SNP (Single Nucleotide Polymorphism) molecular marker combination related to pig body length character and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker combination related to a pig body length character and application, and belongs to the technical field of biology. The SNP molecular marker combination is located on a pig X chromosome and comprises SNP1, SNP2 and / or SNP3; the SNP1 locus, the SNP2 locus and the SNP3 locus are respectively positioned at the 467th basic group, the 473rd basic group and the 752nd basic group of the pig BHLHB9 gene; the basic group of the SNP1 site is C / T missense mutation. The SNP molecular marker related to the pig body length character is disclosed for the first time, is used for distinguishing individuals with medium and short body lengths and individuals with long body lengths, is suitable for domestic pig breeds and foreign pig breeds, and is wide in pig breed application range. The method for detecting the genotype of the SNP molecular marker disclosed by the invention can be used for quickly and effectively predicting the body length of a pig in an early stage at low cost, has a wide application prospect in the aspect of improvement of a pig variety strain, and can obtain an excellent economic value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a SNP molecular marker combination related to pig body length traits and an application thereof. Background Art

[0002] Body length is one of the most commonly used indicators to measure body shape traits, and its heritability is 0.29-0.55, which is a medium or above heritability trait. In livestock, body length traits are closely related to the meat production of animals. It can be used as an important indicator to improve livestock production performance and has important economic value. At the same time, the variation of body length is also one of the most important quantitative traits in the process of species evolution, and there are large differences between species and different breeds within species. As one of the main centers of domestication of domestic pigs, China has formed many distinctive local breeds driven by long-term selective evolution, which have the characteristics of high reproductive efficiency, strong disease resistance, and good meat quality. However, compared with foreign commercial pigs, they generally show the characteristics of small size. The huge differences in body size between different pig breeds provide an ideal research model for revealing the molecular mechanism of their body shape development and the genetic improvement of local pig breeds. In this context, the development of rapid, simple and effective molecular genetic markers for pig body length traits can be applied to large-scale population detection, early selection and breeding, increase the selection pressure for body length, and accelerate the progress of genetic improvement.

[0003] BHLHB9 (basic helix-loop-helix domain containing, class B, 9) is an important transcription factor in the B class protein of the basic / helix-loop-helix transcription factor family. It exists in the cytoplasm and nucleus and plays an important role in cell proliferation, determination and differentiation. Its functional domain contains a basic region and two α-helical regions (helix-loop-helix, HLH) connected by a loop region of variable length. Usually, the HLH domain first forms homologous or heterologous dimers or polymers, and then the basic region binds to DNA. Studies have shown that BHLHB9 may be an important hub gene involved in bone development. It is highly expressed in proliferating growth plate chondrocytes, and its expression pattern in vitro is consistent with that of the early cartilage marker gene collagen III. In humans and mice, the deletion of four gene fragments including BHLHB9 will cause human Xq22.1 deletion syndrome such as postnatal growth retardation and weight loss, indicating that BHLHB9 may be related to fetal body shape. However, there is still a large gap in the research on the role of BHLHB9 gene in the development of pig body length. In addition, it is still unclear whether there are molecular markers in the pig BHLHB9 gene that can be used for pig body length trait selection. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP molecular marker combination and application related to pig body length traits to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a SNP molecular marker combination associated with pig body length, wherein the SNP molecular marker combination is located on the pig chromosome X, and includes SNP1, SNP2 and / or SNP3;

[0007] The SNP1, SNP2 and SNP3 sites are located at the 467th base, the 473rd base and the 752nd base of the pig BHLHB9 gene, respectively;

[0008] There is a C / T missense mutation in the base of the SNP1 site.

[0009] The second technical solution of the present invention is a primer set for specifically amplifying the SNP molecular marker combination, comprising an upstream primer as shown in SEQ ID NO.1 or SEQ ID NO.3;

[0010] and a downstream primer as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0011] The third technical solution of the present invention is a method for identifying pig body length using the SNP molecular marker combination, detecting the genotype of the single nucleotide polymorphism site corresponding to the SNP molecular marker combination in the DNA of the pig sample to be identified, and judging the pig body length-related traits according to the genotype of the SNP molecular marker combination.

[0012] A fourth technical solution of the present invention is the application of the SNP molecular marker combination in breeding of pig body length-related traits.

[0013] A fifth technical solution of the present invention is the application of the SNP molecular marker combination in identifying or assisting in identifying traits related to pig body length.

[0014] The sixth technical solution of the present invention is the use of the SNP molecular marker combination in the preparation of products for identifying or assisting in the identification of pig body length-related traits.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] The present invention discloses for the first time a SNP molecular marker related to the pig body length trait, which is used to distinguish between individuals with medium-short body length and long body length, and is applicable to domestic and foreign pig breeds, and is adaptable to a wide range of pig breeds. The method for detecting the genotype of the SNP molecular marker of the present invention can predict the pig body length early, quickly, at low cost and effectively, and has broad application prospects in the improvement of pig breeds and strains, and can achieve excellent economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The number of overlapping genes (C) detected by the two methods of Fst (A) and RNA-seq (B) in Example 1 of the present invention.

[0019] Figure 2 This is the Fst graph for the comparison group of large and small body length pig breeds.

[0020] Figure 3 Figure 2 shows the haplotype distribution heat map (A) and linkage disequilibrium analysis diagram (B) within the 5 kb region above and below the BHLHB9 gene in small and large pigs, as well as the partial nucleotide sequence and predicted amino acid sequence (C) of the CDS region of the BHLHB gene. Blue letters mark the SNPs of synonymous mutations and the predicted amino acid changes, red letters mark the SNPs of missense mutations and the predicted amino acid changes. The pie chart shows the changes in allele frequencies in small pigs (left) and large pigs (right).

[0021] Figure 4 They are the 467th and 473rd bases in the CDS region of the BHLHB9 gene of Wuzhishan pig and Large White pig.

[0022] Figure 5 This is the sequencing peak diagram of the 752nd base mutation position in the CDS region of the BHLHB9 gene of Wuzhishan pig and Large White pig.

[0023] Figure 6 Schematic diagram of ApaI endonuclease analysis.

[0024] Figure 7 The results of agarose electrophoresis after ApaI digestion of the two genotypes. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0031] The embodiment of the present invention provides a SNP molecular marker combination associated with pig body length, wherein the SNP molecular marker combination is located on the pig chromosome X, and includes SNP1, SNP2 and / or SNP3;

[0032] The SNP1, SNP2 and SNP3 sites are located at the 467th base, the 473rd base and the 752nd base of the pig BHLHB9 gene, respectively;

[0033] There is a C / T missense mutation in the base of the SNP1 site.

[0034] It should be noted that the SNP1, SNP2 and SNP3 loci are completely linked.

[0035] The present invention has discovered for the first time that the fully linked SNP molecular marker in the pig BHLHB9 gene is associated with the pig's body length trait, and can therefore be used in pig marker-assisted selection breeding, that is, it provides a new use of the fully linked SNP molecular marker in the pig BHLHB9 gene in marker-assisted breeding of pig body length traits. The invention is not only simple to operate, fast in typing, accurate in results and low in cost, but also greatly improves the efficiency of trait selection, helps shorten the breeding cycle, and meets the actual needs of large-scale molecular marker-assisted selection.

[0036] It should be noted that the reference genome of the present invention is the pig reference genome (Sus scrofa) version 11.1.

[0037] In some specific embodiments, the SNP 1 has genotypes TT and CC.

[0038] The embodiment of the present invention also provides a primer set for specifically amplifying the SNP molecular marker combination, including an upstream primer as shown in SEQ ID NO.1 or SEQ ID NO.3;

[0039] and a downstream primer as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0040] The embodiment of the present invention also provides a method for identifying pig body length using the SNP molecular marker combination, detecting the genotype of the single nucleotide polymorphism site corresponding to the SNP molecular marker combination in the pig sample DNA to be identified, and judging the pig body length-related traits according to the genotype of the SNP molecular marker combination.

[0041] In some specific embodiments, the method for detecting the genotype of the single nucleotide polymorphism site corresponding to the SNP molecular marker combination in the pig sample DNA to be identified is: using the pig sample DNA to be identified as a template, amplifying using the primer set, then using the RFLP restriction endonuclease ApaI to digest the amplified product, and determining the genotype of the SNP molecular marker combination based on the digestion product.

[0042] In some specific embodiments, the method for determining the genotype of the SNP molecular marker combination based on the enzyme cleavage product is: if the enzyme cleavage product is 419bp, the genotype of the SNP1 site is TT; if the enzyme cleavage product is 271bp and 148bp, the genotype of the SNP1 site is CC.

[0043] In some specific embodiments, the method for determining pig body length-related traits is: if the genotype of SNP1 is TT, the pig to be identified is a medium-short body length individual;

[0044] If the genotype of SNP1 is CC, the pig to be identified is a long-bodied individual.

[0045] The embodiment of the present invention also provides the application of the SNP molecular marker combination in breeding of pig body length-related traits.

[0046] The embodiment of the present invention also provides the use of the SNP molecular marker combination in identifying or assisting in identifying pig body length-related traits.

[0047] The embodiment of the present invention also provides the use of the SNP molecular marker combination in the preparation of products for identifying or assisting in identifying pig body length-related traits.

[0048] The objective of the present invention is achieved through the following technical solutions:

[0049] First, by comprehensively analyzing the selection signal data and transcriptome data of large and small pig groups, the present invention successfully screened out that the BHLHB9 gene has the largest differentiation index in different groups, which can be used as a key candidate gene affecting the body length trait of pigs. Further, the region about 5kb upstream and downstream of BHLHB9 mRNA was detected, and 95 SNPs were found, and the entire region was in a state of complete linkage disequilibrium. In the CDS region of the BHLHB9 gene, 9 SNPs were found, of which 6 were synonymous mutations and 3 were missense mutations.

[0050] Then, using the genomes of 10 Wuzhishan pigs and 10 Large White pigs as templates, the partial base sequence of the pig BHLHB9 gene was sequenced using the PCR technology Singer. The sequence results were analyzed using chromasPro v1.41 software to find SNP mutation sites, and the SNP sites that affect the body length trait of pigs were screened out. Among them, there were CT missense mutations at sites 94980080, 94980086 and 94980365 of the X chromosome. When the site genotype is C, the pig's long body length trait is more obvious, and when the site genotype is T, the pig's short body length trait is more obvious.

[0051] Further, the present invention uses large-scale SNP typing work, with 20 Wuzhishan pigs (average body length), 10 Bama Xiang pigs (average body length), 6 Tibetan pigs (average body length), 10 Diannan small-eared pigs (average body length), and 30 Ding'an pigs (average body length) as the medium-short body length variety verification group, and 30 Duroc pigs (average body length), 14 Pietrain pigs (average body length), 20 Large White pigs (average body length) and 31 Landrace pigs (average body length) as the long body length variety verification group (the experimental pig body length data comes from "Chinese Livestock and Poultry Genetic Resources·Pig Records" May 2011), and it is found that the average body length of the CC genotype group is significantly longer than the average body length of the TT genotype group. Then the linear model of the GenABLE software package in R language was used for analysis and it was found that these SNP sites were significantly associated with the individual body length trait. The SNP allele frequency analysis was conducted using the local Chinese pig breeds Wuzhishan pig, Bama Xiang pig, Tibetan pig, Diannan small-eared pig and Dingan pig as medium-short body length pig breeds, and the long-bodied Duroc pig, Pietrain pig, Large White pig and Landrace pig. It was found that the gene frequency of the three SNP loci allele C in the local Chinese pig breeds was low, all below 0.10, while the gene frequency in the long-bodied pig group was extremely high, all above 0.93. This situation further confirmed the above-mentioned association analysis point of view: these three SNP loci are significantly associated with the pig body length trait, among which C is positively correlated with the long body length trait, and T is positively correlated with the medium-short body length trait.

[0052] Example 1

[0053] Acquisition of SNP molecular markers related to body length traits

[0054] This study analyzed the selection signal of the resequencing results of the smallest local pig breeds, Wuzhishan pig and Bama Xiang pig, and the relatively large foreign pig breeds, Large White pig and Duroc pig. The results showed that there were 657 differentially expressed genes in the four comparison groups of Wuzhishan pig vs Large White pig, Wuzhishan pig vs Duroc pig, Bama Xiang pig vs Large White pig, and Bama Xiang pig vs Duroc pig (see Figure 1 In addition, RNA-seq of thoracic vertebral cartilage tissues of Wuzhishan pigs and Large White pigs at the beginning of rapid growth and at the end of growth was performed at 4 months old. The results showed that there were 22 differentially expressed genes in the four comparison groups of 1-month-old Wuzhishan pigs vs Large White pigs, 4-month-old Wuzhishan pigs vs Large White pigs, 1-month-old Wuzhishan pigs vs 4-month-old Wuzhishan pigs, and 1-month-old Large White pigs vs 4-month-old Large White pigs (see Figure 1 The results of the combined analysis of selection signals and RNA-seq showed that there were 3 genes that overlapped with the differentially expressed genes (see Figure 1 Among them, the BHLHB9 gene has the largest differentiation index among groups and may be a key candidate gene involved in the development of pig body length (see Figure 2 ).

[0055] According to the pig BHLHB9 gene sequence (XM_005657883.3) published by NCBI, the BHLHB9 gene is located on the pig X chromosome and has a total length of 1677 bp. A total of 95 SNPs were found in the region about 5 kb upstream and downstream of BHLHB9 mRNA, and there were significantly different genotypes in large and small body length pigs (see Figure 3 A), in addition, the entire region is in complete linkage disequilibrium (see Figure 3 B). Furthermore, 9 SNPs were found in the CDS region of the BHLHB9 gene, of which 6 were synonymous mutations and 3 were missense mutations. The 9 SNPs showed significantly different genotypes and allele frequencies in large and small pigs (see Figure 3 C and Table 1).

[0056] Table 1 SNP sites of BHLHB9 gene

[0057]

[0058]

[0059] Using 10 Wuzhishan pigs and 10 Large White pigs genomes as templates, PCR technology Singer sequencing was used to detect the partial base sequence of the pig BHLHB9 gene. The primer sequences of this example are shown in Table 2. According to the sequencing peak graph, it was confirmed that there were indeed C / T mutations at the three missense mutation positions (see Figure 4 , 5 ).

[0060] Table 2 Specific primers for SNP detection of BHLHB9 gene

[0061]

[0062] Example 2

[0063] Establishment of a method for detecting SNP molecular markers associated with pig body length traits

[0064] 1. Detection of SNP loci at the population level using RFLP restriction endonucleases

[0065] According to the research results of Example 1 and the characteristics of the sequence, the restriction fragment length polymorphism (RFLP) was selected to detect the SNP molecular markers of Example 1 at the population level. A specific primer was used to introduce mutations upstream of the mutation position to create an ApaI endonuclease site (see Table 3). When the base at the first SNP site is C, it can be cut by ApaI, and when it is T, it cannot be cut by ApaI, thereby being used for typing (such as Figure 6 ). PCR amplification was performed using the primers in Table 3.

[0066] Table 3 Specific primers for detecting C / T mutations using ApaI digestion

[0067]

[0068] Table 4 ApaI restriction enzyme digestion reaction system

[0069]

[0070] The reaction system for PCR amplification was: SYBR Green Master Mix: 10 μL, upstream / downstream primers 0.5 μL each, cDNA: 1 μL, RNase-Free ddH2O: 8 μL.

[0071] The reaction program of PCR amplification was: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 72°C for 5 min, for a total of 30 cycles.

[0072] After the amplification is completed, the amplified product is digested using the reaction system in Table 4.

[0073] After enzyme digestion, 10 μL of the digested product was subjected to 2% agarose gel electrophoresis at 110V for 40 min. The specific typing can be performed according to the position of the electrophoresis bands. Figure 7 If the enzyme cleavage product is 419bp, the genotype of the SNP1 site is TT, and the pig to be tested is a medium-short body length individual; if the enzyme cleavage product is 271bp and 148bp, the genotype of the SNP1 site is CC, and the pig to be tested is a long body length individual.

[0074] Furthermore, the SNP allele frequency analysis was performed using the Chinese local pig breeds Wuzhishan pig, Bama Xiang pig, Tibetan pig, Diannan small-eared pig and Dingan pig as medium-short body long pig breeds, and the long body long type Duroc pig, Pietrain pig, Large White pig and Landrace pig. It was found that the gene frequency of the SNP locus allele C was low in Chinese local pig breeds, all below 0.10, while the gene frequency in the long body long type pig group was extremely high, all above 0.93. The specific distribution frequency is shown in Table 5 below.

[0075] Table 5 Genotype frequency and allele frequency of ChrX:94980080, ChrX:94980086 and ChrX:94980365 in different pig breeds

[0076]

[0077]

[0078] 2 Population-level association analysis

[0079] The SNP typing data and body length phenotypic data of the above 171 pigs (body length data are from the "Chinese Livestock and Poultry Genetic Resources·Pig" May 2011) were sorted out, see Table 6.

[0080] Table 6 Phenotypes of body length traits in different pig groups

[0081]

[0082] A linear regression model between the body length phenotype and the genotype frequency of SNP C was further constructed, and the linear regression model between the two was finally obtained as Y=72.101X+83.268, P=0.0003, indicating that the gene frequency in the SNP molecular marker of the present invention is significantly positively correlated with the long body length trait of pigs. In summary, the C→T site is related to the body length trait of pigs and can be used as a marker for the improvement of the breeding population with long body length.

[0083] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A SNP molecular marker combination associated with pig body length, characterized in that: The SNP molecular marker combination is located on the pig chromosome X, including SNP1, SNP2 and / or SNP3; The SNP1, SNP2 and SNP3 sites are located at the 467th base, the 473rd base and the 752nd base of the pig BHLHB9 gene, respectively; There is a C / T missense mutation in the base of the SNP1 site.

2. The SNP molecular marker combination according to claim 1, characterized in that: The SNP 1 has genotypes TT and CC.

3. A primer set for specifically amplifying the SNP molecular marker combination according to claim 1 or 2, characterized in that: comprising an upstream primer as shown in SEQ ID NO.1 or SEQ ID NO.3; and a downstream primer as shown in SEQ ID NO.2 or SEQ ID NO.

4.

4. A method for identifying pig body length using the SNP molecular marker combination of claim 1 or 2, characterized in that: The genotype of the single nucleotide polymorphism site corresponding to the SNP molecular marker combination in the pig sample DNA to be identified is detected, and the pig body length-related traits are determined according to the genotype of the SNP molecular marker combination.

5. The method according to claim 4, characterized in that The method for detecting the genotype of the single nucleotide polymorphism site corresponding to the SNP molecular marker combination in the pig sample DNA to be identified is: using the pig sample DNA to be identified as a template, amplifying using the primer set described in claim 3, then using the RFLP restriction endonuclease ApaI to digest the amplified product, and judging the genotype of the SNP molecular marker combination based on the digestion product.

6. The method according to claim 5, characterized in that The method for determining the genotype of the SNP molecular marker combination according to the enzyme cleavage product is: if the enzyme cleavage product is 419 bp, the genotype of the SNP1 site is TT; if the enzyme cleavage product is 271 bp and 148 bp, the genotype of the SNP1 site is CC.

7. The method according to claim 4, characterized in that The method for determining the pig body length-related traits is as follows: if the genotype of SNP1 is TT, the pig to be identified is a medium-short body length individual; If the genotype of SNP1 is CC, the pig to be identified is a long-bodied individual.

8. Use of the SNP molecular marker combination as claimed in claim 1 or 2 in breeding of pig body length-related traits.

9. Use of the SNP molecular marker combination as claimed in claim 1 or 2 in identifying or assisting in identifying traits related to pig body length.

10. Use of the SNP molecular marker combination as claimed in claim 1 or 2 in the preparation of products for identifying or assisting in the identification of pig body length-related traits.

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