Long white pig growth and reproduction SNP molecular marker and application thereof

Abstract

The application relates to the field of gene molecular breeding, discloses a long white pig growth and reproduction SNP molecular marker and application thereof, and is characterized in that the long white pig SNP molecular marker is composed of 306 SNP molecular markers, the physical position of the 306 SNP molecular markers is determined based on sequence alignment of a pig reference genome Sscrofa11.1; and the SNP molecular marker site information is specifically shown in Table 1 in the description. The long white pig growth and reproduction SNP molecular marker provided by the application can be used for low-cost and large-scale genotyping of long white pigs, is based on liquid phase chip technology, is flexible in site, and can increase new functional marker sites at any time in the later period.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic molecular breeding, and particularly to a growth and reproduction SNP molecular marker of Landrace and application thereof. BACKGROUND

[0002] Landrace has the characteristics of fast growth, high feed conversion rate, high lean meat rate, and so on. In addition, sows of Landrace have more offspring and more milk, and have very high breeding value. Landrace is often used as a female pig breed, and when used for crossbreeding with other pig breeds, can effectively improve the growth and reproduction performance of offspring. In modern pig production, Landrace is indispensable in any crossbreeding combination, and cannot be separated from Landrace in any matching line. Therefore, breeding improvement of Landrace is of great significance to the development of the pig industry.

[0003] With the development of molecular biology technology, molecular markers have been gradually applied to pig breeding. Single nucleotide polymorphism (SNP) as the third generation of genetic markers has been widely used due to its high coverage density, wide distribution, large number, low mutation rate, and high genetic stability, and is 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 on the market are mainly developed based on solid-phase technology, and have high detection accuracy, but have the disadvantages of poor flexibility, strict sample size requirement, and high customization cost. Limited by the high cost of resequencing means, the relatively low site density and flexibility of traditional solid-phase chips, it is difficult to be widely applied in Landrace production practice. Therefore, we developed a high-efficiency, rapid, and low-cost large-scale genotype detection tool based on genotyping by target sequencing (GBTS) technology for important traits of Landrace. For 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, greatly improving the application flexibility of liquid-phase chips. At the same time, liquid-phase chips have higher detection capacity, simple operation, larger reaction space, and in addition, the polymorphic sites upstream and downstream of the target site can also be genotyped when the liquid-phase chip detects the genotype of the target site, which is called multiple single nucleotide polymorphism cluster (mSNP). Based on the multiple single nucleotide polymorphism technology, liquid-phase chips can significantly increase the effective SNP markers without increasing the target site markers, and improve the information content of the chip, so liquid-phase chips have a broader application prospect.

[0004] Locus information is the data foundation for gene chip probe design, and locus selection is the core content and key step in the chip design process, its effectiveness directly determining the practical value of the chip. In pigs, several SNP chips have been developed, among which chips containing about 50,000 markers (50K) are the mainstream.

[0005] Geneseek, Neocate, and Jiangxi Agricultural University have successively developed Porcine SNP 50KBeadchip (Geneseek), KPS Porcine Breeding Chip v2 (Zhongxin No. 1), and Porcine GGP 50Kchip. These three chips are all solid-phase chips, each containing approximately 50,000 SNP markers covering the entire pig genome. These chips are designed for the entire pig breed, encompassing multiple traits across all breeds, but lack precision in identifying specific SNP loci for particular traits in specific breeds. Considering current pig breeding practices, Landrace pigs require extensive selection as maternal lines. For Landrace pig breeding, we focus more on their growth and reproductive performance; therefore, it is necessary to design and develop specialized breeding chips for these two traits specifically for this breed. Summary of the Invention

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

[0007] To achieve the above objectives, an application of Landrace pig SNP molecular markers in the selection of growth and reproductive traits in Landrace pigs is provided. The Landrace pig SNP molecular markers consist of 306 SNP molecular markers, the physical locations of which are determined by sequence alignment based on the pig reference genome Sscrofa11.1. The specific SNP molecular marker site information is 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 includes 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 as described in claim 1.

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

[0011] Application of at least one of the long white pig SNP molecular marker, long white pig SNP liquid phase gene chip and the above-mentioned kit in long white pig cluster analysis and kinship identification.

[0012] Application of at least one of the long white pig SNP molecular marker, long white pig SNP liquid phase gene chip and the above-mentioned kit in long white pig genetic diversity identification.

[0013] Application of at least one of the long white pig SNP molecular marker, long white pig SNP liquid phase gene chip and the above-mentioned kit in long white pig breeding or assisted breeding.

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

[0015] A breeding method of long white pig, characterized in that, comprising the following steps:

[0016] The DNA of the pig to be tested is detected by using at least one of the long white pig SNP molecular marker, long white pig SNP liquid phase gene chip and the above-mentioned kit, and the long white pig is selected for subsequent breeding.

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

[0018] (1) The long white pig growth and reproduction SNP molecular marker provided by the present application can be used for low-cost and large-scale genotyping of long white pigs, based on liquid chip technology, flexible site, and new functional marker sites can be added at any time in the later stage.

[0019] (2) The long white pig whole genome 50k SNP liquid phase chip is developed by using the multiple mononucleotide polymorphism technology. Compared with the solid phase chip, the SNP liquid phase chip designed in the present application can produce multiple SNP markers at a single amplification site (target site), so that the number of detectable SNPs is expanded to 1.5-2 times the number of sites, that is, under the premise of not increasing the cost, the marker information is effectively increased.

[0020] (3) The present application mainly identifies relevant sites through whole genome association analysis for growth and reproduction traits of long white pigs and designs chips. Compared with the same type of 50K chip, not only has better breed representativeness, but also more accurate probe sequences are designed specifically for long white pig breeds. In China, long white pigs are often used as female parents, and their reproduction and growth traits are the two most concerned traits by breeders. The chip designed based on the single breed has strong pertinence and higher accuracy, and the chip cost is lower. From the perspective of breeding or economy, the chip has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The number of SNP markers on different chromosomes for the growth and reproduction of Changbai pigs using 50K liquid phase chips: a. The horizontal axis represents 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 The distribution of all SNP markers on the chromosomes for the growth and reproduction of Changbai pigs using 50K liquid phase chips. The darker the color, the greater the marker distribution density.

[0023] Figure 3 The distribution of all SNP markers on the gene structure for the growth and reproduction of Changbai pigs using 50K liquid phase chips. The horizontal axis represents the gene structure type, and the vertical axis represents the number of sites under this type.

[0024] Figure 4 The MAF value statistics of all sites for the actual detection of 105 Changbai pigs using 50K liquid phase chips for the growth and reproduction of Changbai pigs. The greater 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 scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments.

[0026] Example 1

[0027] In this embodiment, a liquid phase chip for Changbai pig breeding is designed and prepared, and the specific steps are as follows:

[0028] 1) Obtain a set of SNP candidate sites at the whole genome level:

[0029] Step one: To obtain the whole genome sites, the actual production data and chip data (three chip data, respectively, Borealis 50K, Nuygen 50K and Shadow gene 180K) of 2111 Landrace pigs were collected and sorted, and the phenotypes included pig weight, age, production traits (backfat thickness, eye muscle area), and reproductive traits (total litter size, live litter size, total weaning weight). The pig genome data filling platform of South China Agricultural University was used to fill the chip data to the sequencing data level, and after filling, a total of 42523218 sites were included. PLINK software was used to filter SNPs, and according to the genomic data, the following filtering criteria were used: remove SNPs with detection rate <95%, minimum allele frequency <0.05, extreme Hardy-Weinberg equilibrium test P value less than 10-6, and no chromosomal location information and detection rate <90% of individuals. After filtering, a total of 6970664 SNP sites were obtained.

[0030] 2) Screening of SNP sites:

[0031] Based on the filled sequencing data, whole genome association analysis was performed for five traits. Mixed linear model was used to associate phenotypes and SNPs with pig weight and total litter size as covariates. Two production traits were involved in the association analysis of 1941 individuals, with individual weight as a covariate, total litter size and live litter size involved in the association analysis of 1051 individuals, and 805 individuals involved in whole genome association analysis of total weaning weight. The above three reproductive traits were all with individual total litter size as a covariate. According to the results of GWAS, the first 5000 sites with the smallest P value were selected, a total of 23236 SNPs. Among them, there were a total of 24 significant sites.

[0032] According to the pig QTL database, a total of 42163 QTLs of known important traits were selected, and the SNPs at the middle position of each QTL were selected as chip design candidate sites to ensure the wide application of the chip, a total of 31312 SNPs. The 23235 SNPs mined from two growth traits and three reproductive traits in this study and the 31312 SNPs related to multiple traits (including growth, reproduction, meat quality, feed reward and other important economic traits) in public data were combined, a total of 54428 SNPs. To ensure uniform distribution of SNPs on each chromosome, the 54428 SNPs were filtered to ensure that the distance between adjacent SNPs was greater than 1 kb, and the remaining 37115 SNPs were obtained.

[0033] To ensure the compatibility of the chip, some of the Geneseek and SM1 chip sites were combined with the sites in this study. According to the SNP spacing and filling the chromosome gap, a total of 21335 SNPs were selected for combination, and a total of 58450 SNPs were selected. Among them, the significant sites in GWAS and the sites close to the significant sites and the SNP sites in the top 5000 sites within the QTL were selected as the core sites of the chip, as shown in Table 1.

[0034] Table 1 Core SNP molecular marker site information of Changbai pig breeding chip

[0035]

[0036]

[0037]

[0038] 3) Further screening of SNP sites:

[0039] Further optimization of 58450 SNPs, combined with the technical parameters of the Boerdi liquid chip (① Select the probe content between 30%-80%; ② Select the number of homologous regions ≤5; ③ Select the probe region does not contain SSR, N region.) And determine the uniformity of distribution on each chromosome, including the specific interval value and the specific density of the dense distribution at both ends of the chromosome, then design the probe, finally design the site number of the growth and reproduction trait characteristic breeding chip is 52781. Then according to the capture stability, 51421 sites are selected as the final site set, which retains all the core SNP sites. The distribution of all sites on each chromosome is shown in Figure 1 and Figure 2 The distribution of all sites on the gene structure is shown in Figure 3 .

[0040] 4) Preparation of chip:

[0041] Boerdi Biotechnology Co., Ltd. synthesizes liquid phase capture probes based on target sites, and uses Genotyping By Target Sequencing (GBTS) technology to obtain the genotype of all SNPs in the target region by target enrichment. The Genotyping By Target Sequencing technology system is composed of two independent but interrelated technologies: and Both technologies 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, and these probes modified with biotin can hybridize with the target region in the denatured resequencing library to form double-stranded DNA. Streptavidin-coated magnetic beads are used to adsorb the molecules carrying biotin. After elution, amplification and sequencing, the genotypes of the target SNPs are finally obtained. At the same time, specific amplification primers are designed for multiple SNPs to be tested using the Bio-Rad software, which can inhibit the production of non-specific amplification and dimers during PCR reaction, so that thousands of primers can complete multiplex target amplification in the same PCR tube with high uniformity. Then, combined with the construction and sequencing of the second-generation sequencing library, the genotype state of the target site is finally revealed.

[0042] Example 2

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

[0044] 1) Obtain and extract genomic DNA from Changbai pig samples; select a total of 100 ear tissues of Changbai pigs, and use a high-throughput DNA extraction kit to extract sample DNA.

[0045] 2) Quality control of sample DNA: 1% agarose gel electrophoresis is used to detect the integrity of genomic DNA; according to the instrument use instruction, the concentration of DNA is measured by Nanodrop 2000 spectrophotometer to judge the degree of protein and RNA contamination.

[0046] 3) Take the quantitative DNA, and use restriction endonuclease for fragmentation. The broken DNA is end-repaired and connected with A tail. The A-tailed DNA fragments are connected with sequencing adapters using ligase, and then the library is purified using carboxyl-modified magnetic beads. The ligation product is added with Barcode-containing sequencing primers and high-fidelity PCR reaction system for PCR amplification. Different Barcodes are used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification product can be used for probe hybridization experiment. Take 500 ng of the constructed sequencing library, freeze-dry, add probes and hybridization reagents, denature and incubate at 65°C for 2 hours to complete the hybridization reaction. After washing the hybridization product, a round of PCR is performed to complete the construction of the hybridization capture library.

[0047] 4) In the quantitative DNA, add multiplex PCR Panel mix and multiplex PCR enzyme system, and place on the PCR instrument to complete the PCR reaction. After the PCR product is purified by carboxyl magnetic beads, the sequencing primer with Barcode and high-fidelity PCR reaction system are counted again for PCR amplification, and different Barcodes are used to distinguish different samples. After purification by carboxyl magnetic beads, the amplification product is obtained, that is, the multiplex PCR capture and library construction are completed.

[0048] 5) Through the above DNA sample, the corresponding product is used for targeted sequencing library construction, and finally the library preparation of all samples of the project is completed. After the library construction is completed, Qubit 2.0 is used for preliminary quantification, and the effective concentration of the library is accurately quantified by using the method of qPCR to ensure the quality of the library. After the library detection is qualified, the sequencing stage is entered.

[0049] 6) Information analysis is carried out according to the sequencing results, mainly including: data quality control (removing adapters and low-quality data), alignment with reference genome, variant detection and annotation analysis. According to the results in Table 2, the site detection rate of 100 samples of Landrace pigs is between 98.971% and 99.38%, and the average detection rate is 99.22%. The MAF distribution is shown in Table 2.

[0050] Figure 4

[0051] Table 2: SNP site detection results

[0052]

[0053]

[0054]

[0055] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples.​​

Claims

1. A primer set and / or liquid phase probe for detecting a SNP molecular marker of Long White pig in the selection of growth and reproduction traits of Long White pig, characterized in that, The long white pig SNP molecular marker is composed of 306 SNP molecular markers, physical positions of the 306 SNP molecular markers are determined based on sequence alignment of a pig reference genome Sscrofa11.1, and the physical positions are specifically as follows: The SNP molecular marker site information is shown in the above table.

2. A long white 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 the long white pig SNP molecular marker 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 long white pig SNP molecular marker described in claim 1.

4. Application of at least one of the long white pig SNP liquid phase gene chip described in claim 2 and the kit described in claim 3 in long white pig whole genome association analysis.

5. Application of at least one of the long white pig SNP liquid phase gene chip described in claim 2 and the kit described in claim 3 in long white pig cluster analysis.

6. Application of at least one of the long white pig SNP liquid phase gene chip described in claim 2 and the kit described in claim 3 in long white pig genetic diversity identification.

7. Application of at least one of the long white pig SNP liquid phase gene chip described in claim 2 and the kit described in claim 3 in long white pig growth and reproduction trait selection.

8. A method for breeding growth and reproduction traits in Large White pigs, characterized in that, comprising the following steps: detecting DNA of a pig to be tested by using at least one of the long white pig SNP liquid phase gene chip described in claim 2 and the kit described in claim 3, and selecting long white pigs for subsequent breeding.

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