SNP molecular marker related to feed conversion ratio on pig chromosome 11 and application thereof

Abstract

The application discloses a SNP molecular marker related to a feed conversion ratio trait on a pig chromosome 11, which is a G>A mutation located at the position of 34549890bp on the chromosome 11 in the international pig genome version 11.1. The application of the SNP molecular marker to the breeding of a pig line with a low feed conversion ratio and the improvement of the feed conversion ratio of pigs can effectively reduce the feed conversion ratio of pigs, reduce the feed cost and improve the economic benefits of a pig farm. The selection of the advantageous allele A of the SNP molecular marker can increase the frequency of the advantageous allele A generation by generation, reduce the feed conversion ratio of pigs, breed excellent pigs with a low feed conversion ratio, accelerate the genetic improvement of pigs and effectively improve the economic benefits of pig breeding.

Description

Technical Field

[0001] This invention relates to the fields of molecular biotechnology and molecular marker technology, and in particular to a SNP molecular marker on pig chromosome 11 related to the feed conversion ratio trait and its application. Background Technology

[0002] Feed costs account for 60-80% of the total cost of pig farming, making it the largest single expenditure. Therefore, efficiently converting feed into pig weight gain is extremely important for reducing farming costs, and improving feed utilization efficiency is a key way to reduce pig production costs. Reducing the feed conversion ratio (FCR) is the most effective and feasible method to reduce grain consumption and lower pork production costs. The FCR refers to the amount of feed consumed to gain one kilogram of weight in a pig and is a common indicator of feed utilization efficiency. Pig feed utilization efficiency is affected by many factors, including genetics, diet composition, environmental temperature, feeding management, and pig health, but genetic factors are the most significant. Therefore, genetic analysis of the FCR trait in pigs to identify key genes and their key mutations is of great value for the genetic improvement of feed efficiency. In the past, QTL identification was performed using candidate gene methods and QTL mapping. While candidate gene methods are simple and easy to operate, they can only target genes with known biological functions; and the confidence regions of candidate genes identified by QTL mapping are also relatively large, limiting the application of complex trait molecular markers in livestock genetic breeding.

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

[0004] The purpose of this invention is to provide a SNP molecular marker on pig chromosome 11 that is related to feed conversion ratio. The application of this SNP molecular marker in the selection of pig feed conversion ratio traits, the breeding of pig breeds with low feed conversion ratio performance, and the improvement of pig feed conversion ratio traits can effectively reduce the feed conversion ratio of pigs, reduce feed costs, and improve the economic benefits of pig farms.

[0005] According to a first aspect of the present invention, a SNP molecular marker related to feed conversion ratio (FCR) on pig chromosome 11 is provided. This SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 in International Pig Genome Version 11.1. Therefore, by applying this SNP molecular marker in the selection and breeding of pig strains with low FCR performance and in the improvement of pig FCR traits, the FCR of pigs can be effectively reduced, feed costs can be lowered, and the economic benefits of pig farms can be improved.

[0006] According to a second aspect of the present invention, an application of an SNP molecular marker in the preparation of products for identifying the feed conversion ratio trait in pigs is provided. The SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1. Therefore, using this SNP molecular marker, products (such as primer pairs, kits, or probes) for identifying the feed conversion ratio trait in pigs can be prepared. These products allow for selective breeding of pigs with the feed conversion ratio trait, thereby effectively reducing the feed conversion ratio in pigs, lowering feed costs, and improving the economic benefits of pig farms.

[0007] According to a third aspect of the present invention, an application of a SNP molecular marker in the selection of pig feed conversion ratio traits is provided. The SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1. Therefore, by applying this SNP molecular marker in the selection of pig feed conversion ratio traits, the feed conversion ratio of pigs can be effectively reduced, feed costs can be lowered, and the economic benefits of pig farms can be improved.

[0008] According to a fourth aspect of the present invention, there is an application of an SNP molecular marker in the breeding of pig breeds with low feed conversion ratio performance, wherein the SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1, and the application includes the following steps:

[0009] S1: Genotyping of the SNP molecular markers in replacement gilts;

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

[0011] S3: Detect the SNP molecular markers in the piglets born from mating in step S2, retain the AA genotype individuals for breeding, and then breed them to cultivate a pig breed with low feed conversion ratio.

[0012] Therefore, by selecting the dominant allele A of this SNP molecular marker, the frequency of the dominant allele A can be increased generation by generation, reducing the feed conversion ratio of pigs, creating pig breeds with low feed conversion ratio performance, accelerating the progress of pig genetic improvement, and thus effectively improving the economic benefits of pig breeding.

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

[0014] S1: Genotyping of the SNP molecular markers in replacement gilts;

[0015] S2: Select individuals with the AA genotype detected in step S1 as breeding pigs, eliminate individuals with the GG and GA genotypes, and breed these breeding pigs.

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

[0017] Therefore, by selecting the dominant allele A of this SNP molecular marker, the frequency of the dominant allele A can be increased generation by generation, reducing the feed conversion ratio of pigs, genetically improving the feed conversion ratio trait of pigs, accelerating the progress of pig genetic improvement, and thus effectively improving the economic benefits of pig breeding.

[0018] According to a sixth aspect of the present invention, an application of a SNP molecular marker in the detection / screening / identification of feed conversion ratio (FCR) traits in pigs is provided. The SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1. Therefore, by using this SNP molecular marker to detect the genotype of pigs, the FCR trait can be detected, screened, or identified efficiently, rapidly, accurately, and conveniently.

[0019] In some embodiments, the pig is a Duroc pig or its synthetic line.

[0020] In some embodiments, the pig is a Duroc-Landrace-Landrace-Landrace-Landrace-Handcross hybrid.

[0021] According to a seventh aspect of the present invention, a primer pair is provided for identifying a SNP molecular marker associated with the feed conversion ratio trait in pigs, wherein the SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1, and the nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3. Therefore, this primer pair allows for efficient, rapid, accurate, and convenient detection, screening, or identification of the feed conversion ratio trait in pigs, facilitating subsequent breeding efforts.

[0022] According to an eighth aspect of the present invention, a kit is provided for identifying a SNP molecular marker associated with the feed conversion ratio trait in pigs, wherein the SNP molecular marker is a G>A mutation located at position 34549890 bp on chromosome 11 of the international pig genome version 11.1. The kit comprises a primer pair, the nucleotide sequences of which are shown in SEQ ID NO:2 and SEQ ID NO:3. Therefore, this kit allows for efficient, rapid, accurate, and convenient detection, screening, or identification of the feed conversion ratio trait in pigs, facilitating subsequent breeding efforts.

[0023] According to a ninth aspect of the present invention, the primer pair or kit described herein is provided for use in the preparation of products for identifying the feed conversion ratio trait in pigs. Thus, this application allows for the efficient, rapid, accurate, and convenient identification of the feed conversion ratio trait in pigs, facilitating subsequent breeding efforts.

[0024] According to a tenth aspect of the present invention, an application of the primer pair or kit described above in the selection of feed conversion ratio traits in pigs is provided. Using this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established to reduce the feed conversion ratio in pigs, rapidly and accurately improve feed utilization efficiency in pigs through selective breeding, and accelerate breeding progress.

[0025] According to an eleventh aspect of the present invention, the application of the primer pair or kit described above in the breeding of pig breeds with low feed conversion ratio traits is provided. Using this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technique can be established to reduce the feed conversion ratio of pigs and cultivate pig breeds with low feed conversion ratio traits.

[0026] The beneficial effects of this invention are:

[0027] (1) This invention studies and identifies the G>A mutation at position 34549890 bp on chromosome 11 of pigs, a SNP molecular marker affecting feed conversion ratio (FCR). It verifies the effect of this mutation on the FCR trait and ultimately establishes an efficient and accurate marker-assisted breeding technology. This technology is applied to the genetic selection and improvement of pig FCR, thereby increasing feed utilization efficiency, reducing production costs, increasing economic profits, and enhancing core competitiveness. By optimizing the dominant allele AA of this SNP, the frequency of the dominant allele A can be increased generation by generation, reducing the FCR of the core pig herd and accelerating the genetic improvement of the feed utilization efficiency trait, thus effectively improving the economic benefits of breeding pigs.

[0028] (2) This invention provides a primer pair and kit for identifying SNP molecular markers related to feed conversion ratio. With this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established to reduce the feed conversion ratio of pigs, quickly and accurately improve the high feed utilization efficiency of breeding pigs, and accelerate the breeding progress. Attached Figure Description

[0029] Figure 1 This is a partial Manhattan plot of genome-wide association analysis of feed conversion ratio on chromosome 11 in Duroc, Landrace, and Large White pigs: the horizontal axis represents the chromosome number of the pig, the vertical axis represents the -logP value, and the arrow in the figure points to the g.266G>A molecular marker. Detailed Implementation

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

[0031] Example 1: Screening of SNP molecular markers related to material weight ratio

[0032] (1) Laboratory animals

[0033] The experimental pig population used in this invention consisted of 3,312 Duroc Landrace Large White pigs from Guangxi Yangxiang Co., Ltd.

[0034] This experiment selected Duroc, Landrace, and Large White pigs from this resource population. The pigs had free access to feed and water, and the feeding methods and rearing conditions remained consistent throughout the experiment, which was a conventional approach.

[0035] (2) Sample collection

[0036] Collect the above-mentioned piglet tail and ear tissues, soak them in a 75% ethanol solution, and store them in a -20℃ refrigerator for later use.

[0037] (3) Pig genome 50K SNP genotyping

[0038] Ear or tail tissues were collected from each of the 3312 Duroc, Landrace, and Large White pigs in the aforementioned resource population. Whole-genome DNA was extracted using the standard phenol-chloroform method. The concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined using a NanoDrop 2000 / 2000C nucleic acid and protein analyzer. DNA samples that passed the NanoDrop 2000 / 2000C nucleic acid and protein analyzer test were diluted to approximately 50 ng / μL. 6 μL of the extracted DNA sample was then mixed with 2 μL of loading buffer and loaded onto a 1% (w / v) agarose gel. Electrophoresis was performed at 150 V for 25 min. The DNA integrity was observed and photographed using a UV spectrophotometer and gel imaging device.

[0039] DNA samples were sent to Neogene Biotech (Shanghai) Co., Ltd. for genotyping using a 50K SNP microarray (Illumina, USA) on the Illumina Beadstration platform according to the company's standard procedures. The genotyping data from all samples were quality controlled using the checkmarker function in the GenABEL package of the R language, removing samples with a detection rate below 90%, a family Mendelian error rate above 0.1, a minimum allele frequency below 0.01, and a Hardy-Weinberg equilibrium significance level above 10. -6 The SNPs were analyzed, and finally, 45,094 valid genotype data were obtained.

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

[0041] To eliminate population stratification effects, this invention employs a linear mixed model with single-point regression analysis combined with GWAS analysis using the FarmCPU software package in R. The analysis model utilizes the similarity of genomes among individuals to correct for stratification effects. The Bonferrini method is used to determine the significance threshold for the association between SNPs and the feed conversion ratio trait. The genomic significance threshold is 0.05 divided by the number of effective SNP loci, i.e., the genomic significance threshold is 1.11E-06, or 0.05 / 45094 (number of effective SNPs). The chromosomal significance threshold is 1 divided by the number of effective SNP loci, i.e., the chromosomal significance threshold is 2.21E-05, or 1 / 45094 (number of effective SNPs).

[0042] GWAS analysis results are as follows Figure 1 As shown. From Figure 1It is known that there is a site on chromosome 11 of Duroc × Landrace × Large White pig that significantly affects feed conversion ratio (indicated by the arrow in the figure). The significantly associated SNP site is the G>A mutation at position 34549890 bp on chromosome 11 of the international pig genome version 11.1 reference sequence (P value 1.60E-14). This SNP molecular marker is denoted as g.266G>A molecular marker.

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

[0044] As shown in Table 1, the molecular marker g.266G>A was 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 site in pigs, the feed efficiency of the population can be improved, thereby accelerating the breeding process.

[0045] Furthermore, Table 1 shows that the feed conversion ratio (FCR) of genotype AA is lower than that of genotype GA. The average phenotypic value of genotype AA individuals is 0.18 lower than that of genotype GA individuals, and the difference is highly significant (P<0.001). The average phenotypic value of genotype AA individuals is 0.20 lower than that of genotype GG individuals, and the difference is also highly 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, while those with the AA and GA genotypes should be culled. This will gradually increase the frequency of allele A at this locus, which can significantly reduce the FCR of pigs and bring more economic benefits to pig farms.

[0046] Table 1. Correlation analysis between g.266G>A molecular marker and material weight ratio.

[0047]

[0048] Example 2: Target DNA Sequence Amplification and Sequencing

[0049] (1) Primer design

[0050] Primers were designed using the Ensembl website (http: / / asia.ensembl.org / index.html) based on the upstream and downstream sequences of the g.266G>A molecular marker (the G>A mutation at position 34549890 bp on chromosome 11), using the primer design software PrimerPremier 6.0. The nucleotide sequences of the designed primers are shown below:

[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] To a 10 μL reaction mixture, 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. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, followed by 35 cycles of 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 45 s, with a final extension at 72℃ for 5 min.

[0055] (3) DNA sequencing

[0056] DNA sequence sequencing identification: Two reactions were performed to sequence the gene fragment. The obtained sequence was compared with the NCBI genome sequence to identify the mutation at the corresponding SNP site. The sequencing results are shown below (SEQ ID NO:1):

[0057]

[0058]

[0059] Note: M marked in the sequence listing is the mutation site, which is indicated by underline and bold (the G>A mutation base in parentheses indicates an allele mutation). Underline and italics at the beginning and end of the sequence indicate the location of the designed primer sequence.

[0060] As shown in SEQ ID No:1, the M at position 266 indicates a G>A base mutation. Primers can be designed using this nucleotide sequence to detect the genotype of the g.266G>A molecular marker, and then the feed conversion ratio trait in pigs can be further selected based on the detection results; alternatively, primers can be designed using this nucleotide sequence to create corresponding detection kits for direct detection of the g.266G>A molecular marker in pigs, and then the feed conversion ratio trait in pigs can be selected based on the detected genotype.

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

[0062] As shown in Table 1, the phenotypic effect of the dominant allele AA (g.266G>A) is 0.20 lower than that of the inferior allele GG (2.55 vs. 2.75). If we conservatively estimate a 0.1 reduction in feed weight per pig, saving 50 yuan in feed costs, then for the feed weight trait alone, over 100 yuan can be saved per pig. For a large-scale pig farm with tens of thousands of pigs, this translates to an economic benefit exceeding 1 million yuan. Therefore, by using marker-assisted selection or genomic selection to gradually select and retain pigs with the AA genotype within a population, the allele frequency of allele A can be significantly increased, the feed weight ratio of the pig herd can be reduced, and the progress of pig improvement can be accelerated, thereby effectively improving the economic benefits of pig breeding.

[0063] This invention utilizes the detection of the mutation site at position 266 in the SEQ ID NO:1 sequence to perform preliminary association analysis between its genotype and the feed conversion ratio in pigs, providing a novel molecular marker for marker-assisted selection and genomic selection in pigs.

[0064] Example 4: Application of the 4g.266G>A molecular marker in developing pig breeds with low feed conversion ratio.

[0065] S1: Genotyping of replacement gilts using the g.266G>A molecular marker;

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

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

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

[0069] S1: Genotyping of replacement gilts using the g.266G>A molecular marker;

[0070] S2: Select individuals with the AA genotype of the g.266G>A molecular marker detected in step S1 as breeding pigs, eliminate those with 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 mating in step S2. Keep the AA genotype individuals for breeding and then breed them. Keep the dominant AA genotype individuals in the offspring pigs and eliminate the GG and GA genotypes to increase the frequency of the dominant allele A generation by generation, thereby reducing the feed conversion ratio of pigs and improving the feed conversion ratio trait.

Claims

1. The application of the SNP molecular marker related to the feed conversion trait on pig chromosome 11 in the selection of the feed conversion trait of Duroc × Landrace × Yorkshire three-way cross pigs, wherein, The SNP molecular marker is a G>A mutation at position 34549890bp on chromosome 11 of the international pig genome version 11.

1.

2. The use of a SNP molecular marker in breeding Duroc Large White three-way cross pig line with low feed conversion performance, characterized in that, The SNP molecular marker is a G>A mutation at position 34549890bp on chromosome 11 of the international pig genome version 11.1, and the application comprises the following steps: S1: detecting the genotype of the SNP molecular marker in Duliangda three-way cross breeding pigs; S2: selecting and keeping the individuals with AA genotype detected in step S1 as breeding pigs, eliminating the individuals with GG and GA genotypes, and mating the breeding pigs; S3: detecting the SNP molecular marker in the piglets born in step S2, keeping the individuals with AA genotype for breeding, and breeding to obtain a pig line with low feed conversion ratio.

3. The application of SNP molecular marker in the improvement of feed conversion ratio of Duroc × Landrace × Yorkshire pigs, characterized in that, The SNP molecular marker is a G>A mutation at position 34549890bp on chromosome 11 of the international pig genome version 11.1, and the application comprises the following steps: S1: detecting the genotype of the SNP molecular marker in Duliangda three-way cross breeding pigs; S2: selecting and keeping the individuals with AA genotype detected in step S1 as breeding pigs, eliminating the individuals with GG and GA genotypes, and mating the breeding pigs; S3: detecting the SNP molecular marker in the piglets born in step S2, keeping the individuals with AA genotype for breeding, and breeding to obtain a pig line with low feed conversion ratio.

4. The use of a SNP molecular marker in detecting / screening / identifying the feed conversion ratio trait of Duroc Large White Landrace pigs, characterized in that, The SNP molecular marker is a G>A mutation at position 34549890bp on chromosome 11 of the international pig genome version 11.

1.

5. The primer pair for identifying the SNP molecular marker associated with the feed conversion trait of pigs in the preparation of a product for identifying the feed conversion trait of Duroc × Landrace × Yorkshire pigs or in the selection of the feed conversion trait of Duroc × Landrace × Yorkshire pigs or in the breeding of a Duroc × Landrace × Yorkshire pig line with a low feed conversion trait, characterized in that, The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:

3.

6. The use of a kit for identifying a SNP molecular marker associated with the feed conversion trait in identifying the feed conversion trait of Duroc × Landrace × Yorkshire pigs or in the selection of Duroc × Landrace × Yorkshire pigs for the feed conversion trait or in the breeding of a line of Duroc × Landrace × Yorkshire pigs with a low feed conversion trait, characterized by, The kit comprises a primer pair with nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3.

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