A snp marker related to pig backfat thickness and its detection method and application

Abstract

The present application relates to the field of determination or detection method containing nucleic acid, disclose a kind of SNP marker related to pig back fat thickness and its detection method and application, the SNP marker is located at the 134736770th nucleotide of the positive strand of the 7th chromosome of 10.2 version pig reference genome, it is T or C.In the present application, it is found that the SNP marker at the 134736770th site of the positive strand of pig 7th chromosome is significantly related to back fat thickness, the back fat thickness of CC genotype pig is significantly lower than that of other two genotypes, and in the remaining two genotypes, the back fat thickness of TC genotype pig is significantly lower than that of TT genotype.The nucleotide site detection method is simple and fast, and can be used as a molecular genetic marker for pig back fat thickness, for marker-assisted selection, to speed up the genetic breeding work of high lean rate pig.

Description

Technical Field

[0001] This invention relates to the field of methods for determining or detecting nucleic acids, and more specifically to a SNP marker related to backfat thickness in pigs, its detection method, and its application. Background Technology

[0002] Backfat thickness is one of the most important economic traits in pig farming. It generally shows a significant negative correlation with lean meat percentage, making it a crucial indicator for measuring lean meat percentage in modern pig genetic breeding. Developing pig breeds with higher lean meat percentages is currently the mainstream market demand; therefore, the main goal of current pig genetic improvement is to reduce backfat thickness and increase lean meat percentage, thereby increasing the economic value of pork products. Since pig meat production traits are generally measured after slaughter, superior breeding pigs cannot be reused, resulting in significant losses. Furthermore, traditional breeding methods require long breeding cycles and have limited improvement capabilities. Therefore, more efficient phenotyping and breeding techniques are needed to improve pig meat production traits.

[0003] With the continuous development of sequencing technology and the gradual improvement of porcine genome sequences and single nucleotide polymorphism (SNP) marker maps, a large number of genetic markers have been provided for reference in porcine genome selection and genome-wide association study (GWAS) research. GWAS using SNP microarray genotyping technology can quickly and effectively identify SNP loci associated with backfat thickness in pigs. Early prediction of backfat thickness can be made by detecting the genotype of the pig being tested, saving production costs and accelerating genetic breeding efforts. Summary of the Invention

[0004] The purpose of this invention is to provide SNP markers related to backfat thickness in pigs and their applications, and to provide a method for identifying or assisting in the identification of backfat thickness in pigs.

[0005] This invention provides a method for identifying or assisting in the identification of backfat thickness in pigs. The method includes: detecting the genotype of a SNP in the genome of the pig to be tested; identifying the backfat thickness based on the genotype; specifying that the backfat thickness of pigs with the CC genotype is lower than that of pigs with the TC genotype and the TT genotype, with the TC genotype having a lower backfat thickness than the TT genotype; wherein the SNP is nucleotide 134,736,770 on the positive strand of chromosome 7 in the 10.2 version of the pig reference genome (nucleotide 401 of sequence 1 in the sequence listing), and is either T or C (represented by Y in the sequence listing).

[0006] The CC genotype is a homozygous type of the SNP being C;

[0007] The TC genotype is a heterozygous type of the SNP being C and T;

[0008] The TT genotype is the homozygous type of the SNP being T.

[0009] The application of substances that detect the polymorphism or genotype of the SNP in the identification or auxiliary identification of backfat thickness in pigs should also be within the scope of protection of this invention.

[0010] Application of substances for detecting the polymorphism or genotype of the SNP in pig breeding.

[0011] The pig breeding mentioned above refers to the development of pig breeds with high lean meat percentage (low backfat thickness).

[0012] The substance in question is an SNP chip (such as the Neogen_POR80K chip from Neogen Corporation).

[0013] This invention provides a method for breeding pigs, the method comprising: detecting the genotype of the SNP in the genome of the pig to be tested, and selecting pigs with the CC genotype for breeding, wherein the CC genotype is a homozygous type of the SNP being C.

[0014] The pigs to be tested may be Duroc pigs, Landrace pigs, and / or Large White pigs.

[0015] The method for determining the genotype of the pig to be tested in the present invention is as follows: the genomic DNA of the pig to be tested is genotyped using the Neogen_POR80K chip from Neogen Corporation, and the genotyping platform software is GenCall (Version 7.0.0) from Neogen Corporation.

[0016] The beneficial effects of this invention are as follows: It was discovered that the SNP marker at locus 134736770 on the positive strand of pig chromosome 7 is significantly correlated with backfat thickness. Pigs with the CC genotype have significantly lower backfat thickness than those with the other two genotypes. Among the other two genotypes, pigs with the TC genotype have significantly lower backfat thickness than those with the TT genotype. This nucleotide locus detection method is simple and rapid, and can serve as a molecular genetic marker for pig backfat thickness, enabling marker-assisted selection and accelerating the genetic breeding of pigs with high lean meat percentage. Attached Figure Description

[0017] Figure 1 The results of the differential analysis of backfat thickness in pigs with different genotypes in Example 1;

[0018] Figure 2 The correction factor for backfat thickness in pigs;

[0019] Figure 3 The distribution of the WU_10.2_7_134736770T / C genotype in the population;

[0020] Figure 4 Association analysis of the gene WU_10.2_7_134736770 locus with backfat thickness in pigs. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] The test populations were Duroc, Landrace, and Large White pigs from Hebei Meishen Original Breeding Pig Farm, and were used for subsequent phenotypic recording, genotyping, and genome-wide association analysis.

[0024] This invention uses the pig reference genome Sscrofa10.2 sequence information to detect the deoxyribonucleotide information at position 134736770 on the positive strand of chromosome 7 of the pig to determine whether the pig's genotype is TT, TC, or CC. The TT genotype is homozygous for T at position 134736770 on the positive strand of chromosome 7; the CC genotype is homozygous for C at this position; and the TC genotype is heterozygous for both C and T at this position. Statistical results show that the backfat thickness of CC genotype pigs is significantly lower than that of the other two genotypes, while among the other two genotypes, the backfat thickness of TC genotype pigs is significantly lower than that of TT genotype pigs.

[0025] The methods described above can be applied to pig breeding.

[0026] Example 1: The WU_10.2_7_134736770 locus was significantly correlated with the backfat thickness trait in pig herds.

[0027] Experimental materials: 1,173 pigs, all from Hebei Meishen Original Breeding Pig Farm. Among them, there were 23 Duroc boars, 177 Duroc sows, 15 Landrace boars, 363 Landrace sows, 2 Large White boars, and 593 Large White sows.

[0028] I. Measurement of back fat thickness

[0029] When the individual weight of the pigs was within the range of 85-105 kg, the backfat thickness and weight of each individual were measured, and data such as the age at measurement were recorded. The collected data were phenotypically corrected using the genetic assessment trait measurement procedure of the Hebei Provincial Local Standard (DB 13 / T 2065-2014) document "Technical Regulations for the Measurement of Production Performance in Breeding Pig Farms".

[0030] Backfat thickness (BFT) at 100kg body weight: Backfat thickness at 100kg body weight is measured simultaneously with the age at which the animal reaches 100kg body weight. Ultrasound scans are used to measure the backfat thickness at the 3rd-4th intercostal space from the bottom, in millimeters. Finally, it is converted to the live backfat thickness at 100kg body weight using the following correction formula (the calculation method is as follows, and the correction coefficient for correcting backfat thickness is as follows). Figure 2 As shown, Figure 2 The correction coefficients for backfat thickness in boars and sows are given, where A and B are correction coefficients for backfat thickness in different pigs. Corrected backfat thickness (mm) = Measured backfat thickness (mm) × CF; where CF = A ÷ {A + [B × (Measured weight (kg) - 100)]}.

[0031] II. DNA Extraction and SNP Detection

[0032] 1. Genomic DNA extraction

[0033] Ear tissues were collected from 1,173 pigs, minced, lysed by TL, digested with proteinase K, and DNA was extracted using the DP1902 cell / tissue genomic DNA extraction kit from Beijing Biotech Biotechnology Co., Ltd. The DNA was dissolved in the kit's elution buffer EB to obtain genomic DNA from each pig.

[0034] 2. Genotyping

[0035] Genomic DNA from each pig prepared in step 1 was collected, and the genotype of each individual was detected using the Neogen_POR80K chip from Neogen Corporation. The SNP genotyping platform software was Neogen Corporation's GenCall (Version 7.0.0). The SNP locus WU_10.2_7_134736770 was detected. The SNP locus WU_10.2_7_134736770 is located at nucleotide 134736770 on the positive strand of chromosome 7 in the pig reference genome version 10.2, and it is either T or C. The sequence of the SNP locus and the 400 nucleotides before and after it is shown in Sequence 1. The SNP locus is the 401st position in Sequence 1, and it is represented by Y as either T or C.

[0036] 3. Quality control of phenotypic and genotypic data

[0037] (1) Quality control standards for phenotypic data: remove individuals with missing phenotypic values; remove individuals whose deviation from the mean is greater than 3 times the standard deviation.

[0038] (2) SNP chip typing filtering criteria: remove SNP loci with a genotype detection rate of less than 95%; remove individuals with a detection rate of less than 95%; remove individuals with a minimum allele frequency (MAF) of less than 1%; remove SNP loci with a Hardy–Weinberg Equilibrium (HWE) chi-square test p-value of less than 1.0E-4; remove SNP loci on sex chromosomes.

[0039] After quality control, the data of 1,173 pigs were found to be of good quality, including 23 Duroc boars and 171 sows, 12 Landrace boars and 336 sows, and 2 Large White boars and 579 sows.

[0040] SNP test results are as follows Figure 3 As shown, Figure 3 The distribution of the WU_10.2_7_134736770T / C genotype in the population.

[0041] III. Genome-wide association analysis of corrected backfat thickness across multiple varieties

[0042] Statistical analysis was performed using the GAPIT (Version 3) R software package based on a compressed mixed linear model. The statistical analysis model was: Y = Xβ + Zu + e; where Y is the observed phenotypic value; β is the unknown value containing fixed effects, including genetic markers, population structure (Q matrix), and intercept; u is the unknown value of the random additive genetic effects of multiple background QTLs from individuals or lines; X and Z are the known design matrices; and e is the unobserved residual vector.

[0043] The Kruskal-Wallis method was used to test the significance of differences between genotype and phenotypic data using RStudio software. A p-value < 0.01 indicated extremely significant differences. Box plots were generated using the ggplot2, ggpubr, and magrittr functions in R. Differences and associations between genotype and backfat thickness in pigs were analyzed. Results are shown below. Figure 1 and Figure 4 As shown, Figure 1 Results of the differential analysis of backfat thickness in pigs with different genotypes; Figure 4 The statistical results of the association analysis between the gene WU_10.2_7_134736770 locus and backfat thickness in pigs.

[0044] The results showed a highly significant correlation between the SNP locus WU_10.2_7_134736770 (T / C; Chr7:134,736,770) and backfat thickness in pigs (P<0.01). Pigs with the CC genotype had significantly lower backfat thickness than those with the other two genotypes. Among the other two genotypes, pigs with the TC genotype had significantly lower backfat thickness than those with the TT genotype. Therefore, in pig populations, successive generations of breeding individuals with the CC genotype at the WU_10.2_7_134736770 locus can gradually reduce backfat thickness, thereby improving feeding efficiency and increasing economic benefits.

[0045] This invention discovered a significant correlation between the T / C SNP at locus 134736770 on the positive strand of pig chromosome 7 and backfat thickness. Pigs with the CC genotype had significantly less backfat thickness than those with the other two genotypes. Among the other two genotypes, pigs with the TC genotype had significantly less backfat thickness than those with the TT genotype. This nucleotide locus detection method is simple and rapid, and can serve as a molecular genetic marker for pig backfat thickness, enabling marker-assisted selection and accelerating the genetic breeding of pigs with high lean meat percentage.

[0046] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Foshan Kunpeng Modern Agriculture Research Institute Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences <120> A SNP marker related to backfat thickness in pigs, its detection method and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 801 <212> DNA <213> Artificial Sequence <400> 1 actgatttaa ggactgcctt aaccactctt aactaggttt ctggtcttct tttgaggctc 60 aggctcctga atcagtcgca atgtggctct gggatgtaca gggggcacag accgtgggag 120 gtcctagaac tcaatagag aggtctgaac ttgacccaag agacataccc caaggtgtga 180 gagcttctga aacaggacag ggtttatgc aagctgtggt agtgatcatc tttatctatg 240 taaattaata ttttggataa ttgttcatct gggagaccaa ctggaaagat tttaataatt 300 ccaataaata tagaaaccct aagccaagat agggctgggg gaaaaaaaca gcaacatgtt 360 gagacaatct aaagggaaga aaccaaaatg ttataggagt ytgatttga aaaagaatca 420 ggaggcattt atggccccca ctctcatctc taggaaaagt tgtgttagac ccactctatg 480 ctaattactt gttctctcct aattgtgaaa agtgtatgga aagaatggca tacaaagtcc 540 ttttcagaaa ctgaaggcat catgctatta gccaaatccc ctttgattaa aagaagtaca 600 tggtatctat cctcaatgta ggatgaacac tgaactttta tgaactatca gaaaggaggt 660 cttattgct ttattgtcca gtcaagtcct gaaaaagact gctagagcag tcagggtttt 720 gaactaagtg ctgtgaaaat ggccttggct ttttctttct ggaaatgtac cacttgacct 780 tgactgccag gttgtataac a 801

Claims

1. A method of identifying or assisting in the identification of backfat thickness in a pig, characterized in that, The method comprises detecting the genotype of the SNP in the genome of the pig to be tested, identifying the backfat thickness of the pig according to the genotype, the backfat thickness of the pig with CC genotype is lower than that of the pig with TC genotype and that of the pig with TT genotype, the backfat thickness of the pig with TC genotype is lower than that of the pig with TT genotype; the SNP is the nucleotide at position 134736770 on the positive strand of chromosome 7 in the Sscrofa10.2 version of the pig reference genome, which is T or C, The CC genotype is the homozygous type of the SNP being C; The TC genotype is the heterozygous type of the SNP being C and T; The TT genotype is the homozygous type of the SNP being T; The pig to be tested is a Duroc pig, a Landrace pig and / or a Large White pig.

2. Application of a substance for detecting the polymorphism or genotype of the SNP in claim 1 in identifying or assisting in identifying the backfat thickness of a pig, wherein the pig is a Duroc pig, a Landrace pig and / or a Large White pig.

3. Use according to claim 2, characterized in that, The substance is a SNP chip.

4. Application of a substance for detecting the polymorphism or genotype of the SNP in claim 1 in pig breeding, wherein the pig breeding is breeding of a pig variety with low backfat thickness, and the pig is a Duroc pig, a Landrace pig and / or a Large White pig.

5. A method for breeding swine, characterized by, The method comprises detecting the genotype of the SNP in claim 1 in the genome of the pig to be tested, and selecting the pig with CC genotype for breeding, wherein the CC genotype is the homozygous type of the SNP being C, and the pig breeding is breeding of a pig variety with low backfat thickness, and the pig is a Duroc pig, a Landrace pig and / or a Large White pig.

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