of 2111 Landrace pigs (three chip data, namely Boredi 50K, Newgen 50K and Shadow Gene 180K) were collected and sorted. The phenotypes included pig weight, age, production traits (back fat thickness, eye muscle area), and reproductive traits (total number of piglets born, number of piglets alive, and total weaning weight). The chip data was filled to the sequencing data level using the pig genome data filling platform of South China Agricultural University, and a total of 42523218 loci were included after filling. PLINK software was used to filter SNPs. According to the genomic data, the following filtering criteria were used: remove SNP detection rate <95%, minimum allele frequency <0.05, extremely inconsistent Hardy-Weinberg equilibrium test P value less than 10-6, and SNP sites without chromosome location information and individuals with detection rate <90%. After filtering, a total of 6970664 SNP sites were obtained.

[0030] 2) Screening of SNP sites:

[0031] Based on the filled sequencing data, genome-wide association analysis was performed for the five traits. A mixed linear model was used to associate the phenotype with SNP using pig weight and total parity as covariates. There were 1941 individuals involved in the association analysis of the two production traits, with individual weight as a covariate, 1051 individuals involved in the association analysis of total litter size and live piglet number, and 805 individuals involved in the genome-wide association analysis of total weaning weight. The above three reproductive traits all used individual total parity as a covariate. According to the results of GWAS, the top 5000 sites with the smallest P value were selected, totaling 23236 SNPs. There were 24 significant sites.

[0032] According to the pig QTL database, a total of 42,163 QTLs of all known important traits were selected, and the SNPs in the middle of each QTL were selected as candidate sites for chip design to ensure the wide application of the chip, with a total of 31,312 SNPs. The 23,235 SNPs mined for two growth traits and three reproductive traits in this study and the 31,312 SNPs related to multiple traits (including important economic traits such as growth, reproduction, meat quality, and feed conversion) in public data were combined, totaling 54,428 SNPs. In order to ensure that the SNPs are evenly distributed on each chromosome, the 54,428 SNPs were filtered to ensure that the distance between adjacent SNPs was greater than 1 kb, leaving 37,115 SNPs.

[0033] Finally, to ensure the compatibility of the chip, some sites of Geneseek and SMIC-1 chips were extracted and merged with the sites of this study. According to the SNP spacing and filling the chromosome gaps, a total of 21,335 SNPs were selected for merging, totaling 58,450 SNPs. Among them, the significant sites in GWAS and the points close to the significant sites and the SNP sites in the QTL of the top 5000 sites were used as the core sites of the chip, as shown in Table 1.

[0034] Table 1 Core SNP molecular marker loci information of Landrace pig breeding chip

[0035]

[0036]

[0037]

[0038] 3) Further screening of SNP sites:

[0039] The 58,450 SNPs were further optimized, combined with the technical parameters of the Boridi liquid phase chip (① Select probes with a content between 30% and 80%; ② Select the number of homology regions ≤ 5; ③ Select probe regions that do not contain SSR and N regions.) and the uniformity of distribution on each chromosome was determined, including the specific spacing value and the specific density of the denser distribution at both ends of the chromosome. After that, the probe design was carried out, and the number of sites for the growth and reproduction trait characteristic breeding chip was finally designed to be 52,781. Subsequently, based on the capture stability, 51,421 sites were selected as the final site set, in which all core SNP sites were retained. The distribution of all sites on each chromosome is shown in the figure. Figure 1 and Figure 2 The distribution of all sites in the gene structure is shown in Figure 3 shown.

[0040] 4) Preparation of chips:

[0041] Based on the synthesis of liquid capture probes based on the target site by Broady Biotechnology Co., Ltd., the genotypes of all SNPs in the target region are obtained by enriching the target site using the targeted sequencing genotyping (GBTS) technology. The targeted sequencing genotyping technology system consists of two independent but intertwined technologies: and Both technologies can achieve accurate capture of genomic target sites and target regions. According to the principle of DNA complementarity, one or more probes covering the target SNP are designed at each site to be tested. These probes modified with biotin can hybridize with the target region in the denatured resequencing library to form a double strand. The molecules carrying biotin are adsorbed by streptavidin-coated magnetic beads, and the genotype of the target SNP is finally obtained after elution, amplification and sequencing. At the same time, using Boredi software, specific amplification primers are designed for multiple SNP sites to be tested, and non-specific amplification and dimer production are inhibited during the PCR reaction, so that thousands of primers can complete multiple target amplifications in a highly uniform manner in the same PCR tube, and then combined with the second-generation sequencing library construction and sequencing to finally reveal the genotype status of the target site.

[0042] Example 2

[0043] In this example, the 51421 SNP molecular markers screened in Example 1 were used to detect DNA samples of 100 Landrace pigs. The specific steps are as follows:

[0044] 1) Acquisition of Landrace pig samples and extraction of genomic DNA: Ear tissues from 100 Landrace pigs were selected, and sample DNA was extracted using a high-throughput DNA extraction kit.

[0045] 2) Quality control of sample DNA: Use 1% agarose gel electrophoresis to detect the integrity of genomic DNA; according to the instrument instructions, use Nanodrop2000 spectrophotometer to measure the concentration of DNA and determine the degree of protein and RNA contamination.

[0046] 3) Take a quantitative amount of DNA, fragment it using restriction endonucleases, and connect the A tail to the broken DNA after end repair. Use ligase to connect the DNA fragments with the sequencing adapter, and then purify the library using carboxyl-modified magnetic beads. Add sequencing primers with barcodes and a high-fidelity PCR reaction system to the connection product for PCR amplification. Different barcodes are used to distinguish different samples. After purification by carboxyl magnetic beads, the amplified product can be used for probe hybridization experiments. Take 500ng of the constructed sequencing library, lyophilize it, add the probe and hybridization reagent, denature it and incubate it at 65°C for 2 hours to complete the hybridization reaction. After the hybridization product is washed with washing solution, another round of PCR is performed to complete the construction of the hybridization capture library.

[0047] 4) Add multiple PCR Panel mix and multiple PCR amplification enzyme system to the quantitative DNA and place it on a PCR instrument to complete the PCR reaction. After the PCR product is purified using carboxyl magnetic beads, it is again added to the sequencing primer with barcode and the high-fidelity PCR reaction system for PCR amplification. Different barcodes are used to distinguish different samples. After the amplified product is purified by carboxyl magnetic beads, the multiple PCR capture and library construction are completed.

[0048] 5) Use the above DNA samples and corresponding products to construct targeted sequencing libraries, and finally complete the library preparation of all samples in the project. After the library is constructed, Qubit2.0 is used for preliminary quantification, and the effective concentration of the library is accurately quantified using the qPCR method to ensure the quality of the library. After the library is qualified, it enters the sequencing stage.

[0049] 6) Perform information analysis based on sequencing results, mainly including: data quality control (removal of adapters and low-quality data), comparison with reference genome, variant detection and annotation

[0050] The results in Table 2 show that the site detection rate of 100 Landrace pig samples ranged from 98.971% to 99.38%, with an average detection rate of 99.22%. The MAF distribution is as follows Figure 4 shown.

[0051] Table 2 SNP site detection results

[0052]

[0053]

[0054]

[0055] The above shows and describes the basic principle and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments.

Claims

1. An application of a Landrace pig SNP molecular marker in the selection of growth and reproduction traits of Landrace pigs, characterized in that: The Landrace pig SNP molecular markers consist of 306 SNP molecular markers, and the physical positions of the 306 SNP molecular markers are determined by sequence alignment based on the pig reference genome Sscrofa11.1; the SNP molecular marker site information is specifically shown in Table 1 of the specification.

2. A Landrace pig SNP liquid phase gene chip, characterized in that: The SNP liquid phase gene chip comprises a primer set and / or a liquid phase probe for detecting a Landrace pig SNP molecular marker as described in claim 1.

3. A kit, characterized in that: The kit comprises a primer set and / or a liquid phase probe for detecting the Landrace pig SNP molecular marker described in claim 1.

4. Use of at least one of the Landrace pig SNP liquid-phase gene chip as described in claim 2 and the kit as described in claim 3 in genome-wide association analysis of Landrace pigs.

5. Use of at least one of the Landrace pig SNP molecular markers as claimed in claim 1, the Landrace pig SNP liquid phase gene chip as claimed in claim 2, and the kit as claimed in claim 3 in Landrace pig cluster analysis and kinship identification.

6. Use of at least one of the Landrace pig SNP molecular markers as claimed in claim 1, the Landrace pig SNP liquid phase gene chip as claimed in claim 2, and the kit as claimed in claim 3 in the identification of genetic diversity of Landrace pigs.

7. Use of at least one of the Landrace pig SNP molecular marker as claimed in claim 1, the Landrace pig SNP liquid phase gene chip as claimed in claim 2, and the kit as claimed in claim 3 in Landrace pig breeding or assisted breeding.

8. The use according to claim 7, wherein the breeding or assisted breeding includes at least one of assisted major effect gene selection, molecular assisted breeding, whole genome selection breeding, genetic map construction, gene mapping, species evolution analysis and germplasm resource identification.

9. A method for breeding Landrace pigs, characterized in that: The method comprises the following steps: using at least one of the Landrace pig SNP molecular markers as described in claim 1, the Landrace pig SNP liquid gene chip as described in claim 2, and the kit as described in claim 3 to detect the DNA of the pig to be tested, and selecting Landrace pigs for subsequent breeding.

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