SNP markers, amplification primers and their applications affecting production traits of Sujiang pigs

Abstract

The invention discloses a SNP marker, amplification primer and application that affect the production traits of Sujiang pigs. The SNP marker is located on the nucleotide sequence of the DST gene on chromosome 7 of pigs, and is selected from the first mutation site, the second mutation site and / or the third mutation site; the first mutation site is the 29153437th base (rs337582032) G>A mutation of chromosome 7 sequence of the pig reference genome 11.1 version sequence; the second mutation site is the 29154793rd base (rs341009127) G>A mutation of chromosome 7 sequence of the pig reference genome 11.1 version sequence; the third mutation site is the 29261358th base (rs331889218) G>A mutation of chromosome 7 of the pig reference genome 11.1 version sequence. The association analysis shows that the SNP marker is significantly correlated with the production traits of Sujiang pigs, and can be used in molecular marker-assisted selection breeding of Sujiang pigs.

Description

Technical Field

[0001] The invention relates to pig marker-assisted selection (MAS) technology and genetic breeding field, and in particular to SNP markers, amplification primers and applications that affect production traits of Sujiang pigs. Background Art

[0002] Runs of homozygosity (ROH) are a certain number of single nucleotide polymorphisms (SNPs) with a certain density on the genome that are homozygous. ROH information plays an important role in the identification of livestock and poultry kinship, genetic diversity analysis, inbreeding depression assessment, selection signal identification and functional gene screening.

[0003] The selection signals identified in the genome reveal traces of bidirectional selection in domesticated populations. In the process of artificial selection, the genomes of livestock and poultry populations are reshaped, the frequency of ROH in the selected regions increases, and even "ROH islands" are generated. Therefore, by analyzing ROH, selection signals can be identified and functional genes related to economic traits can be screened. There is a linear correlation between harmful homozygous variants and ROH fragments in the genome, and the frequency of homozygous harmful genes at pathogenic loci appearing on ROH is higher than that of normal genes. This is also consistent with the assumption that the mutation sites of harmful genes that cause inbreeding depression generally exist in a homozygous state.

[0004] In August 2013, Sujiang Pig passed the new livestock and poultry variety approval of the National Animal Genetic Resources Committee and obtained a new variety certificate ((Nong 01) New Variety Certificate No. 22). Its meat quality indicators have met the requirements of high-quality pork.

[0005] Based on this, a SNP marker and breeding method that utilizes the ROH information in the Sujiang pig genome to identify and develop molecular markers related to the production traits of Sujiang pigs and apply them to the breeding of high-quality Sujiang pigs, meet people's demand for high-quality pork, and promote the breeding of high-quality breeding pigs in my country is an urgent problem to be solved by the present invention. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to develop molecular markers that can be used to assist in the selection of production traits of Sujiang pigs, thereby providing a new direction for the genetic improvement of production traits of Sujiang pigs.

[0007] In order to achieve the above object, the present invention provides a SNP marker affecting the production traits of Sujiang pigs, wherein the SNP marker is located on the nucleotide sequence of the DST gene on chromosome 7 of the pig, and the site of the SNP marker is selected from at least one of the first mutation site, the second mutation site and the third mutation site; wherein,

[0008] The first mutation site is the G>A mutation at base 29153437 (rs337582032) of chromosome 7 in the pig reference genome version 11.1 sequence, corresponding to base 282 in the sequence shown in SEQ ID No: 11;

[0009] The second mutation site is the G>A mutation at base 29154793 (rs341009127) of chromosome 7 in the pig reference genome version 11.1 sequence, corresponding to base 288 in the sequence shown in SEQ ID No: 12;

[0010] The third mutation site is the G>A mutation at base 29261358 (rs331889218) on chromosome 7 of the pig reference genome version 11.1, corresponding to base 259 in the sequence shown in SEQ ID No:13.

[0011] Among them, SEQ ID No: 11 (403bp) is the sequence amplified by the first primer pair; SEQ ID No: 12 (430bp) is the sequence amplified by the second primer pair, and SEQ ID No: 13 (372bp) is the sequence amplified by the third primer pair, and the above three sites are completely linked, that is, if one mutation is detected, the conditions of the other two mutations can be deduced, and the effect of any one mutation on the production trait is consistent, that is, when rs337582032 is GG, rs341009127 and rs331889218 must be GG. Figure 6 The LD pattern analysis of these three SNP loci shows that the three SNPs are in a completely linked state (r 2 =1).

[0012] Preferably, the production trait associated with the SNP marker disclosed in the present invention is body length and / or live back fat thickness of Sujiang pigs.

[0013] The present invention also provides an amplification primer for the above-mentioned SNP marker, wherein the amplification primer is selected from at least one of a first primer pair, a second primer pair and a third primer pair, the first primer pair is used to amplify a gene fragment containing the first mutation site, the second primer pair is used to amplify a gene fragment containing the second mutation site, and the third primer pair is used to amplify a gene fragment containing the third mutation site; and,

[0014] The first primer pair is shown as SEQ ID No: 1 and SEQ ID No: 2;

[0015] The second primer pair is shown as SEQ ID No: 3 and SEQ ID No: 4;

[0016] The third primer pair is shown as SEQ ID No:5 and SEQ ID No:6.

[0017] It is necessary to further explain that the specific selection of the primer pair here is related to the selection of the aforementioned SNP markers to be detected, that is, the two are in a one-to-one correspondence. When the site of the SNP marker to be detected is the first mutation site, it is necessary to specifically amplify the gene fragment containing the first mutation site. At this time, the amplification primers here use the first primer pair. Similarly, when the site of the SNP marker to be detected is the second mutation site, the second primer pair is used accordingly; when the site of the SNP marker to be detected is the third mutation site, the third primer pair is used accordingly.

[0018] The present invention also provides an application of the SNP marker described above in breeding Sujiang pigs with advantageous production traits. When breeding Sujiang pigs with advantageous production traits, Sujiang pigs in which at least one of the first mutation site, the second mutation site and the third mutation site is a GG genotype are selected.

[0019] That is, by successive breeding of GG individuals at the 29153437th base position (rs337582032) on chromosome 7 of the pig reference genome version 11.1, the GA and AA individuals at this position are eliminated; and / or, by successive breeding of GG individuals at the 29154793th base position (rs341009127) on chromosome 7 of the pig reference genome version 11.1, the GA and AA individuals at this position are eliminated; and / or, by successive breeding of GG individuals at the 29261358th base position (rs331889218) on chromosome 7 of the pig reference genome version 11.1, the GA and AA individuals at this position are eliminated.

[0020] Preferably, the breeding process specifically includes: amplifying the gene fragment containing the site of the SNP marker to obtain an amplification product, performing genotyping on the obtained amplification product using sequencing technology, and screening Sujiang pigs with the GG genotype at the site of the SNP marker.

[0021] Since the SNP-marked site here can be any one or more of the three mutation sites, when the SNP-marked site includes the first mutation site, the amplification primers include the first primer pair shown in SEQ ID No: 1 and SEQ ID No: 2; when the SNP-marked site includes the second mutation site, the amplification primers include the second primer pair shown in SEQ ID No: 3 and SEQ ID No: 4; when the SNP-marked site includes the third mutation site, the amplification primers include the third primer pair shown in SEQ ID No: 5 and SEQ ID No: 6.

[0022] That is, here, according to the selected mutation site, one or more operations can be selected from the following three genotyping operations:

[0023] A. A first primer pair is used to amplify a gene fragment containing the G>A mutation at base 29153437 (rs337582032) of chromosome 7 of the pig reference genome version 11.1, the gene fragment is shown in SEQ ID No: 11, and then genotyping is performed using sequencing technology;

[0024] B. A second primer pair was used to amplify a gene fragment containing the G>A mutation at base 29154793 (rs341009127) of chromosome 7 of the pig reference genome version 11.1, the gene fragment being shown in SEQ ID No: 12, and then genotyping was performed using sequencing technology;

[0025] C. A third primer pair was used to amplify a gene fragment containing the G>A mutation at base 29261358 (rs331889218) on chromosome 7 of the pig reference genome version 11.1, as shown in SEQ ID No: 13, and then genotyping was performed using sequencing technology.

[0026] Through the above technical scheme, the present invention identified three effective molecular marker sites from the pig DST gene, wherein the first molecular marker is located at the G / A mutation site at base 29153437 (rs337582032) on chromosome 7 of the pig reference genome version 11.1, the second molecular marker is located at the G / A mutation site at base 29154793 (rs341009127) on chromosome 7 of the pig reference genome version 11.1, and the third molecular marker is located at the G / A mutation site at base 29261358 (rs331889218) on chromosome 7 of the pig reference genome version 11.1, and a convenient and reliable polymorphism detection method is established for the above three SNPs. By conducting association analysis between the above three SNP loci and the production traits of Sujiang pigs, it was found that their genotypes had a significant effect on the body length and live back fat thickness of Sujiang pigs (P<0.05), so that they can be applied to marker-assisted selection breeding of Sujiang pig production traits based on the above three SNPs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0028] Figure 1This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at the 29153437th base (rs337582032) of chromosome 7 of the pig reference genome version 11.1 in Example 2, wherein the black frame is the G / C mutation site at the 29153437th base (rs337582032) of chromosome 7 of the pig reference genome version 11.1, and the sequencing results of the GG, GA and AA genotypes are shown from top to bottom;

[0029] Figure 2 This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at the 29154793rd base (rs341009127) of chromosome 7 of the pig reference genome version 11.1 in Example 2, wherein the black frame is the G / C mutation site at the 29154793rd base (rs341009127) of chromosome 7 of the pig reference genome version 11.1, and the sequencing results of the GG, GA and AA genotypes are shown from top to bottom;

[0030] Figure 3 This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at the 29261358th base (rs331889218) of chromosome 7 of the pig reference genome version 11.1 in Example 2, wherein the black frame is the G / C mutation site at the 29261358th base (rs331889218) of chromosome 7 of the pig reference genome version 11.1, and the sequencing results of the GG, GA and AA genotypes are shown from top to bottom;

[0031] Figure 4 This is a sequencing result diagram of the amplified product of the gene fragment containing the T / G mutation site at position 29155663 (rs323990131) of chromosome 7 of the pig reference genome version 11.1 in Example 2, wherein the black frame is the T / G mutation site at position 29155663 (rs323990131) of chromosome 7 of the pig reference genome version 11.1, and the sequencing results of the TT, TG and GG genotypes are shown from top to bottom;

[0032] Figure 5 This is a sequencing result diagram of the amplified product of the gene fragment containing the A / T mutation site at position 29202080 (rs345119308) of chromosome 7 of the pig reference genome version 11.1 in Example 2, wherein the black frame is the A / T mutation site at position 29202080 (rs345119308) of chromosome 7 of the pig reference genome version 11.1, and the sequencing results of the AA, AT and TT genotypes are shown from top to bottom;

[0033] Figure 6This is the LD pattern analysis diagram of the three SNP sites rs337582032, rs341009127 and rs331889218 in Example 3. DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0035] The technical solution of the present invention is described in detail below through specific embodiments.

[0036] Example 1: Identification of SNPs in the Sujiang Pig DST Gene

[0037] (1) Detection of whole-genome ROH and high-frequency ROH regions

[0038] Neogen Biotech (Shanghai) Co., Ltd. was commissioned to use GeneSeek GGPPorcine 80K chips to perform genotyping on 365 180±5-day-old Sujiang pigs (refer to the genotype data published in Animal Genetics, 2020, 51(2): 314-318 to complete the genotyping of 365 Sujiang pigs). Plink software was used to perform quality control on the chip data of the Sujiang pig population. The quality control standards were: 1) individual detection rate ≥ 90%; 2) SNP detection rate ≥ 90%; 3) minor allele frequency (MAF) ≥ 0.01; 4) Hardy-Weinberg equilibrium P ≥ 10-6; 5) sex chromosomes were eliminated. 4235 SNPs passed quality control. ROH detection uses Plink software and a sliding window method to detect autosomes. The specific detection parameters are as follows: 1) ROH length is greater than 1Mb; 2) at least 50 consecutive SNPs; 3) the distance between consecutive SNPs is less than 1Mb; 4) a maximum of 5 deletions and 1 heterozygous in ROH is allowed; 5) the window threshold is 0.01; 6) the density is 1SNP / 50kb. A total of 9097 ROHs were detected. The proportion of the number of times each SNP participated in the formation of ROH in the Sujiang pig population to the number of samples was counted, and the top 1% of SNPs constituted a high-frequency ROH region. A total of 7 high-frequency ROH regions were detected, and the results are shown in Table 1.

[0039] Table 1

[0040] chromosome Starting position (bp) End position (bp) Length (bp) 1 25618647 27251841 1633194 2 149771432 151385319 1613887 7 27707775 29599648 1891873 7 104701201 106648050 1946849 8 135768303 138876853 3108550 15 131605148 133461343 1856195 18 44038621 46523411 2484790

[0041] (2) Identification of candidate genes and SNPs

[0042] Via Ensembl ASIAThe high-frequency ROH region was annotated by the database. GO and KEGG analysis were performed on the genes in the high-frequency ROH region, and the candidate gene DST was determined by combining the Pig QTL Database and related literature. The missense mutation of the DST gene was detected using 10× resequencing data of 27 Sujiang pigs (Animal, 2022, 16(8):100591), and SNPs with SIFT values ​​less than 0.05 were selected as candidate SNPs. The obtained candidate SNPs are shown in Table 2.

[0043] Table 2

[0044]

[0045]

[0046] Example 2: Genotyping of 5 SNPs of Sujiang pig DST gene

[0047] (1) Reference sequence download and primer design

[0048] From Ensembl ASIA The pig reference genome version 11.1 DST gene (ENSSSCG00000001499) was downloaded from the database, including the sequence of rs337582032 SEQ ID No: 11, the sequence of rs341009127 SEQ ID No: 12, the sequence of rs331889218 SEQ ID No: 13, the sequence of rs323990131 SEQ ID No: 14, and the sequence of rs345119308 SEQ ID No: 15. PCR primers were designed using Primer 5.0, as shown in Table 3.

[0049] Table 3

[0050]

[0051]

[0052] (2) PCR amplification

[0053] Reaction system: The reaction system is 40 μL, including 2×T8 High-Fidelity Master Mix (TSE111) 20 μL, forward primer (F) 2 μL, reverse primer (R) 2 μL, template DNA 1 μL, and ultrapure water 15 μL.

[0054] Reaction procedure: pre-denaturation at 98°C; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and 72°C for 10 s, for 35 cycles; extension at 72°C for 5 min; storage at 4°C.

[0055] (3) PCR product sequencing

[0056] The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in the figure Figure 1-Figure 5 As shown, Figure 1 This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at base 29153437 (rs337582032) on chromosome 7 of the pig reference genome version 11.1. Figure 2 This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at base 29154793 (rs341009127) of chromosome 7 of the pig reference genome version 11.1. Figure 3 This is a sequencing result diagram of the amplified product of the gene fragment containing the G / A mutation site at base 29261358 (rs331889218) on chromosome 7 of the pig reference genome version 11.1. Figure 4 This is a sequencing result diagram of the amplified product of the gene fragment containing the T / G mutation site at base 29155663 (rs323990131) of chromosome 7 of the pig reference genome version 11.1. Figure 5 This is a diagram of the sequencing results of the amplified product of the gene fragment containing the A / T mutation site at base 29202080 (rs345119308) on chromosome 7 of the pig reference genome version 11.1.

[0057] Example 3: Analysis of genotype frequency and allele frequency of 5 SNPs in the Sujiang pig DST gene

[0058] according to Figure 1-Figure 5The sequencing peak diagram in the genotyped the five SNP sites of the DST gene in the Sujiang pig population. Table 4 shows the genotype frequency and allele frequency analysis results of the five SNP sites in the Sujiang pig population (the specific sample number corresponding to the genotype is in the brackets in Table 4). The G / A mutation site at the 29153437th base (rs337582032) of chromosome 7 of the pig reference genome version 11.1 was successfully typed in 365 individuals in the Sujiang pig population. The results showed that 193 individuals had the GG genotype, 149 individuals had the GA genotype, and 23 individuals had the AA genotype. The G / A mutation site at the 29154793th base (rs341009127) of chromosome 7 of the pig reference genome version 11.1 was successfully typed in 365 individuals in the Sujiang pig population. The results showed that 193 individuals had the GG genotype, 149 individuals had the GA genotype, and 23 individuals had the AA genotype. The T / G mutation site at base 29155663 (rs323990131) on chromosome 7 of the pig reference genome version 11.1 was successfully typed in 365 individuals in the Sujiang pig population. The results showed that 142 pigs had the TT genotype, 170 pigs had the TG genotype, and 53 pigs had the GG genotype. The A / T mutation site at base 29202080 (rs345119308) on chromosome 7 of the pig reference genome version 11.1 was successfully typed in 365 individuals in the Sujiang pig population. The results showed that 154 pigs had the AA genotype, 176 pigs had the AT genotype, and 35 pigs had the TT genotype. The G / A mutation site at base 29261358 (rs331889218) on chromosome 7 of the porcine reference genome version 11.1 was successfully typed in 365 individuals in the Sujiang pig population. The results showed that 193 individuals had the GG genotype, 149 individuals had the GA genotype, and 23 individuals had the AA genotype.

[0059] The above results indicate that the five SNP variant sites have obvious polymorphism in the Sujiang pig population and have potential value for molecular marker opening. At the same time, the LD block diagram of the five SNP sites was analyzed, such as Figure 6 As shown, rs337582032, rs341009127, and rs331889218 are in a complete linkage state (r 2 =1).

[0060] Table 4

[0061]

[0062] Example 4: Association analysis between polymorphism of 5 SNP sites of Sujiang pig DST gene and production traits

[0063] One-way ANOVA in SPSS19.0 software was used to statistically analyze the association between different genotypes in the population and the production traits of Sujiang pigs. The results were expressed as mean standard deviation; LSD method was used for pairwise comparison between groups when variance was equal, and Tamhane's T2 test was used for analysis when variance was unequal, with P<0.05 as the criterion for significant difference. The results of the association analysis are shown in Tables 5-7 (the brackets in Tables 5-7 are the specific sample numbers corresponding to the genotype, and a, b, and c in the same row in Tables 5-7 are the significant difference levels P<0.05). Among them, the SNP loci corresponding to Table 5 are rs337582032 / rs341009127 / rs331889218, the SNP loci corresponding to Table 6 are rs323990131, and the SPN loci corresponding to Table 7 are rs345119308.

[0064] From Table 5, it can be seen that the body length of Sujiang pigs with genotype GG is significantly greater than that of genotype AA (P<0.05), and the back fat thickness of GG Sujiang pigs is significantly less than that of genotype AA (P<0.05), indicating that this SNP significantly affects the body length and back fat thickness of Sujiang pigs. Through auxiliary selection of this SNP locus in Sujiang pigs, the genetic progress of body length and back fat thickness of this group can be accelerated. In breeding work, individuals with GG genotype should be selected.

[0065] From Table 6, it can be seen that the weight, body length, body height and chest width of Sujiang pigs with genotype TT are significantly greater than those with genotype GG (P<0.05), and the back fat thickness of Sujiang pigs with TT is significantly less than that of GG (P<0.05), indicating that this SNP significantly affects the weight, body length, body height, chest width and back fat thickness of Sujiang pigs. The genetic progress of the weight, body length, body height, chest width and back fat thickness of the group can be accelerated by auxiliary selection of this SNP locus in Sujiang pigs. In breeding work, individuals with genotype TT should be selected.

[0066] From Table 7, it can be seen that the body length of Sujiang pigs with genotype AA is significantly greater than that of genotype TT (P<0.05), and the back fat thickness of Sujiang pigs with genotype AA is significantly less than that of genotype TT (P<0.05), indicating that this SNP significantly affects the body length and back fat thickness of Sujiang pigs. Through auxiliary selection of this SNP locus in Sujiang pigs, the genetic progress of body length and back fat thickness of this group can be accelerated. In breeding work, individuals with genotype AA should be selected.

[0067] Table 5

[0068]

[0069]

[0070] Table 6

[0071]

[0072] Table 7

[0073]

[0074] It can be seen from Examples 1-4 that the five SNP loci of the DST gene identified by the present invention have a significant effect on several indicators of the production performance of Sujiang pigs. Through the linkage status of multiple SNPs, the present invention further selects three SNPs of the Sujiang pig DST gene rs337582032, rs341009127, and rs331889218 as molecular markers.

[0075] The present invention identifies a candidate gene DST from the high-frequency ROH region of Sujiang pigs, then identifies potential genetic variation sites that can affect the production traits of Sujiang pigs from the DST gene, then establishes a simple molecular marker detection method for the corresponding genetic variation sites, and verifies the application effect of the molecular markers in the Sujiang pig herd, thereby developing a molecular marker-assisted breeding method that can be applied to at least one trait of body length and live back fat thickness of Sujiang pigs.

[0076] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0077] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0078] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An application of a SNP marker in breeding Sujiang pigs with advantageous production traits, characterized in that: When breeding Sujiang pigs with advantageous production traits, Sujiang pigs whose first mutation site, second mutation site and third mutation site are all GG genotypes are selected; wherein the SNP marker is located on the nucleotide sequence of the DST gene on chromosome 7 of the pig; the first mutation site is the rs337582032 G>A mutation at base 29153437 of chromosome 7 sequence of the pig reference genome version 11.1; the second mutation site is the rs341009127 G>A mutation at base 29154793 of chromosome 7 sequence of the pig reference genome version 11.1; the third mutation site is the rs331889218 G>A mutation at base 29261358 of chromosome 7 of the pig reference genome version 11.1; the first mutation site, the second mutation site and the third mutation site are completely linked inheritance; the production trait is the body length and / or living back fat thickness of the Sujiang pig.

2. The use according to claim 1, characterized in that: The SNP-marked site is selected from at least one of the first mutation site, the second mutation site and the third mutation site. The breeding process specifically includes: amplifying a gene fragment containing the SNP-marked site to obtain an amplified product, performing genotyping on the obtained amplified product using sequencing technology, and screening Sujiang pigs with the SNP-marked site as the GG genotype.

3. The use according to claim 2, characterized in that: When the SNP-marked site includes a first mutation site, the amplification primers include a first primer pair as shown in SEQ ID No:1 and SEQ ID No:2; when the SNP-marked site includes a second mutation site, the amplification primers include a second primer pair as shown in SEQ ID No:3 and SEQ ID No:4; when the SNP-marked site includes a third mutation site, the amplification primers include a third primer pair as shown in SEQ ID No:5 and SEQID No:6.

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