CN108676897B - SNP marker influencing daily gain traits of pigs and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biotechnology and molecular markers, and particularly relates to application of a pig SNP molecular marker in pig daily gain trait research and pig breeding. The pig SNP molecular marker locus is shown in SEQ ID NO.1, is positioned in the 321 st nucleic acid single base mutation of the sequence fragment and is named as: g15588826C > T. The SNP molecular marker corresponds to 15588826 th nucleotide site C > T mutation on the chromosome of the international pig reference genome 10.2 version 3. By optimizing the dominant allele of the SNP molecular marker, the genetic progress of the white-day weight gain character can be increased, the breeding time of the white-day weight gain character is reduced, and the economic benefit of breeding the breeding pigs is effectively improved.

Description

A SNP marker affecting daily weight gain in pigs and its application

technical field

The invention belongs to the technical field of molecular biotechnology and molecular markers, and particularly relates to the application of a porcine SNP molecular marker in the study of pig daily weight gain traits and pig breeding.

Background technique

Daily weight gain is an important indicator for evaluating the growth performance of pigs, so it is the focus of genetic improvement research in breeding pigs. In the past 100 years, the breeding goals of Western pig breeds have often focused on improving lean meat percentage and reducing backfat thickness, which has improved the genetic progress of daily weight gain to a certain extent, but there has been a lack of more direct breeding methods. Traditional breeding methods have little effect on traits that are difficult to phenotype (such as daily gain), and molecular marker assisted selection (MAS) has broken this technical barrier. This method can be used to improve meat quality (such as intramuscular fat) and disease resistance traits (such as anti-diarrhea), which are difficult or expensive to measure in vivo (such as daily gain), and reproduction (such as litter size), etc. Expression of late-limiting traits in life activities. Moreover, MAS is not easily affected by the environment, gender, and age, so it can be selected early, which can shorten the generation interval and improve the efficiency of seed selection (Liu Xiaochun et al., 2008). Therefore, if MAS technology can be used to improve the daily weight gain of breeding pigs, it will have broad application prospects.

The Big White is one of the world's most famous lean pig breeds. In the modern pig breeding system, Dabai is famous for its high fertility, good growth and fattening, and strong adaptability. Due to its excellent comprehensive performance, it can be used as the male or female parent of many synthetic commercial pigs, and has a wide range of applications. Therefore, improving the daily weight gain of large white pigs can effectively shorten the fattening time of commercial pigs and save breeding costs.

SUMMARY OF THE INVENTION

In order to overcome the insufficiency and shortcoming of the genetic selection of daily gain in the prior art, the purpose of the present invention is to provide the application of a porcine SNP molecular marker in the study of porcine daily gain traits and pig breeding.

The object of the present invention is achieved through the following technical solutions:

Application of a porcine SNP molecular marker in the study of pig daily weight gain traits and pig breeding; the SNP site of the porcine SNP molecular marker corresponds to the C>T mutation at 15588826bp on chromosome 3 of the international pig reference genome version 10.2 ; The SNP site of the molecular marker is shown in SEQ ID NO.1, wherein M in the sequence is T or C, resulting in the difference in daily weight gain traits;

The pig is at least one of Dabai and its synthetic line;

The primers used to identify the above-mentioned pig SNP molecular markers are preferably PCR-F and PCR-R;

Upstream primer PCR-F: 5'-CAGGTAGGACACCTCCACT-3';

Downstream primer PCR-R: 5'-TGACAGCAGCACTAAATGAC-3';

The research on the traits of daily gain in pigs is preferably the use of the above-mentioned pig SNP molecular markers to identify the traits of daily weight gain in pigs;

The research on the pig daily weight gain trait preferably also includes utilizing the above-mentioned pig SNP molecular marker to regulate the pig daily weight gain;

Described breeding is preferably molecular marker-assisted breeding;

A method for detecting daily weight gain traits of pigs, comprising the following steps:

Detecting whether the single nucleotide of the above-mentioned pig SNP molecular marker on the pig chromosome 3 is C or T;

The pig is at least one of Dabai and its synthetic line;

A method for genetic improvement of pigs, comprising the following steps:

Determine the above-mentioned porcine SNP molecular markers of the breeding pigs in the core group of breeding pigs, and make corresponding selections according to the porcine SNP molecular markers: Subsequent breeding of breeding pigs CT-type and TT-type individuals at 15588826bp on chromosome 3 of the international pig reference genome version 10.2 , eliminate the CC type individuals at this point; increase the frequency of the allele T at this locus from generation to generation, thereby increasing the daily weight gain of the offspring pigs;

Described breeding pig is at least one of Dabai and its synthetic line;

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention researches and determines the molecular markers related to daily gain, and the use of the molecular markers for marker-assisted selection can greatly increase the daily weight gain of Dabai and its synthetic lines.

(2) The present invention provides a primer pair for identifying a molecular marker that affects pig daily weight gain. Through the molecular marker and the primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established and applied to the daily weight gain of breeding pigs In the genetic improvement of traits, the daily weight gain of pigs can be increased, thereby saving grain, reducing production costs of enterprises, improving enterprise profits, and increasing core competitiveness.

(3) The present invention can increase the genetic progress of the daily weight gain trait of Dabai by optimizing the dominant allele of the molecular marker, and reduce the breeding time of the daily weight gain trait of Dabai, thereby effectively improving the economic benefit of breeding pigs, wherein, by With this molecular marker, the present invention selects all CC-type individuals into TT-type individuals, the average daily weight gain of each pig can be increased by 88.7g, and a large-scale 10,000-head pig farm can increase pork by 62.09t within 70 days. It can be seen that The potential for excellent daily gain performance to provide benefits to the pig industry is enormous.

Description of drawings

Figure 1 is an analysis chart of daily weight gain of pigs of different genotypes.

Figure 2 is a genome-wide association (GWAS) analysis diagram of the daily weight gain trait on chromosome 3 of large white pigs; wherein: the abscissa represents the chromosome number of the pig; the ordinate represents the -logP value.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

In the example, a total of 689 purebred large whites were used; wherein, the age of pigs when the body weight reached 30 kg was measured by the OSBORNE system and recorded as Day1, and the age of pigs when the body weight reached 100 kg was recorded as Day2. The formula for calculating daily weight gain is: 70/(Day2-Day1). The above experiments were carried out in the Shahu Breeding Pig Farm of Guangdong Wen's Food Group Co., Ltd. In the measurement stage, 10 to 12 pigs were housed in each pen (each pig occupies an area of 2 square meters) with free access to food and water, and they were reared in a unified manner. Standard, uniform feeding rations.

(1) The extraction method of DNA from large white pig ear tissue refers to the extraction of whole genome DNA by the labeled phenol-chloroform method. The DNA of purebred large white population was tested for quality and concentration with Nanodrop-ND1000 spectrophotometer. A260/280 ratio of 1.8 to 2.0, A260/230 ratio of 1.7 to 1.9 is judged as qualified. Finally, the qualified DNA samples were uniformly diluted to 50 ng/µl.

(2) 80K SNP genotype detection in pig whole genome: GeneSeek Genomic Profiler Porcine80KSNP typing platform, using Illumina Infinium's instructions and standard procedures for chip hybridization and result scanning. Finally, the genotype data was read by GenomeStudio software. The quality control of the obtained genotype data was carried out with PLINK v1.07, and the detection rate was <99.7%, and the allele frequency (mimor allel frequency, MAF) was <0.01% or deviated from the Hardy-Weinberg Equilibrium (Hardy-Weinberg Equilibrium). , HWE)P≤10 ^-6 SNP markers, the individuals with detection rate <90% and family Mendelian error rate higher than 0.1 were excluded; the remaining 58991 SNP markers and 689 samples of quality control were used for subsequent data analysis.

(3) Genome-wide association (GWAS) analysis: using the GenABEL package in R software to perform GWAS analysis on the daytime gain, the model is: y=1u+Xb+Sc+Za+α. Among them, y is the measured phenotype value and (average daily weight gain value), u is the overall average value, b is the gender and fixed effect value, c is the remaining micro-effects, c—N(0, бc2), α is the random additive genetic effect vector, and α～N(0, бα2) (G is the molecular blood correlation matrix between individuals, бα2 is the additive genetic variance), e is the residual, X, S and Z are the pair of fixed Correlation matrix of effects and random benefits, with herd-year-season effects included in batch effects. The Bonferroni method was used to determine the significance threshold of the genome-wide association analysis. The significant threshold at the genome level was 0.05 divided by the number of effective SNP sites, that is, 0.05/58991=8.46E-7; the significant threshold at the chromosome level was 1 divided by the effective SNP sites. Quantity, ie 1/58991=1.70E-5. The GWAS analysis results are shown in Figure 2. It can be seen from Figure 2 that in Dabai and its synthetic lines, there are loci in chromosome 3 that significantly affect the daily weight gain trait, and the most strongly associated SNP is g.15588826C>T (P=4.97E-6).

(4) Correlation analysis between different genotypes and daily weight gain phenotypes: According to Table 1, the molecular marker SNP site g.15588826C>T was significantly correlated with daily weight gain traits (P<0.001), indicating that this molecule The marker significantly affects the daily weight gain of pigs, and the auxiliary selection of this SNP locus in pigs can improve the daily weight gain of the group, thereby speeding up the breeding process. In addition, according to Table 1 and Figure 1, it can be seen that the average daily weight gain of the TT type is higher than that of the CC and TC types, indicating that the homozygous CC is the most unfavorable for the average daily weight gain. Daily weight gain is an important indicator of growth traits, and high daily gain indicates good growth performance of pigs. Therefore, the growth performance of pigs with CC genotype is the worst. In the process of breeding, we need to eliminate the breeding pigs of CC type and keep the breeding pigs of TT and TC type to increase the allele T of this locus from generation to generation. frequency.

Table 1 Correlation between molecularly marked SNP site g.15588826C>T and daily weight gain

Example 2

(1) The amplification target fragment containing the target fragment of the SNP site significantly related to the daytime weight gain performance is a nucleotide sequence of 695bp in chromosome 3, and the upstream and downstream primers for sequence amplification are:

Upstream primer PCR-F: 5'-CAGGTAGGACACCTCCACT-3';

Downstream primer PCR-R: 5'-TGACAGCAGCACTAAATGAC-3'.

(2) PCR amplification system and conditions

Configure a 20uL system, including 3.5μL of DNA sample, 0.6μL of upstream primer, 0.6μL of downstream primer, 10mL of PCR mix, 5.3μL of ddH ₂ O, and PCR conditions are 98°C pre-denaturation for 2min, 98°C denaturation for 10s, 56.5°C annealing for 15s, 72°C The extension was carried out for 40 s for a total of 35 cycles, and the final extension was 72 °C for 10 min.

(3) DNA sequence sequencing and identification: sequence sequencing was carried out in Shenzhen Huada Gene Technology Co., Ltd., and the gene fragments were tested for positive and negative reactions. The measured sequence was compared with the NCBI genome sequence, and the mutation of the corresponding SNP site was obtained.

The sequencing results are as follows:

Note: The M marked in the sequence table is the mutation site, which is marked with an underline (mutation bases in parentheses are allelic mutations), and the primer sequence is shown in bold at the beginning and end of the sequence.

Example 3 Molecularly labeled SNP site g.15588826C>T effect analysis

The invention provides a SNP marker that can significantly increase the daily gain of large white pigs and their synthetic lines. Using the SNP for marker-assisted selection can greatly increase the daily weight gain breeding process of large white pigs and their synthetic lines. All the CC-type individuals with molecular markers affecting the daily weight gain traits of pigs of the present invention are all selected and bred into TT-type individuals, the average daily weight gain of each pig can be increased by 88.7g, and a large-scale pig farm with 10,000 pigs can increase pork within 70 days. 62.09t, it can be seen that the excellent daily weight gain performance has a huge potential to provide benefits to the pig industry. Among the individuals marked by this SNP, there is a significant difference in the daily weight gain performance between CC type individuals and TT type individuals (P<0.01). By optimizing the dominant allele (T) of the SNP in Dabai and its synthetic line, the The purpose of improving the economic benefits of commercial pigs.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> South China Agricultural University

<120> A SNP marker that affects pig daily weight gain and its application

<130> 1

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 695

<212> DNA

<213> Artificial Sequence

<220>

<223> Porcine SNP Molecular Markers

<400> 1

caggtaggac acctccactt ggctctccag ggactctccc aggtgggttg gtttgagtac 60

ctttgaaaca caatggctgg gttccaagac taagcatctc taaagagcca gttggaagtt 120

gtgttttttg actttgccac cagtcaccca gtgtcatgtc tactgcagtc acaattcaaa 180

ggaagccctg ggttcaagag gaggggacgt ggaccccact tctggctggg cagaataacc 240

ttacaaaaga gcatgtgggc gacatggctg cagagatttt gggaccatgt aatctacacg 300

tgggagactg ttcaacccac tctcccagct caggtggatt agccatcagc catccaaggg 360

cagagggcct gggactgcat ctgcagtctc tccctgctct gtttccctgt taactctgcc 420

atctctttga tggggcgtca ctgctgccac atctggtcct tgcaacctcg gtctggtctt 480

ccacccgaga aggaattgaa ttcagtccag cggccatttc cagagctgtg catgcaagga 540

caggagcgaa acattgcctc aggccctgtg cgggccgaga ggaaagggtg tctgcctgga 600

acgtgccgcg gtgagacgtg tgacaggtgt gcagctgacc gcaacacagg ccagatgtca 660

tttagtgctg ctgtcaggta gaagcaaagc attgg 695

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer PCR-F

<400> 2

caggtaggac acctccact 19

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer PCR-R

<400> 3

tgacagcagc actaaatgac 20

Claims (10)

1. The application of the pig SNP molecular marker in pig daily gain character research and pig breeding is characterized in that: the SNP locus of the pig SNP molecular marker corresponds to the 15588826bp C > T mutation on the chromosome of the international pig reference genome 10.2 version 3.

2. The application of the pig SNP molecular marker in pig daily gain trait research and pig breeding according to claim 1, wherein the pig SNP molecular marker comprises the following components in percentage by weight:

the pig is at least one of Chinese cabbage and a synthetic line thereof.

3. The application of the pig SNP molecular marker in pig daily gain trait research and pig breeding according to claim 1, wherein the pig SNP molecular marker comprises the following components in percentage by weight:

the primers used for identifying the porcine SNP molecular marker in claim 1 are PCR-F and PCR-R;

the upstream primer PCR-F: 5'-CAGGTAGGACACCTCCACT-3', respectively;

the downstream primer PCR-R': 5'-TGACAGCAGCACTAAATGAC-3' are provided.

4. The application of the pig SNP molecular marker in pig daily gain trait research and pig breeding according to claim 1, wherein the pig SNP molecular marker comprises the following components in percentage by weight:

the pig daily gain trait research is to identify the traits of the daily gain of pigs by using the pig SNP molecular markers in claim 1.

5. The application of the pig SNP molecular marker in pig daily gain trait research and pig breeding according to claim 4, wherein the pig SNP molecular marker comprises the following components in percentage by weight:

the pig daily gain trait research also comprises the step of regulating the daily gain of the pig by using the pig SNP molecular marker in claim 1.

6. The application of the pig SNP molecular marker in pig daily gain trait research and pig breeding according to claim 1, wherein the pig SNP molecular marker comprises the following components in percentage by weight:

the breeding is molecular marker assisted breeding.

7. A method for detecting the daily gain character of pigs is characterized by comprising the following steps:

detecting whether the single nucleotide of the porcine SNP molecular marker according to claim 1 on chromosome 3 of a pig is C or T.

8. The method for detecting the daily gain trait of pigs of claim 7, wherein:

the pig is at least one of Chinese cabbage and a synthetic line thereof.

9. A method of genetic improvement in pigs, comprising the steps of:

determining porcine SNP molecular markers according to claim 1 of the porcine in the porcine core group, and making corresponding selections according to the porcine SNP molecular markers: carrying out subculture breeding on the boars to select CT type and TT type individuals at 15588826bp on the chromosome No. 3 of the 10.2 version of the international pig reference genome, and eliminating CC type individuals at the point; so as to increase the frequency of the allele T of the locus generation by generation, thereby increasing the daily gain of the offspring pig.

10. The method of genetic improvement in swine according to claim 9, wherein:

the breeding pig is at least one of white rice and a synthetic line thereof.

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