SNP (Single Nucleotide Polymorphism) marker primer pair related to pig multirib character and application of SNP marker primer pair

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) marker primer pair related to pig multirib traits and application of the SNP marker primer pair. The SNP site is a nucleotide site of chromosome 7 rs701714758 of a reference sequence of an international pig genome version 11.1, has G / A polymorphism, is located in a protein coding region of an ABCD4 gene, and is remarkably associated with rib number characters of Duroc pigs, landrace pigs, large white pigs and Pietrain pigs through verification of whole genome association analysis and meta analysis. The invention further provides a primer pair for detecting the SNP marker, an upstream primer of the primer pair is SEQ ID NO: 2, and a downstream primer of the primer pair is SEQ ID NO: 3. The SNP marker can be applied to marker-assisted selection and whole genome selection of rib number characters of multiple varieties of pigs, can accurately screen a multi-rib number pig group, improves character prediction accuracy, increases pork and rib row yield, meets consumption demands, and has both social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a SNP marker primer pair related to a pig multi-rib trait and application thereof. BACKGROUND

[0003] The vertebrae of a pig is composed of cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and coccyx, wherein the number of thoracic vertebrae and lumbar vertebrae varies among different individuals and different breeds, and the number of thoracic vertebrae directly determines the number of ribs. The number of ribs is also one of the important factors affecting the body structure and carcass length of a pig. Generally, the more the number of ribs, the longer the carcass, and the lean meat ratio and edible part also increase. Studies have shown that the increase in the number of ribs is significantly positively correlated with the body length, lean meat ratio and carcass weight of a pig, which makes the number of ribs an important economic trait for measuring whether the body shape of a pig is excellent or not.

[0004] Systematically analyzing the genetic basis of the variation in the number of ribs of a pig and using molecular marker-assisted breeding technology to breed a multi-rib pig population have significant economic and breeding values for improving breeding efficiency and promoting the high-quality development of the pork industry.

[0005] Previous studies on the number of ribs mainly focused on a single breed, such as Su Huai pig, and the genetic background difference among different breeds may lead to limited applicability of related markers. Therefore, it is necessary to systematically screen and verify genetic markers for multiple commercial pig breeds (including Duroc, Landrace, Large White and Pietrain pigs) so as to establish a molecular detection system for the number of ribs that can be widely applied to multiple breeds. This not only helps to reveal the molecular regulation mechanism of the variation in the number of ribs, but also provides a more universal molecular tool for genetic improvement of a pig population. SUMMARY

[0006] The technical problem to be solved is that the traditional pig rib number breeding is slow and limited to a single breed. The purpose of the present application is to provide a SNP marker primer pair related to a pig multi-rib trait and application thereof. The application of the SNP marker and primer pair in breeding can screen a multi-rib pig population and provide a more universal molecular tool for genetic improvement of a pig population, which can promote the high-quality development of the pork industry.

[0007] The technical scheme is that a SNP marker primer pair related to a pig multi-rib trait is composed of an upstream primer shown in SEQ ID NO: 2 and a downstream primer shown in SEQ ID NO: 3.

[0008] Further, the SNP marker is located at the nucleotide site rs701714758 of pig chromosome 7 in the international pig genome reference sequence version 11.1, and the SNP marker site in SEQ ID NO: 1 is located at position 501, and there is G / A polymorphism.

[0009] Further, the pig is a Duroc pig, a Landrace pig, a Large White pig and a Pietrain pig.

[0010] Further, the process of identifying the above-mentioned SNP marker is as follows: (1) The rib number phenotype data of 1222 Duroc pigs, 508 Landrace pigs, 1018 Large White pigs and 448 Pietrain pigs were collected, and the genomes of these individuals were detected; (2) The whole genome association analysis was carried out in each pig breed using GEMMA v0.98.5 software; (3) The summary statistics of the whole genome association analysis of the four breeds were subjected to meta-analysis using METAL software; (4) The sites with significant association were screened, the phenotype differences of individuals with different genotypes at these sites were observed, and finally the nucleotide site rs701714758 of pig chromosome 7 was selected.

[0011] A method for detecting the SNP marker related to the rib number trait of the pig of the present application comprises PCR amplifying a sequence of the nucleotide site rs701714758 of pig chromosome 7 in the international pig genome reference sequence version 11.1, sequencing the amplification product, and determining the G / A polymorphism of the site.

[0012] Further, the genomic DNA of the Duroc pig, the Landrace pig, the Large White pig and the Pietrain pig is subjected to PCR amplification using the primer pair of the present application.

[0013] Further, the method comprises the following steps: Extract total DNA from pig ear tissue samples; Using the extracted pig genomic DNA as a template, the primer pair provided by the present application is used for PCR amplification; (3) The amplification product is sequenced, the sequencing result is analyzed, and the G / A polymorphism at position 501 of SEQ ID NO: 1 is determined.

[0014] The above-mentioned SNP marker primer pair related to the pig multi-rib trait is used for detecting the pig multi-rib trait and / or pig breeding.

[0015] A method for screening a multi-ribbed pig population, detecting the genotype of the nucleotide site rs701714758 on chromosome 7 of the international pig genome 11.1 version reference sequence, and breeding individuals with the genotype GG at the nucleotide site as reserve breeding pigs.

[0016] Further, the method for detecting the genotype of the nucleotide site rs701714758 on chromosome 7 of the international pig genome 11.1 version reference sequence is selected from PCR or genome sequencing. Beneficial effects

[0017] The SNP marker provided by the present application is significantly associated with the rib number trait of pigs of multiple breeds through verification of a large sample of 1222 Duroc pigs, 508 Landrace pigs, 1018 Large White pigs and 448 Pietrain pigs; the corresponding primer pair has strong amplification specificity and clear target fragments, and the detection method is simple to operate and accurate. When applied to breeding, it can quickly screen GG-type multi-rib individuals, significantly improve breeding efficiency, overcome the problems of long breeding cycle and limited breed adaptability, provide a universal molecular tool for genetic improvement of pigs of multiple breeds, and help improve the quality and efficiency of the pork industry. The SNP marker and primer pair can be applied to breeding to screen multi-rib pig populations, providing a more universal molecular tool for genetic improvement of pig populations and promoting the high-quality development of the pork industry. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The position of the SNP marker related to the rib number trait of pigs in the present application on chromosome 7 of the international pig genome 11.1 version reference sequence has been marked with a vertical line; Figure 2 The phenotype distribution corresponding to different genotypes of the SNP marker in Duroc, Landrace, Large White and Pietrain pig populations, n represents the sample size, and *** indicates that the difference between phenotypes is extremely significant (P<0.001) in t-test. P <0.001); Figure 3 The PCR amplification gel map of individuals with three different genotypes at the rs701714758 site on chromosome 7 in Duroc, Landrace, Large White and Pietrain pig populations, respectively, wherein M represents a DNA marker, 1, 4, 7 and 10 are individuals with genotype AA, 2, 5, 8 and 11 are individuals with genotype GA, and 3, 6, 9 and 12 are individuals with genotype GG; Figure 4 A genotyping diagram example of the rs701714758 site on chromosome 7 in Duroc, Landrace, Large White and Pietrain pig populations, wherein A represents the SNP site with GG genotype, B represents the SNP site with GA type, and C represents the SNP site with AA type, and the site has been marked with a red square. DETAILED DESCRIPTION

[0019] The present application provides a SNP marker primer pair related to the pig multi-rib trait and application thereof, in order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0020] Example 1 Source of test animals: Shiji Guigang Genetic Core Farm of Guigang City, Guangxi Zhuang Autonomous Region Extraction of pig genomic DNA: Collect ear tissue samples of individuals with GG, GA and AA genotypes at rs701714758 site on chromosome 7 in Duroc, Landrace, Large White and Pietrain breeds, a total of 12 samples for individual DNA extraction.

[0021] The extraction steps are as follows: ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse PW respectively, and mix thoroughly; ② Take about 100 mg of ear tissue sample and crush it into a cell suspension, place it in a 2 mL EP tube, then centrifuge at 10,000 rpm (~11200xg) for 1 min, discard the supernatant, add 200 μL of buffer GA, and shake until completely suspended; ③ Add 20 μL of proteinase K solution, mix well, and place at 56 ℃ until the tissue sample is dissolved, and briefly centrifuge to remove water droplets on the inner wall of the tube cap; ④ Add 200 μL of buffer GB, mix thoroughly by inverting, and place at 70 ℃ for 10 min, the solution should be clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap; ⑤ Add 200 μL of anhydrous ethanol, mix thoroughly by shaking for 15 sec, at this time a flocculent precipitate may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap; ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm (~13,400xg) for 30 sec, discard the waste, and place the adsorption column CB3 back into the collection tube; ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400xg) for 30 sec, discard the waste, and place the adsorption column CB3 into the collection tube; ⑧ Add 600 μL of rinse PW to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400xg) for 30 sec, discard the waste, and place the adsorption column CB3 into the collection tube; ⑨ Repeat step ⑧; ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for 2-5 min to thoroughly dry any residual washing liquid in the adsorption material; ⑪ Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE dropwise to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution into the centrifuge tube. The quality and concentration of DNA were determined using a Nanodrop-2000 spectrophotometer. All DNA concentrations were diluted to 50 ng / μL and stored at -20 ℃ for later use.

[0022] PCR amplification and sequencing of the target fragment: PCR amplification was performed using genomic DNA from various pig breeds as templates. The reaction system included 3 μL of DNA template, 1 μL each of the primers shown in SEQ ID NO:2 and SEQ ID NO:3, and 21 μL of PCR mix. The amplification program was as follows:

[0023] The PCR products were subjected to 1% agarose gel electrophoresis with a DL2000 marker. The product fragment size was approximately 343 bp. The electrophoresis results are as follows: Figure 3 As shown. The remaining amplification products were sequenced, and the sequencing results were compared and verified for sequence accuracy using DNAman software. The rs701714758 site was genotyped using Chromas v2.6.6 software.

[0024] Genotypic and phenotypic association analyses were performed on Duroc, Landrace, Large White, and Pietrain pig populations using a general linear model in R 4.5.2 software. The model was: Y = μ + S + FYS + G + e Where Y is the number of ribs in an individual, determined using computed tomography (CT) without slaughter; μ is the mean number of ribs in the population; S is the fixed effect of sex; FYS is the fixed effect of birth place, year, and season; G is the fixed effect of SNP markers; and e is the residual.

[0025] Results: Table 1 shows the effects of different genotypes at the rs701714758 locus on the rib number phenotype in different pig breeds. The results showed highly significant differences in rib number among individuals with the three genotypes at the rs701714758 locus in Duroc, Large White, and Pietrain breeds. P <0.001). Individuals with the GG type had significantly more ribs than those with the GA and AA types. P<0.001), and the number of ribs in GA type individuals is significantly greater than that in AA type individuals ( P <0.001). In the Landrace pig population, individuals with the GG rib type had significantly more ribs than those with the GA and AA rib types. P <0.001). Therefore, selecting individuals with the GG type at the rs701714758 locus in Duroc, Landrace, Large White, and Pietrain pig populations can increase the number of ribs in these pig populations, thereby breeding pig populations with more ribs, which can improve pork production and breeding efficiency.

[0026] Table 1. Association analysis results between chromosome 7 rs701714758 locus and rib number in Duroc, Landrace, Large White, and Pietrain pig populations.

[0027] Note: The numbers below the genotype are the corrected mean ± standard error. Different letters in the superscript of the numbers in the same row indicate extremely significant differences. P <0.001) The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pair of SNP marker primers associated with the multiribbed trait in pigs, characterized in that, It consists of the upstream primer shown in SEQ ID NO:2 and the downstream primer shown in SEQ ID NO:

3.

2. The SNP marker primer pair related to the multiribbed trait in pigs according to claim 1, characterized in that, The SNP marker is located at nucleotide position rs701714758 on chromosome 7 of the international pig genome version 11.1 reference sequence. The SNP marker position described in SEQ ID NO:1 is located at position 501 and exhibits G / A polymorphism.

3. The application of the SNP marker primer pair associated with the multi-ribbed trait in pigs according to claim 1 or 2 in the detection of multi-ribbed traits in pigs and / or in pig breeding.

4. The application according to claim 3, characterized in that, The pigs mentioned are Duroc, Landrace, Large White, and Pietrain.

5. A method for screening multi-ribbed pig populations, characterized in that, The genotype of the rs701714758 nucleotide site on chromosome 7 of the pig was detected in the international pig genome version 11.1 reference sequence, and individuals with the GG genotype at this nucleotide site were selected as replacement breeding pigs.

6. The method according to claim 5, characterized in that, The genotype of the rs701714758 nucleotide site on chromosome 7 of the pig in the International Swine Genome Version 11.1 reference sequence was determined by either PCR or genome sequencing.

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