CN107937559B - SNP (single nucleotide polymorphism) site related to economic traits of pigs and application thereof - Google Patents
SNP (single nucleotide polymorphism) site related to economic traits of pigs and application thereof Download PDFInfo
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Abstract
The invention relates to an SNP locus, and particularly discloses an SNP locus (Chr 7: 34960267) related to a plurality of important economic traits of pigs and application thereof. According to the invention, through measuring and recording a plurality of important economic traits of the Duroc pigs and carrying out GWAS research on 3702 Duroc pig bodies by utilizing an optimized GBS technology, an SNP (Chr 7: 34960267) site which is obviously related to the important economic traits (backfat thickness, eye muscle area and lean meat percentage) of the pigs is obtained. And (3) counting the SNP frequency of the SNP locus in the re-sequenced local pig breeds (Jinhua pigs, Wuzhishan pigs, Luchuan pigs, Meishan pigs, Rongchang pigs, Laiwu pigs, Erhualian pigs, Hetao pigs and Min pigs) and commercial pig breeds (Duroc pigs, Changbai pigs, Yorkshire pigs and Duchang pigs), finding that the SNP frequency distribution has obvious difference between the local pig breeds and the commercial pig breeds, G in the commercial pigs is a dominant allele, C in the local pigs is a dominant allele, and providing scientific basis for the marker-assisted selection of the pigs.
Description
Technical Field
The invention relates to an SNP locus, in particular to an SNP locus related to a plurality of important economic traits of pigs and application thereof.
Background
China is a big pig-raising country, the market demand for the pork yield and quality is increasing, the pork yield is increased, the pork carcass quality is improved, and the method becomes a work which is continuously explored by breeding scientists for a long time. Early breeding efforts focused primarily on phenotypic selection in swine, and with the continued advancement of genomic work and the widespread development of genetic markers, molecular selection is becoming a reliable and efficient selection method.
A Single Nucleotide Polymorphism (SNP) marker is a third generation SNP site and refers to a polymorphism generated by mutation of a single base on a genome DNA sequence, and the mutation comprises single base transversion, conversion, insertion and deletion. The SNP has the advantages of large amount, high frequency, low mutation rate and the like, and is widely applied to genome analysis, biological information automatic detection, genetic research of simple and complex diseases, livestock breeding markers, global ethnic genetics and other researches. SNP locus assisted selective breeding is to select target characters on a molecular level, can not be influenced by environment, and reduces linkage drag through genetic background selection, thereby accelerating the breeding process and precision.
Genome-wide association assays (GWAS) are important methods for livestock and poultry economic trait genetic improvement and mechanism analysis. With the development of the second-generation sequencing technology, the whole genome re-sequencing and simplified genome sequencing technology becomes a powerful tool for high-throughput SNP typing, GBS (Genotyping-by-sequencing) is a classic representation of simplified genome sequencing, is an efficient whole genome SNP typing method, can directly identify and type SNP from a population, can obtain SNP typing information with different values from tens of thousands to hundreds of thousands at a lower cost, and is widely applied to researches such as SNP locus development, population genetic analysis, whole genome association analysis, genome selection breeding and the like of animals and plants (De Donato et al, 2013; Elshire et al, 2011; He et al, 2014).
The back fat thickness of the pig indicates the amount of fat, and the thicker the back fat thickness, the lower the lean meat percentage, and conversely, the higher the lean meat percentage. The lean meat percentage refers to the amount of lean meat contained in the pig, the ratio of the lean meat, and the lean meat percentage of good pigs is higher. The pig eye muscle area refers to the cross section area of the longest muscle of a pig, and the higher the lean meat percentage of a pig individual is, the larger the eye muscle area is. The three characters have strong correlation with the meat production performance of the domestic pigs, and have important research significance in breeding.
If an SNP locus or an SNP molecular marker which is significantly related to the three economic traits can be screened, the method is favorable for providing a favorable theoretical basis for marker-assisted selective breeding of pigs.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an SNP site related to a plurality of important economic traits of pigs and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in one aspect, the invention provides a SNP site related to economic traits of pigs, which is the gene group version of Chr7 of Ensembl Scrofa 10.2: 34960267, the allele at the site is G and C, and has three genotypes of G/G, C/C and G/C.
According to the invention, multiple important economic traits of the duroc pigs are measured and recorded, GBS sequencing is carried out on 3757 duroc boars, 102,254 SNPs are identified to cover the whole genome of the pigs, 66,737 GWAS researches for multiple important economic traits of 3702 duroc pig pure population are remained after strict quality control, multiple important economic traits of the pigs are obtained, and an SNP (Chr 7: 34960267) site which is obviously related to the multiple important economic traits of the pigs (backfat thickness, eye muscle area and lean meat percentage) is obtained. And (3) counting the SNP frequency of the SNP locus in the re-sequenced local pig breeds (Jinhua pigs, Wuzhishan pigs, Luchuan pigs, Meishan pigs, Rongchang pigs, Laiwu pigs, Erhualian pigs, Hetao pigs and Min pigs) and commercial pig breeds (Duroc pigs, Changbai pigs, Yorkshire pigs and Duchang pigs), and finding that the SNP frequency distribution of the SNP locus has obvious difference in the local pig breeds and the commercial pig breeds, G in the commercial pigs is a dominant allele, and C in the local pigs is a dominant allele.
Wherein, the commercial pig has the advantages of faster weight gain and higher lean meat percentage compared with the local pig.
Further, the economic trait is backfat thickness, eye muscle area and/or lean meat percentage. Namely, the allele of the SNP locus is obviously related to the three economic traits.
The SNP site is located at the 101 th base in the nucleotide sequence shown in SEQ ID NO. 1.
In another aspect, the invention provides the application of the SNP locus in marker-assisted selection breeding of pigs.
The application is embodied in a method for carrying out auxiliary breeding on pigs by utilizing the SNP loci, and the method can comprise the following steps:
(1) detecting the genotype of the sample pig at the SNP locus;
(2) selecting a sample pig with the dominant allele genotype to breed the dominant strain.
Preferably, selecting the pig with G/G homozygous genotype for dominant line breeding.
Wherein the dominant line is mainly characterized by high lean meat percentage.
Further, the step (1) can be performed by direct sequencing, or by amplifying a fragment containing the SNP site and detecting the amplified fragment, for example, designing a primer, amplifying a fragment containing the SNP site from the sequence shown in SEQ ID No.1, and detecting the allele at the site.
The invention also provides application of the SNP locus in identifying commercial pig/local pig economic trait dominant strains.
On the other hand, primer pairs for amplifying the SNP site fragments of the invention also belong to the protection scope of the invention. The primer pair has the following functions: amplifying a fragment containing the SNP locus from the sequence shown in SEQ ID No. 1.
The invention has the beneficial effects that:
the invention is suitable for the pig Chr 7: 34960267, carrying out genotyping on the SNP locus, carrying out correlation analysis on the SNP locus and a plurality of important economic traits (backfat thickness, eye muscle area and lean meat percentage) of the pig, and finding out that the SNP frequency distribution has obvious difference in local pig species and commercial pig species, G in the commercial pig is a dominant allele, C in the local pig is a dominant allele, thereby providing scientific basis for marker-assisted selection of the pig.
Drawings
FIG. 1 is a Manhattan plot of pig 100kg body weight backfat thickness (A), 100kg body weight eye muscle area (B) and 100kg body weight lean (C) GWAS results.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 pig economic traits Whole genome Association analysis
1. Test materials
Production performance records of 33,960 individuals (12,987 boars and 20,973 sows) born in 8-2016 and 1-2007 were collected for the Duroc pig pure population as a study subject and used in the present invention. Conventional performance measurements were performed strictly in accordance with pig farm internal specifications, including 100kg body weight Backfat (BF), 100kg body weight eyemuscle area (LMA), and 100kg body weight Lean (LMP).
2. Test method
2.1 general determination of Properties
Backfat thickness, eye muscle area and lean meat percentage of 100kg body weight: when the weight of the pig reaches 100 +/-5 kg, carrying out final measurement; the backfat thickness and the eye muscle area between the reciprocal 3-4 ribs were measured using an Aloka SSD-500 model B-ultrasonic instrument, and the lean meat percentage was estimated. During measurement, the probe die and the part to be measured are compact without heavy pressure; the straight plane of the probe is vertical to the longitudinal axis plane of the median line of the pig back, and can not be obliquely cut; when identifying the ultrasonic image, a strong echo belt generated by a skin interface, an interesterification connective tissue and a longisimus muscle membrane of the back is firstly determined, and then a strong echo image of muscle membranes around the eye muscle is determined to determine an eye muscle area belt.
2.2 GBS technology-based pig whole genome SNP (Single nucleotide polymorphism) typing method
The enzyme digestion effect of 36 common type II restriction enzymes and 24 double enzyme digestion combinations in the pig genome is predicted by simulating enzyme digestion of the pig genome; according to research purposes and population characteristics, an EcoRI-Msp I double enzyme digestion combination is selected for GBS library construction of pigs, GBS experiments and analysis processes are optimized, and a pig whole genome SNP typing method based on a GBS technology is established. By GBS sequencing of a wenchok duroc 3757 head duroc pig, 102,254 SNPs were identified that covered the entire genome of the pig.
2.3 Whole genome Association analysis
The genome-wide association analysis is carried out on 3 traits of the Duroc pig population, namely 100kg body weight backfat thickness (BF), 100kg body weight eye muscle area (LMA) and 100kg body weight Lean Meat Percentage (LMP).
2.4SNP quality control
In order to obtain a reliable GWAS result, the invention adopts the following conditions for quality control: (1) MAF is more than or equal to 0.05; (2) HWE is more than or equal to 10E-6; (3) the number of two homozygote individuals of each SNP is more than or equal to 30.
2.5 SNP loci significantly related to economic traits
And (3) detecting significant sites at the genome level, performing Bonferroni correction by adopting independent mark numbers, and calculating the independent mark numbers by using a PLINK indep-pair command to obtain a p value of 0.05/14,084-3.55 × 10 at 5% significant level of the Bonferroni genome level-6And the p-value threshold of the potential association is 1.0/14,084-7.10 × 105。
According to the standard, an SNP locus (P) which is significant to 5% of genome level corrected by Bonferroni and reaches various economic traits of pigs is obtained<10-5.45)。
3. Results and analysis
The invention takes 3702 Duroc populations as objects, 102,254 SNPs obtained by optimized GBS sequencing are used for carrying out GWAS analysis on 3 important traits of the pigs, and an SNP (Chr 7: 34960267) site which is obviously related to three production traits of 100kg weight backfat thickness, 100kg weight eye muscle area and 100kg weight lean meat percentage of the pigs is determined, as shown in figure 1.
Example 2 frequency distribution of SNP (Chr 7: 34960267) in different breeds of pigs
1. Test materials
Local pig breeds: 6 Laiwu pigs, 5 Erhualian pigs, 6 Hetao pigs, 6 Min pigs, 5 Jinhua pigs, 6 Wuzhishan pigs, 6 Luchuan pigs, 14 Meishan pigs and 9 Rongchang pigs. The commercial pig breeds comprise: 16 Duroc pigs, 12 white pigs, 8 Yorkshire pigs and 36 big Duchan pigs.
2. Test method
2.1 extraction of genomic DNA
The genomic DNA was extracted using the GIAamp DNA Mini kit from QIAGEN, and the specific procedures were as follows:
(1) adding 180 mul of ATL buffer solution into a 1.5ml centrifuge tube, adding 20 mul of proteinase K, and uniformly mixing;
(2) taking about 20mg of ear tissue samples, putting the ear tissue samples into the solution, and digesting for 8 hours at 55 ℃;
(3) adding 3 mul of RNase A into the digested tissue fluid, and placing in a constant-temperature water bath kettle at 37 ℃ for 30min to degrade RNA in the tissue fluid;
(4) adding 200 μ l of AL solution into the solution, performing vortex oscillation, mixing, and digesting in 70 deg.C water bath for 10 min; after the centrifugal tube returns to the room temperature, adding 200 mu l of absolute ethyl alcohol, and uniformly mixing by vortex oscillation again;
(5) adding all the above solutions into DNA adsorption column, standing at room temperature for 2min, and centrifuging at 13000rpm for 1 min;
(6) after the centrifugation is finished, the filtrate is discarded, the adsorption column is placed into another new collection tube of 2ml, 500 mu lPW1 solution is added, and the centrifugation is carried out for 1min at 13000 rpm;
(7) after the centrifugation is finished, the adsorption column is put into another new collection tube of 2ml, 500 mul PW2 solution is added, and centrifugation is carried out for 3min at 13000 rpm;
(8) pouring off the solution in the collecting pipe, wiping off the liquid at the pipe orifice of the collecting pipe with a paper towel, putting the adsorption column in the collecting pipe again, and centrifuging at 13000rpm for 2 min;
(9) opening the cover of the DNA adsorption column, placing into a numbered 1.5ml centrifuge tube, and standing at room temperature for 2min to volatilize the residual ethanol in the tube;
(10) adding 120-150 μ l AE buffer solution into the center of the adsorption column, standing at room temperature for 2min, and centrifuging at 13000rpm for 1min to obtain the solution as genome DNA. Detecting the qualified genome DNA by 1% agarose gel electrophoresis, and quantifying the concentration by using a NanoDrop; the concentration was then diluted to 50 ng/. mu.l in bulk for further experiments.
2.2 frequency of SNP (Chr 7: 34960267) in Whole genome re-sequencing data of various pigs
The genomic DNA of various pigs is sequenced, and the frequency distribution of SNP (Chr 7: 34960267) in the resequencing data of the local pig species and the commercial pig species is counted.
3. Results and analysis
The results of the frequency distribution of SNPs in different local and commercial pig breeds of SNP (Chr 7: 34960267) are shown in Table 1, and there is a significant difference between local and commercial pig breeds. G in commercial pigs is the dominant allele, and C in endemic pigs is the dominant allele.
TABLE 1 SNP (Chr 7: 34960267) SNP frequency in different local and commercial pig species
An SNP marker associated with a plurality of economic traits of pigs is obtained, and in a population with low lean meat percentage, the lean meat percentage of the population after breeding can be improved by selecting individuals of allele G for breeding.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of agriculture in China
<120> SNP site related to economic traits of pigs and application thereof
<130>KHP171115707.0
<160>1
<170>SIPOSequenceListing 1.0
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
ctcaatgatt gtttcagcta ctgttgattt ttaataaaat atgtgtaact tgcaaattag 60
cacattttta ttacttatcc tttaagcagc attgtattct ctgtgtaaag tgtttgggag 120
aattctcagt gatttagcca cccgcacgca aggggaccca gccacccctg gtcatgtgta 180
tgcagaattg tttgtgttca c 201
Claims (3)
1. The application of the SNP locus related to the economic character of the pig in the auxiliary breeding of the pig is provided, wherein the breeding aim is to breed excellent characters with low backfat thickness, large eye muscle area and/or high lean meat percentage; the SNP locus is Chr7 of genome version EnsemblScrofa 10.2: 34960267, the alleles at the locus are G and C, wherein the pig with the G allele has lower backfat thickness, larger eye muscle area and/or higher lean meat percentage compared to the pig with the C allele.
2. Use according to claim 1, characterized in that it comprises the following steps:
(1) detecting the genotype of the sample pig at the SNP locus;
(2) selecting the pig with G/G homozygote type to breed dominant strain; the dominant line shows high lean meat percentage.
3. The use of claim 2, wherein the step (1) is performed by direct sequencing or by amplifying a fragment containing the SNP site and detecting the amplified fragment.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103497994A (en) * | 2013-09-09 | 2014-01-08 | 安徽省农业科学院畜牧兽医研究所 | Molecule marking method for pig backfat thickness property |
| CN104480108A (en) * | 2014-12-12 | 2015-04-01 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying pig back fat thickness and special primer pair thereof |
| CN105255870A (en) * | 2015-10-30 | 2016-01-20 | 中国农业大学 | SNP (Single Nucleotide Polymorphism) molecular marker related with back fat thickness trait of pig and detection method of SNP molecular marker |
| CN105969845A (en) * | 2016-04-27 | 2016-09-28 | 华中农业大学 | Molecular marker of loin-eye area character-related gene SVEP1 and application of molecular marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497994A (en) * | 2013-09-09 | 2014-01-08 | 安徽省农业科学院畜牧兽医研究所 | Molecule marking method for pig backfat thickness property |
| CN104480108A (en) * | 2014-12-12 | 2015-04-01 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying pig back fat thickness and special primer pair thereof |
| CN105255870A (en) * | 2015-10-30 | 2016-01-20 | 中国农业大学 | SNP (Single Nucleotide Polymorphism) molecular marker related with back fat thickness trait of pig and detection method of SNP molecular marker |
| CN105969845A (en) * | 2016-04-27 | 2016-09-28 | 华中农业大学 | Molecular marker of loin-eye area character-related gene SVEP1 and application of molecular marker |
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