Use of molecular markers associated with average daily feed intake in pigs from 30 kg to 120 kg

Abstract

The present application relates to the application of molecular markers related to the average daily feed intake of pigs in the 30kg-120kg stage. Disclosed are a method for identifying the average daily feed intake of pigs in the 30kg-120kg stage and a breeding method for breeding pigs with high average daily feed intake in the 30kg-120kg stage. The method identifies the average daily feed intake of pigs in the 30kg-120kg stage by detecting the genotype of the pig at the 270804825 locus of chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1, and the pig with CC genotype is higher than the pig with CT genotype, and the pig with CT genotype is higher than the pig with TT genotype. In actual breeding work, the test pig with C270804825T locus genotype CC can be selected as the parent for breeding, which is of great significance for selecting pigs with high feed intake and high feed utilization efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of molecular markers related to the average daily feed intake of pigs in the 30kg-120kg stage. Background Technology

[0002] Since feed costs can account for about 70% of pig farming costs, breeding companies are increasingly focusing on breeding pig breeds with high feed utilization efficiency. For example, in the current breeding goals of Danyu Company, the feed conversion ratio is more than 30% important. Average daily feed intake refers to the average daily feed intake of pigs during the fattening stage, i.e., the stage where the body weight is 30kg-120kg. The heritability of average daily feed intake was between 0.2 and 0.3, and it was strongly genetically correlated with feed utilization efficiency (Li et al. 2022, Reference: Li W, Wang Z, Luo S, Wu J, Zhou L, Liu J. Genome-Wide Association Analysis and Genetic Parameters for Feed Efficiency and Related Traits in Yorkshire and Duroc Pigs. Animals (Basel). 2022 Jul 26; 12(15): 1902.) and pig resilience (Homma et al. 2021, Reference: Homma C, Hirose K, Ito T, Kamikawa M, Toma S, Nikaido S, Satoh M, Uemoto Y. Estimation of genetic parameter for feed efficiency and resilience traits in three pig breeds. Animal. 2021 Nov; 15(11): 100384.). Therefore, many companies select pigs with high average daily feed intake for breeding to improve feed utilization efficiency.

[0003] Measuring average daily feed intake requires specialized equipment and takes a long time, increasing breeding costs and making it very difficult. The rapid development of molecular biotechnology and pig genome association analysis has provided methods for the discovery and development of molecular markers for average daily feed intake, and has also made it possible to develop more accurate and faster breeding methods for quantitative traits such as average daily feed intake. Some studies have already explored genes related to average daily feed intake. For example, Zhang et al. (2021, reference: Zhang Z, Zhong H, Lin S, Liang L, Ye S, Xu Z, Ji C, Zhang Z, Zhang D, Zhang X. Polymorphisms of AMY1A gene and their association with growth, carcass traits and feed intake efficiency in chickens. Genomics. 2021 Mar; 113(2):583-594.) conducted a genome-wide association analysis (GWAS) on feed utilization efficiency traits in poultry and found that the AMY1A gene may affect average daily feed intake. Li et al. (2021, reference: Li W, Wang Z, Luo S, Wu J, Zhou L, Liu J. Genome-Wide Association Analysis and Genetic Parameters for Feed Efficiency and Related Traits in Yorkshire and Duroc Pigs. Animals (Basel). 2022 Jul Studies in 26;12(15):1902.) have shown that MC4R and INSR genes can be candidate genes for feed utilization efficiency, such as average daily feed intake. However, since average daily feed intake is a trait controlled by multiple genes, its breeding-related molecular markers need further development. Summary of the Invention

[0004] The purpose of this invention is to provide the application of molecular markers related to the average daily feed intake of pigs in the 30kg-120kg stage;

[0005] Another object of the present invention is to provide a method for identifying or assisting in the identification of the average daily feed intake of pigs in the 30kg-120kg stage;

[0006] Another objective of this invention is to provide a breeding method for selecting pigs with a high average daily feed intake in the 30kg-120kg range.

[0007] This invention is implemented as follows:

[0008] The application of molecular markers associated with average daily feed intake in pigs during the 30kg-120kg weight stage is any one of the following A1 to A4:

[0009] A1. To detect or assist in the detection of the average daily feed intake of pigs in the 30kg-120kg stage;

[0010] A2. Identify and assist in identifying the average daily feed intake of pigs in the 30kg-120kg stage;

[0011] A3. Prepare products for detecting or assisting in the detection of average daily feed intake in pigs weighing 30kg-120kg;

[0012] A4. Products for the preparation, identification, and auxiliary identification of average daily feed intake in pigs weighing 30kg-120kg;

[0013] The molecular marker corresponds to the SNP site at position 270804825 on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1, and the sequence is the nucleotide at position 214 bp of the DNA molecule shown in SEQ ID NO:1, and the nucleotide type is C or T.

[0014] A method for identifying or assisting in the identification of the average daily feed intake of pigs in the 30kg-120kg stage includes the following steps:

[0015] S1 extracts genomic DNA from the pigs to be tested as a template;

[0016] S2 designed primers targeting the 214bp site of the DNA molecule shown in SEQ ID NO.1 for PCR amplification;

[0017] S3 detects the genotype at the 214bp site of the sequence of SEQ ID NO.1 of the pig to be tested;

[0018] S4 uses the genotype obtained in step S3 to identify or assist in identifying the average daily feed intake of the pigs in the 30kg-120kg stage. Pigs with the CC genotype have higher intake than those with the CT genotype, and pigs with the CT genotype have higher intake than those with the TT genotype.

[0019] Preferably, the sequences of the primers described in step S2 are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0020] A breeding method for selecting pigs with high average daily feed intake in the 30kg-120kg range involves identifying the genotype of the pigs to be tested using the aforementioned method, and selecting the pigs with the CC genotype as parents for breeding. The CC genotype is a homozygous type where the 214th bp of the sequence SEQ ID NO.1 is C.

[0021] Preferably, the aforementioned application of molecular markers related to the average daily feed intake of pigs in the 30kg-120kg stage, wherein the product is a reagent kit.

[0022] The average daily feed intake (ADFI) for pigs in the 30kg-120kg weight range described in this invention is the ratio of total feed intake to growth time during this weight range. The purpose of the above-mentioned pig breeding method is to select breeding pigs with higher average daily feed intake in the 30kg-120kg weight range. The SNP mentioned refers to a component located in the reference genome.

[0023] In Sus_scrofa.Sscrofa11.1, position 270804825 on chromosome 1 is named C270804825T. Those skilled in the art will understand that SEQ ID NO:1 consists of the nucleotide sequence at position 214 of the SNP site and its surrounding nucleotide sequence. The quantity of nucleotides surrounding the SNP site should not be considered a limiting factor in the scope of this invention; it can be 25bp, 35bp, 45bp, 65bp, 85bp, 100bp, 150bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, or 1000bp before and after the SNP site, or any other arbitrary value. Its function is to assist in locating the SNP on chromosome 1 of the pig reference genome.

[0024] The front and back directions mentioned in this article should be defined as front or back in a direction generally accepted by those skilled in the art, such as the 5'-3' direction.

[0025] The identification, auxiliary identification, or improvement of pig feed utilization efficiency traits mentioned in this article include, but are not limited to, identifying or assisting in the identification of test pigs with the aforementioned SNP genotype CC for feeding or breeding.

[0026] In the above method, a portion of the pig genome containing the aforementioned SNP can be amplified by PCR. For example, sequencing the PCR product containing SEQ ID NO:1 and detecting the type of deoxyribonucleotide at position 214 of SEQ ID NO:1 can determine the genotype of the SNP site in the pig genome to be tested.

[0027] This invention uses gene sequencing to detect the C270804825T genotype. Only a PCR reaction is required; sequencing alone can determine the genotype of an individual. Genotyping is highly accurate, and the testing cost is low, making it highly valuable for breeding practice. Using this method to select pigs for the average daily feed intake trait can increase the average daily feed intake of CC genotype pigs by approximately 0.12 kg / day compared to TT genotype pigs. Applying the method provided by this invention to pig breeding allows for early screening of potential pigs, effectively alleviating the problem of long selection times for superior breeding pigs in actual production, reducing breeding costs, and effectively improving feed utilization efficiency in actual pig production. The detection method of this invention is simple to operate, inexpensive, highly accurate, and can achieve automated direct detection.

[0028] This invention will play a significant role in pig breeding.

[0029] The beneficial effects of this invention are as follows: This invention discovers the C / T locus at position 270804825 on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1, which has a significant impact on the average daily feed intake of pigs in the 30kg-120kg stage. Based on this SNP locus, a method for identifying or assisting in the identification of the average daily feed intake of pigs in the 30kg-120kg stage is provided. This provides a new molecular breeding marker for marker-assisted breeding of the average daily feed intake trait in pigs in the 30kg-120kg stage, which is beneficial to improving breeding efficiency, effectively shortening the breeding cycle, and is of great significance for selecting superior pig breeds. Attached Figure Description

[0030] Figure 1 This is a QQ diagram representing the data structure of genome-wide association analysis in this invention embodiment;

[0031] Figure 2 This is a Manhattan diagram of genome-wide association analysis in an embodiment of the present invention. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] The data in the following examples were processed using SAS 9.0 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 (*) indicates a significant difference.

[0035] All animals in the following examples were sourced from Henan Yifa Animal Husbandry Co., Ltd.

[0036] The average daily feed intake of pigs in the 30kg-120kg stage in the following examples refers to the average daily feed intake from the time the pigs reach 30kg to the time they reach 120kg.

[0037] Example 1: Determination of SNP sites on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1

[0038] Whole-genome sequencing and genome-wide association analysis (GWAS) of the average daily feed intake trait in pigs weighing 300 kg to 120 kg were performed. The model used was: Y = μ + G + B + P + e, where Y is the trait value; μ is the population mean; G is the genotype effect; B is the batch effect; and P is the farm-year-season effect. Twenty SNP loci significantly associated with average daily feed intake in the 30 kg to 120 kg weight range were identified, such as... Figure 1 and Figure 2 As shown. The inventors designed primers for amplification within a 200bp range upstream and downstream of the five most prominent sites, and found that only the sequence containing one site on stain 1 could be amplified well.

[0039] After amplification, alignment was performed using Seqman software, resulting in a differentially expressed SNP locus, named C270804825T. This locus is located at nucleotide 270804825 on chromosome 1 of the porcine reference genome Sus_scrofa.Sscrofa11.1, which is nucleotide 214 of sequence SEQ ID NO:1. The nucleotide type of C270804825T is either C or T, and the genotype is CC, TT, or CT. The CC genotype at C270804825T is homozygous for C. The TT genotype at C270804825T is homozygous for T. The CT genotype at C270804825T is heterozygous for both T and C.

[0040] Example 2: Correlation analysis of the C270804825T locus on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1 with the average daily feed intake of pigs weighing 30kg-120kg.

[0041] To determine whether the C270804825T locus on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1 is related to the average daily feed intake of pigs in the 30kg-120kg stage, 1188 Large White pigs were used as experimental materials. The genotype of the SNP locus in Example 1 and the average daily feed intake of each individual in the 30kg-120kg stage were measured, and correlation analysis was performed.

[0042] I. Genotype Identification

[0043] 1. PCR amplification

[0044] Based on the SNP information on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1, a pair of primers was designed as follows:

[0045] Upstream primer F: 5'-TCTCTTGAGGAGAGTCTCTTGCC-3' (SEQ ID NO:2);

[0046] Downstream primer R: 5'-GGCTTCCGAGTTCTGCTATC-3' (SEQ ID NO:3).

[0047] Using the genomic DNA of each Large White pig as a template, PCR amplification was performed using the above-mentioned materials to obtain PCR products.

[0048] 2. Cloning, sequencing, and sequence analysis

[0049] The PCR products from each individual were purified using an agarose gel extraction kit (Tiangen Biotech Co., Ltd.). The recovered DNA fragments were ligated into the pGEM-T vector (Promega), and the ligation product was transformed into *E. coli* DH5α competent cells (Mingri Biotech (Beijing) Co., Ltd.). Positive clones were screened based on the carbenicillin resistance marker on the vector to obtain recombinant plasmids containing the recovered fragments. The nucleotide sequence of this recombinant plasmid vector was determined using the T7 and SP6 promoter sequences as primers (Invitrogen (Shanghai) Trading Co., Ltd.).

[0050] The genotypes of the pigs to be tested are as follows:

[0051] CC genotype: If the PCR product obtained by amplifying the genomic DNA of the pig to be tested using upstream primer F and downstream primer R contains only a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 214 of SEQ ID NO:1, and does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 214 of SEQ ID NO:1, then the genotype of the C270804825T position on chromosome 1 of the pig reference genome Sus_scrofa.Sscrofa11.1 of the pig to be tested is CC.

[0052] TT genotype: If the PCR product obtained by amplifying the genomic DNA of the pig to be tested using upstream primer F and downstream primer R does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 214 of SEQ ID NO:1, but only contains a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 214 of SEQ ID NO:1, then the genotype of the C270804825T position on chromosome 1 of the pig reference genome Sus_scrofa.Sscrofa11.1 of the pig to be tested is TT.

[0053] CT genotype: If the PCR product obtained by amplifying the genomic DNA of the pig to be tested using upstream primer F and downstream primer R contains both a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 214 of SEQ ID NO:1, and a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 214 of SEQ ID NO:1, then the genotype of the C270804825T site on chromosome 1 of the pig reference genome Sus_scrofa.Sscrofa11.1 of the pig to be tested is CT.

[0054] The results are shown in Table 2. Genotyping of the C270804825T locus in 1188 Large White pigs showed that 239 pigs had the CC genotype, 427 had the CT genotype, and 522 had the TT genotype. The genotype and allele frequencies of the C270804825T locus in the tested pig population are shown in Table 1. As can be seen from Table 1, the genotype frequency of the TT homozygous type was significantly higher than that of the CC homozygous type, and the T allele was the dominant gene.

[0055] Table 1. Genotype and allele frequencies of the C270804825T locus in the tested pig population.

[0056]

[0057] II. Association Analysis between Pig Genotype and Average Daily Feed Intake in Pigs Weighing 30kg-120kg

[0058] The average daily feed intake of pigs in the 30kg-120kg range was measured using an Osborn automated measuring device. The association between genotype and phenotype was analyzed using SAS software. Duncan's multiple test was used for significance testing (P<0.05). Data are expressed as mean ± standard error, and P<0.05 was considered significant.

[0059] The results are shown in Table 2. The C270804825T locus had a significant effect on the average daily feed intake of pigs in the 30kg-120kg stage. Pigs with the CC genotype had a higher average daily feed intake in the 30kg-120kg stage than pigs with the CT genotype (P<0.05), and pigs with the CT genotype had a higher average daily feed intake in the 30kg-120kg stage than pigs with the TT genotype. Furthermore, pigs with the CC genotype had a significantly higher average daily feed intake in the 30kg-120kg stage than pigs with the TT genotype.

[0060] In actual pig breeding, the average daily feed intake in the 30kg-120kg stage can be selected based on the C270804825T locus typing results.

[0061] Table 2. Association analysis of single nucleotide polymorphisms on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1 with average daily feed intake for individuals weighing 30kg-120kg.

[0062]

[0063] Note: Different lowercase letters in the table indicate significant differences (P<0.05), and the same letter indicates no significant differences (P>0.05). Values ​​are expressed as mean ± standard error.

[0064] In summary, determining the genotype of the C270804825T locus on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1 can help identify the average daily feed intake of pigs weighing 30kg-120kg. Pigs with the CC genotype have a higher average daily feed intake in this range than those with the CT genotype, and pigs with the CT genotype have a higher average daily feed intake in this range than those with the TT genotype. In practical breeding work, pigs with the CC genotype at the C270804825T locus can be selected as parents for breeding. This is of great significance for selecting pigs with high feed intake and high feed utilization efficiency.

[0065] SEQ ID NO:1

[0066]

[0067] The W is either C or T.

[0068] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a reagent for detecting molecular markers related to average daily feed intake in pigs weighing 30kg-120kg, characterized in that, The application is any one of the following A1 to A4: A1. To detect or assist in the detection of the average daily feed intake of pigs in the 30kg-120kg stage; A2. Identify and assist in identifying the average daily feed intake of pigs in the 30kg-120kg stage; A3. Prepare products for detecting or assisting in the detection of average daily feed intake in pigs weighing 30kg-120kg; A4. Products for the preparation, identification, and auxiliary identification of average daily feed intake in pigs weighing 30kg-120kg; The molecular marker corresponds to the SNP site at position 270804825 on chromosome 1 in the pig reference genome Sus_scrofa.Sscrofa11.1, and the sequence is the nucleotide at position 214 bp of the DNA molecule shown in SEQ ID NO:1, and the nucleotide type is C or T.

2. A method for identifying or assisting in the identification of the average daily feed intake of pigs in the 30kg-120kg stage, characterized in that, Includes the following steps: S1 extracts genomic DNA from the pigs to be tested as a template; S2 designed primers targeting the 214bp site of the DNA molecule shown in SEQ ID NO.1 for PCR amplification; S3 detects the genotype at the 214bp site of the sequence of SEQ ID NO.1 of the pig to be tested; S4 uses the genotype obtained in step S3 to identify or assist in identifying the average daily feed intake of the pigs in the 30kg-120kg stage. Pigs with the CC genotype have higher intake than those with the CT genotype, and pigs with the CT genotype have higher intake than those with the TT genotype.

3. The method according to claim 2, characterized in that, The sequences of the primers described in step S2 are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. A breeding method for selecting pigs with high average daily feed intake in the 30kg-120kg weight range, characterized in that... The genotype of the pig to be tested is identified according to the method of claim 2 or 3, and the pig to be tested with the CC genotype is selected as the parent for breeding, wherein the CC genotype is a homozygous type where the 214th bp of the SEQ ID NO.1 sequence is C.

5. The application of the molecular markers related to the average daily feed intake of pigs in the 30kg-120kg stage according to claim 1, characterized in that, The product in question is a reagent kit.

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