CN115094149A - A SNP marker related to porcine backfat thickness and its detection method and application - Google Patents

Abstract

The invention relates to the field of assay or detection methods comprising nucleic acid, and discloses a SNP marker related to the thickness of pig backfat, a detection method and application thereof, the SNP marker is located in the sense strand of chromosome 7 of the pig reference genome of version 10.2 Nucleotide at position 134736770, which is T or C. In the present invention, it is found that the SNP marker at the 134736770th position of the sense strand of the pig chromosome 7 is significantly correlated with the thickness of the back fat. , the backfat thickness of TC genotype was significantly lower than that of TT genotype. The nucleotide site detection method is simple and quick, and can be used as a molecular genetic marker for the thickness of pig backfat for marker-assisted selection, thereby accelerating the genetic breeding of pigs with high lean meat rate.

Description

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

technical field

The present invention relates to the field of assay or detection methods comprising nucleic acid, in particular to a SNP marker related to pig backfat thickness, a detection method and application thereof.

Background technique

Pig backfat thickness is one of the most important economic traits in pig breeding. Backfat thickness generally has a significant negative correlation with lean meat percentage, so this trait is also an important calculation index to measure pig lean meat percentage in modern pig genetics and breeding work. Breeding pig breeds with higher lean meat rate is the mainstream demand in the current market. Therefore, the main goal of current pig genetic improvement is to reduce the backfat thickness of live pigs and increase the lean meat rate, thereby increasing the economic value of pork products. Since the meat production traits of pigs generally need to be measured after slaughtering, the excellent breeding pigs cannot continue to be used, resulting in great losses. At the same time, the traditional breeding methods require a long breeding cycle and a limited degree of improvement. Therefore, more efficient phenotypic determination and Breeding techniques to improve pig meat production traits.

With the continuous development of sequencing technology, the gradual improvement of porcine genome sequence and single nucleotide polymorphism (Single NucleotidePolymorphism, SNP) marker map, for the porcine genome selection and genome-wide association analysis (Genome-Wide Association Study, GWAS) research Provides a large number of genetic markers that can be referenced. Using SNP chip typing technology for GWAS can quickly and effectively discover SNP loci related to the thickness of pig backfat, and early prediction of pig backfat thickness through the detection of the pig genotype to be tested can save production costs and accelerate genetics. Breeding selection work.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide SNP markers related to porcine backfat thickness and applications thereof, and to provide a method for identifying or assisting in identifying porcine backfat thickness.

The invention provides a method for identifying or assisting the identification of pig backfat thickness. The method includes: detecting the genotype of SNP in the genome of a pig to be tested, identifying the pig backfat thickness according to the genotype, and the CC genotype pig backfat thickness is lower than TC genotype pig backfat thickness and TT genotype pig backfat thickness, TC genotype pig backfat thickness is lower than TT genotype; the SNP is located in the 134736770th nucleus of the sense strand of chromosome 7 of the pig reference genome of version 10.2 nucleotide (nucleotide 401 of Sequence 1 in the Sequence Listing), which is T or C (represented by Y in the Sequence Listing),

The CC genotype is that the SNP is homozygous for C;

The TC genotype is that the SNP is a heterozygous type of C and T;

The TT genotype is homozygous for the SNP being T.

The application of the substance for detecting the polymorphism or genotype of the SNP in identifying or assisting the identification of pig backfat thickness should also fall within the protection scope of the present invention.

The application of the substance for detecting the polymorphism or genotype of the SNP in pig breeding.

Wherein, the pig breeding is to breed pigs with high lean meat ratio (pig with low backfat thickness).

Wherein, the substance is a SNP chip (eg Neogen_POR80K chip of Neogen company).

The present invention provides a method for breeding pigs. The method comprises: detecting the genotype of the SNP in the genome of the pig to be tested, and selecting pigs with CC genotype for breeding, the CC genotype being homozygous for the SNP being C type.

The test pigs can be Duroc pigs, Landrace pigs and/or Large White pigs.

The method for determining the genotype of the pig to be tested in the method of the present invention is as follows: use the Neogen_POR80K chip of Neogen company to type the genomic DNA of the pig to be tested, and the typing platform software is GenCall (Version 7.0.0) of Neogen company.

The beneficial effects of the invention are as follows: it is found that the SNP marker at the 134736770 position of the sense strand of the pig chromosome 7 is significantly correlated with the back fat thickness, and the back fat thickness of CC genotype pigs is significantly lower than that of the other two genotypes, while the other two genotypes have significantly lower back fat thickness. Among the genotypes, the backfat thickness of TC genotype was 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 the thickness of pig backfat for marker-assisted selection, thereby accelerating the genetic breeding of pigs with high lean meat rate.

Description of drawings

Fig. 1 is the difference analysis result of different genotypes and pig backfat thickness in Example 1;

Fig. 2 is the correction coefficient of pig backfat thickness;

Fig. 3 is the distribution of locus WU_10.2_7_134736770T/C genotype in the population;

Figure 4 shows the association analysis between the gene WU_10.2_7_134736770 locus and pig backfat thickness.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The populations to be tested were from Duroc, Landrace and Large White pig populations in the North American Shen breeding farm, and were used for subsequent phenotypic recording, genotyping and genome-wide association analysis.

According to the sequence information of the pig reference genome Sscrofa10.2, the invention detects the deoxynucleotide information of the 134736770th position of the sense chain of chromosome 7 of the pig to be tested, and determines whether the genotype of the pig to be tested is TT, TC or CC; the TT genotype is pig 7 The 134736770 deoxynucleotide T homozygous in the sense strand of chromosome No. 1; CC genotype is homozygous for base C at this site; TC genotype is heterozygous for both bases C and T at this site. Statistical results showed that the back fat thickness of CC genotype pigs was significantly lower than that of the other two genotypes, while in the other two genotypes, the back fat thickness of TC genotype pigs was significantly lower than that of TT genotype.

The method described above can be applied to the breeding of pigs.

Example 1: The WU_10.2_7_134736770 locus was significantly associated with the backfat thickness trait in pig herds.

Experimental materials: All 1,173 pigs were from the original breeding farm of Beishen, Hebei. 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.

1. Measurement of backfat thickness

When the individual body weight of the above-mentioned pigs is in the range of 85-105 kg, the backfat thickness and body weight of each individual are measured respectively, and the data such as the measured age are recorded. The collected data were corrected for phenotypic data using the genetic evaluation trait determination procedures of the Hebei Provincial Local Standard (DB 13/T 2065-2014) document "Technical Regulations for Determination of Production Performance in Breeding Farms".

100kg body weight in vivo backfat thickness (Backfat thickness, BFT): when measuring 100kg body weight day-age, 100kg body weight in vivo backfat thickness is measured at the same time. The backfat thickness at the 3rd to 4th intercostal space from the bottom was measured by B-scan, in millimeters. Finally, according to the following correction formula, it is converted into a living body backfat thickness of up to 100kg (the calculation method is as follows, the correction factor for correcting backfat thickness is shown in Figure 2, Figure 2 is the correction factor for male and sow backfat thickness, where A , B are the correction coefficients for different pig backfat thicknesses. Corrected backfat thickness (mm)=measured backfat thickness (mm)×CF; wherein, CF=A÷{A+[B×(measured body weight (kg)-100)] }.

2. DNA extraction and SNP detection

1. Genomic DNA extraction

The pig ear tissues of the 1,173 pigs were collected, cut into pieces, lysed by TL and 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 solubilized with elution buffer EB to obtain the genomic DNA of each pig.

2. Genotyping

Take the genomic DNA of each pig prepared in step 1, and use the Neogen_POR80K chip of Neogen company to detect the genotype of each individual. The SNP typing platform software is GenCall (Version 7.0.0) of Neogen company. WU_10.2_7_134736770 is detected, wherein the SNP site WU_10.2_7_134736770 is located at the 134736770 nucleotide of the sense strand of chromosome 7 of the pig reference genome of version 10.2, which is T or C, the SNP site and its front and back are 400 The sequence composed of nucleotides is shown in sequence 1, and the SNP site is the 401st position of sequence 1, which is represented by Y as T or C.

3. Quality control of phenotypic and genotype data

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

(2) Filtering criteria for SNP microarray typing: remove SNP loci with a genotype detection rate less than 95%; remove individuals with a detection rate less than 95%; remove Minimum Allele Frequency (MAF) less than 1% of individuals; SNP sites with a Hardy-Weinberg Equilibrium (HWE) chi-square test P value less than 1.0E-4 were eliminated; SNP sites on sex chromosomes were eliminated.

After quality control, 1,173 pigs were found to have good data quality, including 23 Duroc boars, 171 sows, 12 Landrace boars, 336 sows, 2 Large White boars, and 579 sows. .

The SNP detection results are shown in Figure 3, which is the distribution of the locus WU_10.2_7_134736770T/C genotype in the population.

3. Genome-wide association analysis of multiple breeds with corrected backfat thickness

The R language package GAPIT (Version 3) statistical analysis software was used for statistical analysis based on the compressed mixed linear model. The statistical analysis model is: Y=Xβ+Zu+e; where Y is the observed phenotypic value; β is the unknown value with fixed effects, including genetic markers, population structure (Q matrix) and intercept; u is derived from Unknown values of random additive genetic effects of multiple background QTLs for an individual or line; X and Z are known design matrices; e is the vector of unobserved residuals.

The Kruskal-Wallis method was used to test the significance of differences between genotype data and phenotype data using Rstudio software. P-value < 0.01 indicated that the difference was extremely significant. The ggplot2, ggpubr and magrittr function packages of R language were used to draw boxplots, and statistical Difference analysis and association analysis between genotype and pig backfat thickness. The results are shown in Figures 1 and 4. Figure 1 shows the difference analysis results between different genotypes and pig backfat thickness; Figure 4 shows the statistical results of the association analysis between the gene WU_10.2_7_134736770 locus and pig backfat thickness.

The results showed that the SNP site WU_10.2_7_134736770 (T/C; Chr7:134,736,770) was significantly correlated with pig backfat thickness (P<0.01). The backfat thickness of CC genotype pigs was significantly lower than that of the other two genotypes, while among the other two genotypes, the backfat thickness of TC genotype pigs was significantly lower than that of TT genotype pigs. It can be seen that in the pig population, the CC type individuals at the WU_10.2_7_134736770 locus can be gradually reduced, thereby improving the feeding efficiency and increasing economic benefits.

The present invention finds that the T/C SNP at the 134736770th position of the sense strand of the pig chromosome 7 is significantly correlated with the thickness of the back fat. The backfat thickness of TC genotype pigs was significantly lower than that of TT genotype. The nucleotide site detection method is simple and quick, and can be used as a molecular genetic marker for the thickness of pig backfat for marker-assisted selection, thereby accelerating the genetic breeding of pigs with high lean meat rate.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> Foshan Kunpeng Institute of Modern Agriculture

Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences

<120> A SNP marker related to pig backfat thickness and its detection method and application

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gtcctagaac tcaaatagag aggtctgaac ttgacccaag agacataccc caaggtgtga 180

gagcttctga aacaggacag ggttttatgc aagctgtggt agtgatcatc tttatctatg 240

taaattaata ttttggataa ttgttcatct gggagaccaa ctggaaagat tttaataatt 300

ccaataaata tagaaaccct aagccaagat agggctgggg gaaaaaaaca gcaacatgtt 360

gagacaatct aaagggaaga aaccaaaatg ttataggagt ytgattttga 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

ctttattgct ttattgtcca gtcaagtcct gaaaaagact gctagagcag tcagggtttt 720

gaactaagtg ctgtgaaaat ggccttggct ttttctttct ggaaatgtac cacttgacct 780

tgactgccag gttgtataac a 801

Claims (6)

1. A method for identifying or aiding in the identification of pig backfat thickness, said method comprising: detecting the genotype of SNP in a genome of a pig to be detected, and identifying the thickness of the backfat of the pig according to the genotype, wherein the thickness of the CC genotype backfat of the pig is lower than the thickness of the TC genotype backfat of the pig and the thickness of the TT genotype backfat the pig, and the thickness of the TC genotype backfat of the pig is lower than the TT genotype; the SNP is 134736770 th nucleotide of the sense strand of No. 7 chromosome of 10.2 porcine reference genome, which is T or C,

the CC genotype is homozygote of the SNP which is C;

the TC genotype is a heterozygous type of the SNP which is C and T;

the TT genotype is homozygous for the SNP which is T.

2. Use of a substance that detects a polymorphism or genotype of a SNP as set forth in claim 1 for identifying or aiding in identifying swine backfat thickness.

3. Use according to claim 2, wherein the substance is a SNP chip.

4. Use of a substance for detecting a polymorphism or genotype of the SNP according to claim 1 in pig breeding.

5. The use of claim 4, wherein the pig breeding is a pig breed for raising pigs with high lean meat percentage.

6. A method of breeding pigs, the method comprising: detecting the genotype of the SNP in claim 1 in the genome of a pig to be detected, and selecting the pig with CC genotype for breeding, wherein the CC genotype is homozygous for the SNP.

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