A method for identifying or assisting in the identification of the porcine eye muscle area and its application

Abstract

The present invention belongs to the field of detection methods involving nucleic acids, and relates to a method for identifying or assisting in the identification of the porcine eye muscle area and its application. The method for identifying or assisting in the identification of the porcine eye muscle area includes: detecting the genotype of SNP markers in the genome of a pig to be tested, and identifying the porcine eye muscle area according to the genotype; the SNP marker is located at the 47,077,245th nucleotide on chromosome 17 of the pig in the 10.2 version of the pig reference genome, which is T or C; the eye muscle area of pigs with the TC genotype is higher than that of pigs with the TT genotype and the CC genotype, and the eye muscle area of pigs with the TT genotype is higher than that of pigs with the CC genotype. The present invention provides an SNP locus related to the porcine eye muscle area, and it is found that this SNP is located at the 47,077,245th nucleotide locus on chromosome 17 of the pig, and is significantly associated with the porcine eye muscle area. By detecting different genotypes of this SNP marker, it can be used for the genetic improvement of the porcine eye muscle area and the improvement of pork quality.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid detection methods and relates to a method for identifying or assisting in identifying the area of ​​pig eye muscles and its application. Background Art

[0002] Pork is the main source of animal protein in human diet, and its consumption accounts for more than half of meat consumption. Research on pork production and quality has always been the focus of animal husbandry scholars. Loin area is an important indicator for measuring pork performance. Scientific research on pig loin area is of great significance for improving pork quality.

[0003] Pig growth and development are mainly determined by individual genetic factors, among which pig eye muscle area is a trait with high heritability, which is directly related to the lean meat rate of pigs and is also one of the most important selection indicators in pig genetic breeding. With the advent of some advanced instruments and equipment for direct in vivo scanning and measurement of eye muscle area, a more accurate method is provided for the determination of eye muscle area phenotype, which also provides a key preliminary guarantee for the selection of this trait.

[0004] SNP (single nucleotide polymorphism) is a mutation caused by a single base and is widely distributed in the genome, so it is very important to screen and discover SNP sites. In recent years, more and more SNP sites have been used in related studies such as gene function discovery, disease control and growth performance association analysis, and related SNP detection technologies are also constantly improving and developing. Summary of the invention

[0005] The purpose of the present invention is to provide a method for developing and utilizing SNP markers associated with pig eye muscle area, and further to provide a method and application for identifying or assisting in identifying pig eye muscle area.

[0006] The present invention provides a method for identifying or assisting in identifying the eye muscle area of ​​a pig, the method comprising: detecting the genotype of a SNP marker (named as WU_10.2_17_47077245) in a pig genome to be tested; the SNP marker is located at the 47,077,245th nucleotide of chromosome 17 of the pig reference genome of version 10.2 (the 101st nucleotide of the sequence shown in sequence 1, represented by y in the sequence table), which is T or C; the eye muscle area of ​​a pig with TC genotype is higher than that of a pig with TT genotype and a pig with CC genotype, and the eye muscle area of ​​a pig with TT genotype is higher than that of a pig with CC genotype; the CC genotype is a homozygous type in which the SNP is C; the TC genotype is a heterozygous type in which the SNP is C and T; the TT genotype is a homozygous type in which the SNP is T.

[0007] The SNP marker WU_10.2_17_47077245 is the 101st nucleotide of the sequence shown in Sequence 1 on Sus scrofa chromosome 17 of the 10.2 version of the pig reference genome, which is T or C (represented by Y in the sequence listing).

[0008] The application of a substance for detecting the polymorphism or genotype of the said SNP marker in identifying or assisting in identifying the longissimus muscle area of pigs shall also fall within the protection scope of the present invention.

[0009] Wherein, the said substance is an SNP chip.

[0010] The application of a substance for detecting the polymorphism or genotype of the said SNP marker in pig breeding shall also fall within the protection scope of the present invention.

[0011] Wherein, the said substance is an SNP chip.

[0012] The above SNP chip is the Neogen_POR80K chip of Neogen Corporation

[0013] Wherein, the said pig breeding is to breed pig varieties with large longissimus muscle area.

[0014] The present invention provides a pig breeding method, which comprises: detecting the genotype of the said SNP marker in the genome of a pig to be tested, and selecting pigs with homozygous CC genotype and TT genotype for crossbreeding.

[0015] Wherein the said pig breeding is to breed pig varieties with large longissimus muscle area.

[0016] The above pigs (pigs to be tested) are Landrace pigs, Yorkshire pigs and Duroc pigs.

[0017] In the present invention, according to the sequence information of the pig reference genome Sscrofa 10.2, the information of the 47,077,245th deoxynucleotide on the sense strand of Sus scrofa chromosome 17 of a pig to be tested is detected to determine whether the genotype of the pig to be tested is TT, TC or CC; the TT genotype is the homozygous type of base T at the 47,077,245th position on the sense strand of Sus scrofa chromosome 17; the CC genotype is the homozygous type of base C at this site; the TC genotype is the heterozygous type in which both bases C and T appear at this site. Statistical results show that the longissimus muscle area of pigs with TC genotype is significantly higher than that of individuals with the other two genotypes, and among the remaining two genotypes, the longissimus muscle area of pigs with TT genotype is significantly higher than that of pigs with CC genotype.

[0018] The method for determining the genotype of a pig to be tested in the method of the present invention is as follows: In this experiment, the Neogen_POR80K chip technology of Neogen Corporation is used to genotype the genomic DNA of a pig to be tested, and the genotyping platform software for SNPs is GenCall (v7.0.0).

[0019] The present invention discovers that the T / C SNP at the 47,077,245th site on the sense strand of Sus scrofa chromosome 17 is significantly associated with the loin eye area. The loin eye area of pigs with the TC genotype is significantly higher than that of individuals with the other two genotypes, and the loin eye area of pigs with the TT genotype is significantly higher than that of pigs with the CC genotype. The detection method of this nucleotide site is simple and fast, and can be used as a molecular genetic marker for the loin eye area of pigs, accelerating the genetic breeding work of pigs with high lean meat percentage.

[0020] The present invention takes pigs as the research object. Through genotyping and genome-wide association analysis, the SNP locus WU_10.2_17_47077245 related to the loin eye area of pigs is obtained. It is found that this SNP is located at the 47,077,245th nucleotide site of Sus scrofa chromosome 17 and is extremely significantly associated with the loin eye area of pigs. By detecting different genotypes of this SNP marker, it can be used for the genetic improvement of the loin eye area of pigs and improve the pork quality. Brief Description of the Drawings

[0021] Figure 1 It is an association analysis diagram of the differences in loin eye area among three genotypes of the SNP locus WU_10.2_17_47077245;

[0022] Figure 2 It is the allele frequency and genotype frequency of the SNP locus WU_10.2_17_47077245 in the population;

[0023] Figure 3 It is the difference in loin eye area among individuals with different genotypes of the SNP locus WU_10.2_17_47077245. Detailed Embodiments

[0024] The present invention will be further described in detail below in combination with the detailed embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0025] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] The following implementation cases are used to illustrate the present invention, but not to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0027] Example 1

[0028] 1. Experimental animals.

[0029] A total of 1,173 Landrace, Yorkshire, and Duroc pigs from Hebei Meishen Purebred Pig Farm were selected for the following experiment. Among them, there were 23 Duroc boars, 178 Duroc sows, 15 Landrace boars, 363 Landrace sows, 2 Yorkshire boars, and 592 Yorkshire sows.

[0030] 2. Phenotypic measurement.

[0031] The phenotypic data was corrected using the genetic evaluation trait measurement procedures of the "National Swine Genetic Evaluation Program" (Document No. 60

[2000] of the National Animal Husbandry and Veterinary General Station). The correction method for the loin eye area at 100 kg body weight: While measuring the live body fat thickness, the loin eye area at the same position was measured using B-ultrasound scanning. It was converted to the loin eye area at 100 kg body weight according to the following correction formula: Corrected loin eye area (cm 2 ) = Actual loin eye area (cm 2 ) + {[100 - Actual body weight (kg)] × Actual loin eye area (cm 2 )} / [Actual body weight (kg) + 70].

[0032] 3. SNP detection.

[0033] 3.1 Genomic DNA extraction: Ear tissue samples of the target pig population (the above 1,173 pigs) were collected and stored at low temperature in PBS buffer. The DP1902 model cell / tissue genomic DNA extraction kit from Beijing Bioteke Corporation was used to extract DNA from the ear tissues of the pigs to be tested for later use.

[0034] 3.2 Genotyping: The genomic DNA of the pigs to be tested extracted in step 3.1 was taken, and the Neogen_POR80K chip from Neogen Corporation was used to detect the genotype of each individual, obtaining the genotype of the SNP locus WU_10.2_17_47077245. The SNP locus WU_10.2_17_47077245 is the 101st nucleotide of the sequence shown in Sequence 1 on chromosome 17 of the pig in the 10.2 version of the pig reference genome, which is T or C (represented by y in the sequence list).

[0035] 3.3 Quality control of phenotypic data and genotypic data: a. Quality control criteria for phenotypic data: Remove individuals with missing phenotypic values; remove individuals with a deviation from the mean greater than 3 standard deviations. b. Filtering criteria for SNP chip genotyping: Remove SNP loci with a genotype detection rate less than 95%; remove individuals with a detection rate less than 95%; remove individuals with a minimum allele frequency (MAF) less than 1%; remove SNP loci with a P-value of the chi-square test for Hardy–Weinberg Equilibrium (HWE) less than 1.0E-4; remove SNP loci on the sex chromosomes.

[0036] 4 Genome-wide association analysis of eye muscle area.

[0037] The analysis software package used in this invention for genome-wide association analysis is the R language package GAPIT Version 3 (developed by the laboratory of Teacher Zhiwu Zhang at the University of Washington). The statistical model of this software package is the compressed mixed linear model. The design purpose of GAPIT is to accurately perform GWAS and genomic prediction on large datasets. The mixed linear model (MLM) includes fixed and random effects. The model takes population structure as a fixed effect and includes individuals in the random effect to construct the individual kinship matrix. Its statistical analysis model is: 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 effect of multiple background QTLs from individuals or lines; X and Z are known design matrices; e is the unobserved residual vector.

[0038] Subsequently, the Kruskal-Wallis method was used to compare the differences in eye muscle area among different genotypes for genotypic data and phenotypic data using Rstudio software. P-value < 0.01 indicates a highly significant difference. Box plots were drawn using the ggplot2, ggpubr, and magrittr function packages of the R language. The results are as Figures 1-3 shown, Figure 1 Association analysis chart of the differences in eye muscle area of three genotypes of SNP locus WU_10.2_17_47077245 in pigs; Figure 2 Allele frequency and genotype frequency of SNP locus WU_10.2_17_47077245 in the population; Figure 3 Differences in eye muscle area among individuals with different genotypes of SNP locus WU_10.2_17_47077245.

[0039] From Figures 1-3It can be seen that there are significant differences in the loin eye area among the three genotypes of pigs. The loin eye area of pigs with the TC genotype is significantly higher than that of individuals with the other two genotypes, and the loin eye area of pigs with the TT genotype is significantly higher than that of pigs with the CC genotype. The WU_10.2_17_47077245 locus can be used as a genetic marker for the loin eye area of pigs and applied to molecular marker-assisted selection for loin eye area traits, thereby increasing the accuracy of breeding pigs with high lean meat percentage and improving their genetic progress. The present invention discovers that the T / C SNP at the 47,077,245th locus on the sense strand of chromosome 17 of pigs is significantly correlated with the loin eye area. The loin eye area of pigs with the TC genotype is significantly higher than that of individuals with the other two genotypes, and the loin eye area of pigs with the TT genotype is significantly higher than that of pigs with the CC genotype. The detection method of this nucleotide locus is simple and fast, and can be used as a molecular genetic marker for the size of the loin eye area of pigs, accelerating the genetic breeding work of pigs with high lean meat percentage.

[0040] The present invention takes pigs as the research object. Through genotyping and genome-wide association analysis, the SNP locus WU_10.2_17_47077245 related to the loin eye area of pigs is obtained, and it is found that this SNP is located at the 47,077,245th nucleotide locus on chromosome 17 of pigs and is extremely significantly correlated with the loin eye area of pigs. By detecting different genotypes of this SNP marker, it can be used for the genetic improvement of the loin eye area of pigs and improve the pork quality.

[0041] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A method for assisting in the identification of porcine eye muscle area, characterized in that, the method includes: detecting the genotype of SNP markers in the genome of the porcine to be tested, and identifying the porcine eye muscle area according to the genotype; the SNP marker is the nucleotide at position 47,077,245 on chromosome 17 of the pig in the 10.2 version of the pig reference genome, the 101st nucleotide of the sequence shown in Sequence 1, which is T or C; the eye muscle area of pigs with the TC genotype is higher than that of pigs with the TT genotype and the CC genotype, and the eye muscle area of pigs with the TT genotype is higher than that of pigs with the CC genotype; the porcine to be tested are Landrace pigs, Large White pigs and Duroc pigs.

2. Application of a substance for detecting the genotype of the SNP marker described in claim 1 in assisting in the identification of porcine eye muscle area, where the pigs are Landrace pigs, Large White pigs and Duroc pigs.

3. According to the application described in claim 2, characterized in that, the substance is an SNP chip.

4. Application of a substance for detecting the genotype of the SNP marker described in claim 1 in pig breeding, where the pig breeding is to cultivate pig breeds with large eye muscle area, and the pigs are Landrace pigs, Large White pigs and Duroc pigs.

5. According to the application described in claim 4, characterized in that, the substance is an SNP chip.

6. A pig breeding method, characterized in that, the method includes: detecting the genotype of the SNP marker described in claim 1 in the genome of the porcine to be tested, and selecting pigs with the homozygous CC genotype and the TT genotype for crossbreeding, where the pig breeding is to cultivate pig breeds with large eye muscle area, and the pigs are Landrace pigs, Large White pigs and Duroc pigs.

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