SNP (Single Nucleotide Polymorphism) molecular marker related to feed-weight ratio character on pig chromosome 11 and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a material weight ratio character on a pig chromosome 11. The SNP molecular marker is Ggt located at the 34549890bp position on the chromosome 11 of an international pig genome 11.1 version; a mutation. Through the application of the SNP molecular marker in pig feed-weight ratio character breeding, breeding of pig strains with low feed-weight ratio performance and pig feed-weight ratio character improvement, the feed-weight ratio of pigs can be effectively reduced, the feed cost is reduced, and the economic benefits of pig farms are improved. By breeding the dominant allele A of the SNP molecular marker, the frequency of the dominant allele A can be increased generation by generation, the feed-weight ratio of the pigs is reduced, excellent pigs with low feed-weight ratio characters are bred, and the genetic improvement progress of the pigs is accelerated, so that the economic benefit of breeding of the breeding pigs is effectively improved.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biotechnology and molecular marker technology, and in particular to an SNP molecular marker related to feed conversion ratio trait on porcine chromosome 11 and its application. Background Art

[0002] The feed cost accounts for 60 - 80% of the total cost of pig farming, which is the largest single expenditure. Therefore, efficiently converting feed into pig weight gain is extremely important for reducing breeding costs, and improving the feed utilization efficiency of pigs is the key way to reduce the production cost of pig farming. Reducing the feed conversion ratio is the most effective and practical method to reduce grain consumption and pork production cost. The feed conversion ratio, which means the amount of feed consumed for a pig to gain one kilogram in weight, is a common indicator to measure feed utilization efficiency. The feed utilization efficiency of pigs is affected by many factors such as genetics, diet composition, environmental temperature, feeding management, and pig health status, but genetic factors are the main reason. Therefore, conducting genetic analysis on the feed conversion ratio trait of pigs, identifying the key genes and their key mutations affecting the feed conversion ratio, has important value for the genetic improvement of feed efficiency. In the past, people used the candidate gene method and QTL mapping method to identify QTL. Although the candidate gene method has the advantages of simple method and convenient operation, it can only target genes with known biological functions; while the confidence region of the candidate genes identified by the QTL mapping method is also relatively large, which limits the application of molecular markers for complex traits in livestock genetic breeding.

[0003] With the development of high-throughput sequencing technology, by detecting single nucleotide polymorphisms (SNPs) on the whole porcine genome and combining with genome-wide association study (GWAS) technology, the key genes and their key molecular markers affecting pig feed utilization efficiency can be identified, which will help to reveal the genetic mechanism underlying the formation of pig feed utilization efficiency traits. Furthermore, applying the genes and molecular markers that improve feed utilization efficiency to marker-assisted selection and genomic selection breeding technologies will greatly accelerate the genetic progress of pig feed utilization efficiency, improve breeding efficiency, reduce the production cost of enterprises, and thus improve the economic benefits of the pig farming industry. Summary of the Invention

[0004] The object of the present invention is to provide an SNP molecular marker related to feed conversion ratio on porcine chromosome 11, and its application in the breeding of pig feed conversion ratio traits, cultivating pig strains with low feed conversion ratio performance, and improving pig feed conversion ratio traits, which can effectively reduce the feed conversion ratio of pigs, reduce feed costs, and improve the economic benefits of pig farms.

[0005] According to the first aspect of the present invention, there is provided an SNP molecular marker related to feed conversion ratio on porcine chromosome 11, and this SNP molecular marker is a G>A mutation located at the 34,549,890bp position on chromosome 11 of the International Pig Genome Version 11.1. Thus, through the application of this SNP molecular marker in the breeding of porcine feed conversion ratio traits and the cultivation of porcine strains with low feed conversion ratio performance, as well as in the improvement of porcine feed conversion ratio traits, the feed conversion ratio of pigs can be effectively reduced, the feed cost can be reduced, and the economic benefits of pig farms can be improved.

[0006] According to the second aspect of the present invention, there is provided an application of an SNP molecular marker in the preparation of a product for identifying porcine feed conversion ratio traits, and this SNP molecular marker is a G>A mutation located at the 34,549,890bp position on chromosome 11 of the International Pig Genome Version 11.1. Thus, through this SNP molecular marker, a product for identifying porcine feed conversion ratio traits (such as primer pairs, kits or probes, etc.) can be prepared, and through this product, the porcine feed conversion ratio traits can be selected and bred, thereby effectively reducing the feed conversion ratio of pigs, reducing the feed cost, and improving the economic benefits of pig farms.

[0007] According to the third aspect of the present invention, there is provided an application of an SNP molecular marker in the breeding of porcine feed conversion ratio traits, and this SNP molecular marker is a G>A mutation located at the 34,549,890bp position on chromosome 11 of the International Pig Genome Version 11.1. Thus, through the application of this SNP molecular marker in the breeding of porcine feed conversion ratio traits, the feed conversion ratio of pigs can be effectively reduced, the feed cost can be reduced, and the economic benefits of pig farms can be improved.

[0008] According to the fourth aspect of the present invention, there is provided an application of an SNP molecular marker in the cultivation of porcine strains with low feed conversion ratio performance, and this SNP molecular marker is a G>A mutation located at the 34,549,890bp position on chromosome 11 of the International Pig Genome Version 11.1. This application includes the following steps:

[0009] S1: Detect the genotype of the above-mentioned SNP molecular marker in replacement pigs;

[0010] S2: Select the individuals with the AA genotype detected in step S1 as breeding pigs, eliminate the GG and GA genotypes, and breed these breeding pigs;

[0011] S3: Detect the above-mentioned SNP molecular marker in the piglets born from the breeding in step S2, retain the AA genotype individuals for breeding, and cultivate a porcine strain with low feed conversion ratio performance.

[0012] Therefore, by selecting the dominant allele A of the SNP molecular marker, the frequency of the dominant allele A can be increased from generation to generation, the feed-to-weight ratio of pigs can be reduced, and pig breeds with low feed-to-weight ratio performance can be developed, thereby accelerating the progress of pig genetic improvement and effectively improving the economic benefits of pig breeding.

[0013] According to a fifth aspect of the present invention, there is provided an application of a SNP molecular marker in improving the feed-to-weight ratio trait of pigs, wherein the SNP molecular marker is a G>A mutation located at the 34549890bp position on chromosome 11 of the international pig genome version 11.1, and the application comprises the following steps:

[0014] S1: Detecting the genotype of the SNP molecular markers in the replacement pigs;

[0015] S2: selecting the individuals with the AA genotype detected in step S1 as breeding pigs, eliminating the GG and GA genotypes, and breeding the breeding pigs;

[0016] S3: Detect the SNP molecular markers in the piglets born from mating in step S2, keep the individuals with AA genotype, and then breed them, retain the dominant genotype AA in the offspring pigs, eliminate the GG and GA genotypes, so as to increase the frequency of the dominant allele A from generation to generation, thereby reducing the feed-to-weight ratio of pigs and improving the feed-to-weight ratio trait.

[0017] Therefore, by selecting the dominant allele A of the SNP molecular marker, the frequency of the dominant allele A can be increased from generation to generation, the feed-to-weight ratio of pigs can be reduced, the feed-to-weight ratio trait of pigs can be genetically improved, the progress of pig genetic improvement can be accelerated, and the economic benefits of pig breeding can be effectively improved.

[0018] According to the sixth aspect of the present invention, there is provided an application of a SNP molecular marker for detecting / screening / identifying the feed-to-weight ratio trait of pigs, wherein the SNP molecular marker is a G>A mutation located at the 34549890bp position on chromosome 11 of the international pig genome version 11.1. Thus, by detecting the genotype of the pig through the SNP molecular marker, the feed-to-weight ratio trait of the pig can be detected, screened or identified efficiently, quickly, accurately and conveniently.

[0019] In certain embodiments, the pig is a Duroc pig and its synthetic strains.

[0020] In certain embodiments, the pig is a Duroc-Changda triple hybrid pig.

[0021] According to the seventh aspect of the present invention, a primer pair for identifying SNP molecular markers related to the feed conversion ratio trait in pigs is provided. The SNP molecular marker is a G>A mutation at the 34,549,890 bp position on chromosome 11 of the international pig genome version 11.1. The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3. Thus, through this primer pair, the feed conversion ratio trait in pigs can be detected, screened or identified efficiently, rapidly, accurately and conveniently, facilitating the subsequent breeding work.

[0022] According to the eighth aspect of the present invention, a kit for identifying SNP molecular markers related to the feed conversion ratio trait in pigs is provided. The SNP molecular marker is a G>A mutation at the 34,549,890 bp position on chromosome 11 of the international pig genome version 11.1. The kit contains a primer pair, and the nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3. Thus, through this kit, the feed conversion ratio trait in pigs can be detected, screened or identified efficiently, rapidly, accurately and conveniently, facilitating the subsequent breeding work.

[0023] According to the ninth aspect of the present invention, there is provided an application of the primer pair or the kit in the preparation of a product for identifying the feed conversion ratio trait in pigs. Thus, through this application, the feed conversion ratio trait in pigs can be identified efficiently, rapidly, accurately and conveniently, facilitating the subsequent breeding work.

[0024] According to the tenth aspect of the present invention, there is provided an application of the primer pair or the kit in the breeding of pigs with the feed conversion ratio trait. Through this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established, reducing the feed conversion ratio of pigs, rapidly and accurately improving the breeding of pigs with high feed utilization efficiency, and accelerating the breeding progress.

[0025] According to the eleventh aspect of the present invention, there is provided an application of the primer pair or the kit in the cultivation of pig lines with low feed conversion ratio traits. Through this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established, reducing the feed conversion ratio of pigs and cultivating pig lines with low feed conversion ratio traits.

[0026] Advantages of the present invention:

[0027] (1) The present invention studies and determines that the SNP molecular marker related to the feed conversion ratio in pigs is located at the G>A mutation at the 34,549,890 bp position on chromosome 11 of pigs, verifies its influence on the feed conversion ratio trait, and finally establishes an efficient and accurate molecular marker-assisted breeding technology, which is applied to the genetic selection and improvement of improving the feed conversion ratio in pigs, thereby improving the feed utilization efficiency of the pig population, reducing the production cost of enterprises, increasing the economic profit of enterprises, and enhancing the core competitiveness. By optimizing the dominant allele genotype AA of this SNP, the frequency of the dominant allele A can be gradually increased from generation to generation, the feed conversion ratio of the core group of pigs can be reduced, and the genetic improvement progress of the feed utilization efficiency trait of pigs can be accelerated, thus effectively improving the economic benefits of breeding pigs.

[0028] (2) The present invention provides a primer pair and a kit for identifying SNP molecular markers related to the feed conversion ratio. Through this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established, the feed conversion ratio of pigs can be reduced, and the improvement and selection of high feed utilization efficiency of breeding pigs can be carried out quickly and accurately, accelerating the breeding progress. Brief Description of the Drawings

[0029] Figure 1 It is a partial Manhattan plot of the genome-wide association analysis of the feed conversion ratio trait on chromosome 11 of Duroc×Landrace×Yorkshire pigs: Among them, the abscissa represents the chromosome number of pigs, the ordinate represents the -logP value, and the molecular marker g.266G>A is indicated by the arrow in the figure. Detailed Embodiments

[0030] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0031] Example 1 Screening of SNP Molecular Markers Related to the Feed Conversion Ratio

[0032] (1) Experimental Animals

[0033] The experimental pig population used in the present invention is 3,312 Duroc×Landrace×Yorkshire pigs of Guangxi Yangxiang Co., Ltd.

[0034] In this experiment, Duroc×Landrace×Yorkshire pigs were selected from this resource population. The pigs were fed and watered freely, and the entire feeding method, breeding conditions, etc. were always kept consistent, which is a conventional method.

[0035] (2) Sample Collection

[0036] The tail tips and ear tissues of the above-mentioned piglets were collected and soaked in an ethanol solution with a volume fraction of 75%, and stored in a -20°C refrigerator for later use.

[0037] (3) Genotyping of 50K SNPs in the Pig Genome

[0038] For each individual in the above-mentioned resource population of 3,312 Duroc×Landrace×Yorkshire pigs, ear tissue or tail tissue was collected, and genomic DNA was extracted using the standard phenol-chloroform method. The concentration and OD ratios (OD260 / 280, OD260 / 230) of the DNA in each sample were accurately measured using a Nanodrop 2000 / 2000C nucleic acid and protein detector. The DNA samples that passed the detection by the NanoDrop 2000 / 2000C nucleic acid and protein detector were diluted to approximately 50 ng / μL according to the detected concentration. Then, 6 μL of the extracted DNA sample to be tested was mixed with 2 μL of Loading Buffer and loaded onto a 1% agarose gel by mass / volume ratio, and electrophoresis was performed at 150 V for 25 min. Observation and photography were carried out under an ultraviolet spectrophotometer and a gel imaging device to observe the integrity of the DNA.

[0039] The DNA samples were sent to Neogene Biotechnology (Shanghai) Co., Ltd., and genotyping of the porcine whole-genome 50K SNP chip (Illumina, USA) was performed on the Illumina Beadstration platform according to the company's standard procedures. The checkmarker in the R language GenABEL package was used to perform quality control on the 50K SNP chip scanning genotyping data of all samples, and SNPs with an individual detection rate lower than 90%, a pedigree Mendelian error rate higher than 0.1, a minor allele frequency less than 0.01, and a Hardy-Weinberg equilibrium significance level higher than 10 -6 were excluded, and finally, effective genotype data of 45,094 SNPs were obtained.

[0040] (4) Genome-wide association (GWAS) analysis

[0041] To eliminate the population stratification effect, the present invention used a linear mixed model single-point regression analysis combined with the R language FarmCPU software package for GWAS analysis. In the analysis model, the stratification effect was corrected using the genomic similarity between individuals. The Bonferrini method was used to determine the significance threshold for the association degree between SNPs and the feed conversion ratio trait. The genome-wide significant threshold was 0.05 divided by the number of effective SNP loci, that is, the genome-wide significant level threshold was 1.11E-06, that is, 0.05 / 45,094 (the number of effective SNPs); the chromosome-level significant threshold was 1 divided by the number of effective SNP loci, that is, the chromosome-level significant level threshold was 2.21E-05, that is, 1 / 45,094 (the number of effective SNPs).

[0042] The results of the GWAS analysis are as Figure 1 shown. From Figure 1It can be seen that there is a locus on chromosome 11 of Duroc×Landrace×Yorkshire pigs that significantly affects the feed conversion ratio (indicated by the arrow in the figure). The significantly associated SNP locus is a G>A mutation at position 34,549,890 bp on chromosome 11 of the International Pig Genome Version 11.1 reference sequence (P value is 1.60E-14), and this SNP molecular marker is denoted as the g.266G>A molecular marker.

[0043] 5) Association analysis between different genotypes and feed conversion ratio phenotypes

[0044] As can be seen from Table 1, the g.266G>A molecular marker is extremely significantly correlated with the feed conversion ratio trait (P<0.001), indicating that this molecular marker significantly affects the feed conversion ratio of pigs. By selecting this SNP locus in pigs, the feed efficiency of this population can be improved, and thus the breeding process can be accelerated.

[0045] In addition, it can also be seen from Table 1 that the feed conversion ratio of the AA genotype is lower than that of the GA genotype. The average phenotypic value of the AA genotype individuals is 0.18 lower than that of the GA genotype individuals, and the difference is extremely significant (P<0.001); the average phenotypic value of the AA genotype individuals is 0.20 lower than that of the GG genotype individuals, and the difference is extremely significant (P<0.001). Therefore, the AA genotype is the dominant genotype, and A is the dominant allele. In breeding, pigs with the AA genotype should be gradually retained, and pigs with the AA and GA genotypes should be eliminated to gradually increase the frequency of allele A at this locus, which can significantly reduce the feed conversion ratio of pigs and bring more economic benefits to breeding enterprises.

[0046] Table 1 Correlation analysis between the g.266G>A molecular marker and feed conversion ratio

[0047]

[0048] Example 2 Amplification and sequencing of the target DNA sequence

[0049] (1) Primer design

[0050] Based on the upstream and downstream sequences of the position where the g.266G>A molecular marker is located (the G>A mutation at position 34,549,890 bp on chromosome 11) through the Ensembl website (http: / / asia.ensembl.org / index.html), primers were designed using the primer design software primer premier 6.0. The nucleotide sequences of the designed primers are as follows:

[0051] P001-F (SEQ ID NO:2): 5’-CTCTACAGGCCAGGAGGTAAA-3’,

[0052] P002-R (SEQ ID NO:3): 5’-TTACAGTTGGTTCCCTGATTCTC-3’;

[0053] (2) PCR amplification

[0054] Add 1 μL of DNA template, 3.4 μL of double-distilled water, 5 μL of 2× Tag PCR StanMix with Loading Dye, and 0.3 μL each of primers P001-F and P002-R into a 10 μL reaction system. The PCR reaction conditions are as follows: pre-denaturation at 94 °C for 5 min, then denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 45 s, for 35 cycles, and finally extension at 72 °C for 5 min.

[0055] (3) DNA sequence determination

[0056] DNA sequence sequencing identification: perform two reactions for the forward and reverse of the gene fragment. Compare the obtained sequence with the NCBI genomic sequence to obtain the mutations at the corresponding SNP sites. The sequencing results are as follows (SEQ ID NO:1):

[0057]

[0058]

[0059] Note: M marked in the sequence list is the mutation site, shown as underlined + bold (the mutated base of G>A in parentheses is an allelic gene mutation), and the primer sequence positions are shown as underlined + italic at the beginning and end of this sequence.

[0060] The M at the 266th base in the sequence shown in SEQ ID No:1 represents the G>A base mutation. Through this nucleotide sequence, primers can be designed to detect the genotype of the g.266G>A molecular marker, and then further select and breed pigs based on the detection results for the feed conversion ratio trait; or design primers based on this nucleotide sequence to prepare a corresponding detection kit for directly detecting the g.266G>A molecular marker in pigs, and then select and breed pigs for the feed conversion ratio trait based on the detected genotype.

[0061] Example 3 Analysis of the effect of the g.266G>A molecular marker

[0062] According to Table 1, the phenotypic effect of the dominant allele genotype AA of the g.266G>A molecular marker has a feed conversion ratio 0.20 lower than that of the recessive allele genotype GG (2.55 vs. 2.75). Conservatively estimating that a reduction of 0.1 in the feed conversion ratio per pig saves 50 yuan in feed costs, then for each pig, more than 100 yuan in costs can be saved only for the trait of feed conversion ratio. For a large-scale pig farm with a population of 10,000 pigs, the economic benefit can be increased by more than 1 million yuan. Therefore, through marker-assisted selection or genomic selection, gradually selecting and retaining pigs with the genotype AA within the population can significantly increase the allele frequency of allele A, reduce the feed conversion ratio of the pig population, accelerate the improvement progress of pigs, and thus effectively improve the economic benefit of breeding breeding pigs.

[0063] The present invention provides a new molecular marker for marker-assisted selection and genomic selection of pigs by detecting the 266th base mutation site in the SEQ ID NO:1 sequence and preliminarily conducting an association analysis between its genotype and the feed conversion ratio of pigs.

[0064] Example 4 Application of the g.266G>A molecular marker in breeding pig lines with low feed conversion ratio performance

[0065] S1: Detect the genotype of the g.266G>A molecular marker in replacement pigs;

[0066] S2: Select individuals with the genotype AA of the g.266G>A molecular marker detected in step S1 as breeding pigs, and breed these breeding pigs;

[0067] S3: Detect the genotype of the g.266G>A molecular marker in the piglets born from the breeding in step S2, retain the AA genotype individuals for breeding, eliminate the GG and GA genotypes, and then breed to cultivate a pig line with low feed conversion ratio performance.

[0068] Example 5 Application of the g.266G>A molecular marker in improving the feed conversion ratio trait of pigs

[0069] S1: Detect the genotype of the g.266G>A molecular marker in replacement pigs;

[0070] S2: Select individuals with the genotype AA of the g.266G>A molecular marker detected in step S1 as breeding pigs, eliminate the GG and GA genotypes, and breed these breeding pigs;

[0071] S3: Detect the genotype of the g.266G>A molecular marker in the piglets born from the breeding in step S2. Retain the individuals with the AA genotype for breeding, and then conduct breeding. Retain the individuals with the dominant genotype AA in the offspring pigs, and eliminate the GG and GA genotypes to gradually increase the frequency of the dominant allele A, thereby reducing the feed conversion ratio of pigs and improving the feed conversion ratio trait.

Claims

1. A SNP molecular marker on chromosome 11 of pigs related to feed-to-weight ratio, characterized in that: The SNP molecular marker is a G>A mutation located at position 34549890bp on chromosome 11 of the international porcine genome version 11.

1.

2. Use of the SNP molecular marker described in claim 1 in the preparation of a product for identifying the pig feed-to-weight ratio trait or in the selection of pig feed-to-weight ratio traits.

3. The use of the SNP molecular marker described in claim 1 in breeding pig strains with low feed-to-weight ratio performance, characterized in that: The application comprises the following steps: S1: Detecting the genotype of the SNP molecular marker as claimed in claim 1 on the replacement pig; S2: selecting the individuals with the AA genotype detected in step S1 as breeding pigs, eliminating the GG and GA genotypes, and breeding the breeding pigs; S3: Detect the SNP molecular markers as described in claim 1 on the piglets born from mating in step S2, save the AA genotype individuals for breeding, and then breed them to cultivate a pig strain with low feed-to-weight ratio.

4. The use of the SNP molecular marker described in claim 1 in improving the feed-to-weight ratio of pigs, characterized in that: The application comprises the following steps: S1: Detecting the genotype of the SNP molecular marker as claimed in claim 1 on the replacement pig; S2: selecting the individuals with the AA genotype detected in step S1 as breeding pigs, eliminating the GG and GA genotypes, and breeding the breeding pigs; S3: Detect the SNP molecular markers as described in claim 1 in the piglets born from mating in step S2, retain the individuals with AA genotype, and then breed them, retain the dominant genotype AA in the offspring pigs, eliminate the GG and GA genotypes, so as to increase the frequency of the dominant allele A from generation to generation, thereby reducing the feed-to-weight ratio of pigs and improving the feed-to-weight ratio trait.

5. Use of the SNP molecular marker described in claim 1 in detecting / screening / identifying the feed-to-weight ratio trait of pigs.

6. The use according to any one of claims 2 to 5, characterized in that: The pigs are Duroc pigs and their synthetic strains.

7. The use according to claim 6, characterized in that: The pig is a Duchang-Changda three-way hybrid pig.

8. A primer pair for identifying SNP molecular markers associated with pig feed-to-weight ratio, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:

3.

9. A kit for identifying SNP molecular markers associated with pig feed-to-weight ratio traits, characterized in that: The kit comprises the primer pair described in claim 8.

10. Use of the primer pair described in claim 8 or the kit described in claim 9 in the preparation of a product for identifying the feed-to-weight ratio trait of pigs, in the selection of pigs for the feed-to-weight ratio trait, or in the breeding of pig strains with low feed-to-weight ratio trait.

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