SNP (Single Nucleotide Polymorphism) molecular marker related to pig growth traits as well as detection primer, kit and application thereof

Abstract

The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to an SNP molecular marker related to pig growth traits as well as a detection primer, a kit and application thereof. The SNP molecular marker is located at the 1001st site of a nucleotide sequence as shown in SEQ ID NO.1 and has CC, CT and TT genotypes; the day age of the CC genotype pig individuals reaching 100 kg and / or the day age of the CC genotype pig individuals reaching 120 kg are obviously lower than those of the CT genotype pig individuals and the TT genotype pig individuals. By detecting the genotype of the molecular marker, rapid, accurate and high-throughput identification or evaluation of pigs with the weight of 100 kg and / or the day age of 120 kg can be achieved on the molecular level, the accuracy and reliability of breeding selection are improved, the breeding period of excellent breeding pigs in production is shortened, the breeding efficiency and precision are improved, and the breeding cost is reduced. And the SNP molecular marker has potential important value for genetic improvement of pig growth traits.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted breeding, and particularly relates to an SNP molecular marker related to porcine growth traits, a detection primer and a kit thereof, and applications thereof. Background Art

[0002] Growth traits are one of the important economic traits in pig breeding, and are often used as one of the core breeding objectives for genetic improvement of pigs. Improving the growth traits of pigs is of great significance for enhancing the overall economic benefits and promoting the healthy and sustainable development of the pig industry. In production, the age at 100 kg body weight and the age at 120 kg body weight are the key indicators for measuring the growth traits and growth rate of pigs; among them, the age at 100 kg body weight (Days to 100 kg, D100) refers to the age when the body weight of a pig reaches 100 kg after birth, and the age at 120 kg body weight (Days to 120 kg, D120) refers to the age when the body weight of a pig reaches 120 kg after birth; the smaller the age, the faster the pig grows to the target weight and the better the growth performance. In pig breeding, reducing the D100 and D120 days of breeding pigs can not only shorten the fattening time of commercial pigs, improve the breeding efficiency of pigs, but also greatly save feed and labor costs. Therefore, how to effectively improve the growth rate of pigs, shorten the D100 and D120 days, and make an early judgment is the main goal of current pig breeding work.

[0003] Early breeding work mainly focused on phenotypic selection of pigs, which was often measured when pigs were sold. There were problems such as a long measurement cycle, large workload, and high cost, which greatly increased the breeding cost and lengthened the generation interval, making the progress of traditional breeding work slow; these problems greatly restricted the selection of replacement breeding pigs with excellent growth traits in production. With the continuous advancement of genome work, the construction of porcine genetic linkage maps, and the extensive development of technologies such as genetic markers, molecular marker-assisted selection technology is gradually becoming a reliable and effective selection method due to its advantages of being unrestricted by time and geographical factors and having an accurate, rapid, and efficient selection process. Molecular marker-assisted selection technology is to find molecular markers closely associated with target traits, further identify and select the molecular marker loci of candidate individuals, so as to achieve the selection of superior genotypes of target traits, and then early selection of the growth rate of pigs can be carried out, thereby breeding breeding pigs with excellent growth traits. Therefore, the discovery of molecular markers associated with growth traits and the establishment of marker-assisted selection methods will greatly solve the problem of difficult selection of pig growth traits.

[0004] Single nucleotide polymorphism (SNP) molecular markers refer to DNA sequence polymorphisms caused by single nucleotide variations at the genomic level, including single-base transversions, transitions, insertions, and deletions. They are the most numerous and widely distributed molecular markers in the genome, with advantages such as easy genotyping, good genetic stability, and easy automation and batch detection. They are considered the most valuable new generation of genetic markers. However, the research on SNP molecular markers in pig breeding is relatively scarce. Currently, there are few research reports on SNP markers related to pig growth traits with clear functions and directly applicable to breeding detection. Therefore, exploring new SNP markers related to the age at 100 kg body weight and / or the age at 120 kg body weight in pigs and applying them is of great significance for pig production and breeding. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an SNP molecular marker related to pig growth traits. By detecting the genotype of this molecular marker, it is possible to rapidly, accurately, and high-throughput identify or evaluate the age at 100 kg body weight and / or the age at 120 kg body weight in pigs at the molecular level, which is beneficial to improving the accuracy and reliability of breeding selection, shortening the selection cycle of excellent breeding pigs in actual production, accelerating the breeding efficiency and precision, and this SNP molecular marker has potential important value for the genetic improvement of pig growth traits. Therefore, the present invention provides the application of this SNP molecular marker, its detection primers, or kits in the identification of pig growth traits or breeding. The present invention is specifically realized through the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the application of an SNP molecular marker related to pig growth traits, its detection primers, or kits in the identification of pig growth traits or breeding; the SNP molecular marker is located at the 1001st base of the nucleotide sequence shown in SEQ ID NO.1, Y is selected from C or T, and the SNP molecular marker has CC, CT, and TT genotypes;

[0007] The growth traits are the age at 100 kg body weight and / or the age at 120 kg body weight. Among them, the age at 100 kg body weight and / or the age at 120 kg body weight of pig individuals with the CC genotype is significantly lower than that of pig individuals with the CT genotype and the TT genotype.

[0008] In the second aspect of the present invention, there is provided a detection primer for detecting the above-mentioned SNP molecular marker. The detection primer includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.3.

[0009] The third aspect of the present invention provides a kit for detecting the SNP molecular marker as described above, and the kit includes the detection primers as described above.

[0010] Further, the kit further includes PCR amplification reagents, and the PCR amplification reagents include Taq DNA polymerase, dNTP, and buffer reagents.

[0011] The fourth aspect of the present invention provides a method for identifying porcine growth traits, including the following steps:

[0012] Extract the genomic DNA of the porcine individual to be tested;

[0013] Use the detection primers shown in SEQ ID NO.2-3 to amplify the genomic DNA by polymerase chain reaction (PCR) technology to obtain an amplification product;

[0014] Detect the genotype of the 301st base of the amplification product. The 301st base has CC, CT, and TT genotypes, and identify the growth traits of the porcine individual to be tested according to the genotype; wherein, the growth traits are the age at 100 kg body weight and / or the age at 120 kg body weight, and the porcine individual with the CC genotype reaches the age at 100 kg body weight and / or the age at 120 kg body weight significantly lower than that of the porcine individual with the CT genotype and the TT genotype.

[0015] Further, the reaction system for PCR amplification is calculated as 50 μL and includes: 2×GS taq PCRMix 25.0 μL, upstream primer 2 μL, downstream primer 2 μL, genomic DNA 2 μL, and deionized water 19.0 μL.

[0016] Further, the reaction program for PCR amplification includes: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, extension at 72°C for 10 s, for a total of 35 cycles; running at 72°C for 5 min.

[0017] The fifth aspect of the present invention provides a method for genetic improvement of porcine growth traits, including the following steps:

[0018] Extract the genomic DNA of the porcine individual to be tested;

[0019] Use the detection primers shown in SEQ ID NO.2-3 to amplify the genomic DNA by polymerase chain reaction (PCR) technology to obtain an amplification product;

[0020] Detect the genotype of the 301st base of the amplification product. The 301st base has CC, CT, and TT genotypes, and retain the porcine individual with the CC genotype as breeding pigs.

[0021] The advantages and positive effects of the present invention are as follows:

[0022] 1. The SNP molecular marker provided by the present invention is closely related to the traits of the age at 100 kg body weight and the age at 120 kg body weight in pigs. By detecting the genotype of this molecular marker, rapid, accurate, and high-throughput identification or evaluation of target traits can be achieved at the molecular level. Through the genetic background selection of SNP variation sites in the present invention, it is beneficial to reduce the interference of environmental factors, improve the accuracy and reliability of breeding selection, and is also beneficial to predicting the growth rate by detecting relevant SNP molecular markers in the early stage of breeding, screening out individuals with excellent growth traits in advance, greatly shortening the breeding cycle of excellent breeding pigs in actual production, and accelerating the breeding efficiency and precision.

[0023] 2. The SNP molecular marker of the present invention has potential important value for the genetic improvement of pig growth traits. By retaining individuals with the CC genotype for breeding production during the breeding process, the proportion of excellent genes in the offspring can be gradually increased, which is beneficial to reducing the age at 100 kg body weight and the age at 120 kg body weight in the population, enhancing the growth rate of the population, and improving growth traits; or, by using the SNP variation site of the present invention as the target for genetic engineering breeding and modifying the CT and TT genotypes into the CC genotype by means of gene mutation, etc., individuals enriched with excellent genes can be achieved, which is beneficial to providing a rapid and effective improvement approach for pig growth traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is the Manhattan plot of the genome-wide association analysis of the age at 100 kg body weight in the embodiment of the present invention;

[0026] Figure 2 It is the Manhattan plot of the genome-wide association analysis of the age at 120 kg body weight in the embodiment of the present invention;

[0027] Figure 3 It is the phenotypic values of the age at 100 kg body weight of different genotypes of the SNP molecular marker in the natural population of 593 Duroc pigs in the embodiment of the present invention;

[0028] Figure 4 It is the phenotypic values of the age at 120 kg body weight of different genotypes of the SNP molecular marker in the natural population of 593 Duroc pigs in the embodiment of the present invention;

[0029] Figure 5 This is the agarose gel electrophoresis diagram of the SNP molecular marker amplification product in the embodiment of the present invention;

[0030] Figure 6 This is the comparison diagram of the sequencing results of the SNP molecular marker amplification product in the embodiment of the present invention;

[0031] Figure 7 This is the nucleotide sequence sequencing peak diagram of different genotypes of the SNP molecular marker in the embodiment of the present invention. Detailed implementation manners

[0032] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] Based on the information contained in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for schematically illustrating specific aspects of the present invention. In fact, various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are all covered within the scope of the appended claims.

[0034] In order to better understand the present invention rather than limit its scope, all numbers representing amounts, percentages and other values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired properties. Each numerical parameter should be regarded as obtained at least according to the reported significant figures and by the conventional rounding method.

[0035] The meanings of the terms "include", "comprise", "contain", "have" and other similar words are non-restrictive, that is, other steps and other components can be added without affecting the result. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0036] To make the above objectives, features and advantages of the present invention more obvious and understandable, the following will give a detailed description of the specific implementation manners of the present invention.

[0037] Among the many factors affecting the production efficiency of pig breeding, the genetic factors of breeding pigs play a leading role. Genome-wide association studies (GWAS) use single nucleotide variations (SNPs) within the genome as molecular markers to perform association analysis on the genotype and phenotype data of the molecular markers, thereby identifying genetic variant loci associated with target traits. GWAS can quickly and comprehensively screen out genes or mutation sites related to traits and functions, providing an effective technical means for analyzing the candidate gene mapping of complex quantitative traits.

[0038] In this invention, the growth traits of 592 Duroc pigs were recorded and measured. The age at 100 kg body weight and the age at 120 kg body weight were used as phenotypic data. Combining with the sequencing data of 592 Duroc pigs, the SNP genotyping data obtained by whole-genome sequencing was used as genetic variant data to conduct genome-wide association analysis (GWAS), and an SNP variant locus significantly associated with both the age at 100 kg body weight and the age at 120 kg body weight was identified. This locus is located at the 58,393,394th base of chromosome 14 of the pig reference genome Sscrofa11.1. Specific primers were designed for this locus, and the PCR amplification products were sequenced and verified, showing that it has C / T polymorphism, and the alleles have three genotypes: CC, TT, and CT. In the pig population, this SNP variant is closely related to the phenotypic traits of the age at 100 kg body weight and the age at 120 kg body weight. The phenotypic values corresponding to different genotypes are significantly different. The age at 100 kg body weight and the age at 120 kg body weight of pigs carrying the CC genotype are significantly lower than those of individuals with the CT genotype and the TT genotype. Therefore, the 58,393,394 (C / T) variant locus of this invention can be independently developed into an SNP molecular marker for assisted breeding, providing a new and reliable molecular marker for breeding breeding pigs with stable genetics and excellent growth traits, and enriching the genetic resources for breeding pigs with high growth traits.

[0039] The physical location of the above-mentioned SNP molecular marker is as follows: it is located at the 58,393,394th base of chromosome 14 of the pig (Sus scrofa) reference genome Sscrofa11.1 version. For the nucleotide sequence of the pig reference genome Sscrofa11.1 version, see NCBI accession number: GCF_000003025.6 (access link: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000003025.6 / ). For the nucleotide sequence of chromosome 14, see NCBI accession number: NC_010456.5. The corresponding locus number of the SNP molecular marker on Ensembl is rs320657871.

[0040] An embodiment of the present invention provides a SNP molecular marker associated with pig growth traits, and the use of a detection primer or a kit thereof in pig growth trait identification or breeding; the SNP molecular marker is located at the 1001th base of the nucleotide sequence shown in SEQ ID NO.1, Y is selected from C or T, and the SNP molecular marker has CC, CT and TT genotypes;

[0041] The growth traits are reaching 100 kg body weight at day 1 and / or reaching 120 kg body weight at day 1, wherein the growth traits of pigs with CC genotype reaching 100 kg body weight at day 1 and / or reaching 120 kg body weight at day 1 are significantly lower than those of pigs with CT genotype and TT genotype.

[0042] Growth traits such as reaching 100kg body weight at age and reaching 120kg body weight at age are important economic traits in the pig breeding process. The SNP molecular marker provided by the present invention is closely related to the aforementioned growth traits of pigs. By detecting the genotype of the molecular marker, rapid, accurate, and high-throughput identification or evaluation of the target traits at the molecular level can be achieved; specifically, when the SNP variant site in the pig to be tested is detected to be the CC genotype, it can be judged that the pig has a reduced 100kg body weight at age and 120kg body weight at age traits, and the pig has a faster growth rate. When the SNP variant site in the pig to be tested is detected to be the TT genotype, it can be judged that the pig has an increased 100kg body weight at age and 120kg body weight at age traits, and the pig has a slower growth rate. When the SNP variant site in the pig to be tested is detected to be the CT genotype, its traits are intermediate. The present invention is conducive to reducing the interference of environmental factors and improving the accuracy and reliability of breeding selection by selecting the genetic background of SNP variant sites. It is also conducive to predicting the growth rate of pigs at any stage in the breeding process, especially in the early stage or even the embryonic stage, by detecting related SNP molecular markers, and screening out individuals with excellent growth traits in advance, greatly shortening the selection cycle of excellent breeding pigs in actual production, and accelerating breeding efficiency and accuracy. In addition, the discovery of the SNP molecular marker has potential important value for the genetic improvement of pig growth traits. By retaining individuals with CC genotypes for seed production during the breeding process, and then gradually increasing the proportion of excellent genes in the offspring, it is conducive to reducing the weight of the group to 100 kg and the weight of 120 kg per day, enhancing the growth rate of the group, and improving growth traits; or, by using the SNP variant sites of the present invention as targets for genetic engineering breeding, using gene mutations and other methods to improve CT and TT genotypes to CC genotypes, and then achieving individuals enriched with excellent genes, it is conducive to providing a fast and effective improvement path for pig growth traits.

[0043] In the present invention, the age at 100 kg body weight and the age at 120 kg body weight are preferably the corrected data. Referring to the agricultural industry standard "NY / T 822-2019 Specification for Determination of Breeding Pig Performance", the determination and correction are carried out according to the following method: When the pig to be determined reaches the target body weight, it is measured on an empty stomach, and the actual body weight and the actual age at the time of measurement are recorded. The calculation is carried out according to the following formula:

[0044] Age at 100 kg body weight = actual age + (100 - actual body weight) × (actual age - A) / actual body weight, where A is the correction coefficient, 55.29 for boars and 49.36 for sows;

[0045] Age at 120 kg body weight = actual age + (120 - actual body weight) × (actual age - B) / actual body weight, where B is the correction coefficient, 50.13 for boars and 46.14 for sows.

[0046] The detection of the SNP molecular marker genotype of the present invention can adopt the methods commonly used in the prior art, such as gene chip technology, Kompetitive Allele Specific PCR (KASP) technology, Taqman probe technology, High Resolution Melting (HRM) method, Allele Specific PCR (AS-PCR) technology, direct sequencing method, Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry technology, etc.

[0047] The present invention preferably adopts the direct sequencing method, which includes the steps of extracting genomic DNA of the pig to be tested, PCR amplifying the fragment to be detected, and sequencing the fragment to be detected. In the sequencing peak map, when the SNP molecular marker genotype is homozygous, it shows a single peak, and when it is heterozygous, it shows an overlapping peak, which is easy to distinguish different genotypes. That is, when the sequencing site shows a single peak of nucleotide C, the genotype of the pig to be tested is CC genotype; when the sequencing site shows a single peak of nucleotide T, the genotype of the pig to be tested is TT genotype; when the sequencing site shows an overlapping peak of nucleotide C and T, the genotype of the pig to be tested is CT genotype.

[0048] Another embodiment of the present invention provides a detection primer for detecting the SNP molecular marker as described above. The detection primer includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer (F) is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer (R) is as shown in SEQ ID NO.3.

[0049] F: 5’-TGAAGGAAAGCGGATACCCA-3’ (see SEQ ID NO.2);

[0050] R: 5’-ATTAACCAATGTGCCACGACA-3’ (see SEQ ID NO.3).

[0051] Using the genomic DNA of the pig individual to be tested as a template, PCR amplification is carried out using the above detection primers. Accurate base information of SNP variant sites can be obtained through means such as sequencing. Its genotype typing effect is good, which is conducive to realizing the rapid and accurate identification of the genotypes of SNP molecular markers, and further predicting the growth performance of the pig individual to be tested.

[0052] Another embodiment of the present invention provides a kit for detecting the SNP molecular marker as described above, and the kit includes the detection primers as described above.

[0053] The advantages of the kit relative to the prior art are the same as those of the detection primers relative to the prior art, and will not be elaborated here.

[0054] Optionally, the kit further includes PCR amplification reagents. The present invention has no special limitation on the source of the PCR amplification reagents, and conventional commercially available products in the art can be used.

[0055] In a typical embodiment, the PCR amplification reagents include Taq DNA polymerase, dNTP, and buffer reagents.

[0056] The present invention has no special limitation on the total amount of PCR amplification reagents and the dosage of detection primers in the kit, and can be set according to the conventional requirements of the kit. Generally speaking, the concentration of the upstream primer and the downstream primer in the kit is preferably 10 - 20 mM, more preferably 10 mM, and this concentration is usually the mother liquor concentration.

[0057] Based on the same inventive concept as above, another embodiment of the present invention provides a method for identifying pig growth traits, including the following steps:

[0058] Extract the genomic DNA of the pig individual to be tested;

[0059] Using the detection primers shown in SEQ ID NO.2 - 3, amplify the genomic DNA by polymerase chain reaction (PCR) technology to obtain an amplification product;

[0060] Detect the genotype of the 301st base of the amplification product. The 301st base has CC, CT, and TT genotypes. Identify the growth traits of the pig individual to be tested according to the genotype; wherein, the growth traits are the age at 100 kg body weight and / or the age at 120 kg body weight. The pig individual to be tested with the CC genotype reaches the age at 100 kg body weight and / or the age at 120 kg body weight significantly lower than the pig individuals to be tested with the CT genotype and the TT genotype.

[0061] Based on the same inventive concept as described above, an embodiment of the present invention further provides a method for genetic improvement of pig growth traits, including the following steps:

[0062] Extract genomic DNA of the pig individual to be tested;

[0063] Use the detection primers shown in SEQ ID NO.2 - 3 to amplify the genomic DNA by polymerase chain reaction (PCR) technology to obtain an amplification product;

[0064] Detect the genotype of the 301st base of the amplification product. The 301st base has CC, CT, and TT genotypes. Retain the pig individuals with the CC genotype as breeding pigs.

[0065] In the present invention, according to the genotype of the SNP molecular marker, individuals with the excellent genotype CC are selected and retained as breeding pigs to participate in breeding, and individuals with CT and TT genotypes at this locus are eliminated. The frequency of the allelic genotype CC at this locus can be increased generation by generation, thereby significantly reducing the age at 100 kg body weight and the age at 120 kg body weight of the population pigs, accelerating the screening of superior pigs with fast growth rate, and improving the growth traits of the population.

[0066] The present invention does not specifically limit the method for extracting genomic DNA of the pig individual to be tested. Commonly used genomic DNA extraction methods or genomic DNA extraction kits in the art can be used, such as the commonly used phenol - chloroform crude extraction method or CTAB extraction method.

[0067] To reduce the harm to pig individuals, the genomic DNA is preferably DNA extracted from pig ear tissue.

[0068] Optionally, the reaction system for the PCR amplification is calculated based on 50 μL and includes: 2×GS taq PCR Mix (purchased from TaKara, product number R300A) 25.0 μL, upstream primer 2 μL, downstream primer 2 μL, genomic DNA 2 μL, and deionized water (ddH2O) 19.0 μL. The PCR reaction procedure includes: pre - denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, extension at 72°C for 10 s, for a total of 35 cycles; running at 72°C for 5 min. The amplification reaction solution is stored at 4°C for standby.

[0069] The following further elaborates the present invention in combination with specific embodiments. For the experimental methods without specific conditions noted in the following embodiments, they are usually in accordance with conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" published by Cold Spring Harbor Laboratory, or usually in accordance with the conditions recommended by the manufacturer.

[0070] This invention is based on a resource population of 592 individuals. By measuring growth traits (age at 100 kg body weight and age at 120 kg body weight), GWAS analysis of growth traits was performed using SNP genotyping data obtained by genome sequencing. Further analysis of the GWAS results was carried out, and finally, it was identified that the SNP locus at position 58393394 on chromosome 14 was significantly associated with both the age at 100 kg body weight and the age at 120 kg body weight in pigs. The polymorphism of this SNP locus is C / T, and the individuals with the CC genotype had significantly lower ages at 100 kg body weight and 120 kg body weight than those with the CT genotype and TT genotype. Using this SNP molecular marker, molecular marker-assisted selection can be carried out simultaneously for the traits of age at 100 kg body weight and age at 120 kg body weight for early breeding selection to improve breeding efficiency.

[0071] 1. Test population

[0072] Taking a population of 592 Duroc pigs from a large-scale commercial pig farm in central China as the research object, including 484 boars and 108 sows. They were raised in an indoor environment with the ambient temperature controlled at 25 °C, separated by gender, 10 pigs in each pen, and the single pen area was 5 m × 4 m; they were fed with a complete corn-soybean meal diet, with ad libitum feeding and drinking throughout the process.

[0073] 2. Measurement of growth traits

[0074] When the pig body weight was close to the target weight (100 kg and 120 kg), body weight measurement and age recording were carried out on an empty stomach. The following formulas were used to correct the age at 100 kg body weight and the age at 120 kg body weight in pigs:

[0075] Age at 100 kg body weight = actual age + (100 - actual body weight) × (actual age - A) / actual body weight, where A is the correction coefficient, 55.29 for boars and 49.36 for sows;

[0076] Age at 120 kg body weight = actual age + (120 - actual body weight) × (actual age - B) / actual body weight, where B is the correction coefficient, 50.13 for boars and 46.14 for sows.

[0077] 3. Genome sequencing of the test population

[0078] Ear tissues of pigs that reached the target weight were collected, and DNA was extracted by the phenol-chloroform crude extraction method. BGI was commissioned to perform whole-genome sequencing with a sequencing depth of 10×.

[0079] 4. Whole-genome SNP genotyping of the test population

[0080] After steps such as reads alignment, sorting, duplicate marking, base quality recalibration, and variant detection on the sequencing data, 9,600,601 SNP sites were initially identified. Subsequently, using the PLINK software, quality control was performed according to the criteria of minor allele frequency less than 0.01, genotype missing rate less than 0.02, and individual missing rate less than 0.03, and finally 8,776,691 high-quality SNP markers were obtained.

[0081] 5. Genome-wide association analysis (GWAS) of growth traits in the experimental population

[0082] Based on the mixed linear model of the GCTA software, GWAS analysis was performed on the age at 100 kg body weight and the age at 120 kg body weight of growth traits. The analysis model is as follows:

[0083] Y = Xβ + Wα + μ + e;

[0084] Among them, Y is the phenotypic value; X is the fixed effect association matrix, β is the fixed effect, including gender and birth weight; W is the SNP marker vector, α is the effect value of the corresponding SNP; μ is the random polygenic effect, obeying μ ~ N(0, KV g ), N is the number of individuals, K is the genotype kinship matrix, Vg is the polygenic additive variance; e is the random residual vector, obeying e ~ N(0, IV e ), N is the number of individuals, I represents the individual identity matrix, V e represents the residual variance.

[0085] The GWAS association analysis results of the age at 100 kg body weight and the age at 120 kg body weight are respectively as Figure 1-2 shown. The abscissa in the figure is the chromosome number, and the ordinate is the P value of the significant locus. It can be seen that the SNP loci most significantly associated with the above two growth traits are both located at the 58,393,394th base on chromosome 14, presenting the largest P value in the figure. Therefore, functional molecular marker development was carried out at this locus.

[0086] The physical location of the above SNP locus is based on the Sus scrofa reference genome version Sscrofa11.1 (the nucleotide sequence can be found in the NCBI accession number: GCF_000003025.6), located at the 58,393,394th base on chromosome 14 (the nucleotide sequence can be found in the NCBI accession number: NC_010456.5). The corresponding locus number in the Ensembl database is rs320657871, with C / T polymorphism, resulting in three allele genotypes at this locus: CC, CT, and TT.

[0087] Taking the base sequence at positions 58392394 to 58394394 on chromosome 14 (14:58392394-58394394) as an example, the SNP locus in this embodiment is located at the 1001st base of the nucleotide sequence shown in SEQ ID NO.1 (5'-3') (see Table 1), that is, the Y base (shown in bold and shaded) is the physical position of the SNP locus, and Y = C or T.

[0088] Table 1 Sequence information of 14:58392394-58394394 containing SNP molecular markers

[0089]

[0090]

[0091] 6. Association analysis of SNP locus genotypes and phenotypic data in the experimental population

[0092] The experimental population was grouped according to the genotypes of the SNP locus at position 58393394 on chromosome 14. For the total sample of 592 individuals, 470 individuals were of the CC genotype, 115 were of the CT genotype, and 7 were of the TT genotype. Using the statistical data of the age at 100 kg body weight and the age at 120 kg body weight as phenotypic values, a grouped t-test was performed on different genotypes, and the P-value was used to indicate the correlation between the variation after the t-test and the phenotype. Less than 0.05 was considered significant (indicated by the symbol *), and less than 0.01 was considered extremely significant (indicated by the symbol **).

[0093] Figure 3-4 The statistical results of the phenotypic data of different genotypes of the SNP locus at position 58393394 on chromosome 14 in the experimental population are shown respectively. The abscissa represents the genotype, and the ordinate represents the age at 100 kg body weight or the age at 120 kg body weight. It can be seen from the figure that the growth traits corresponding to different genotypes are significantly different. Among them, the ages at 100 kg body weight and 120 kg body weight of individuals with the CC genotype are extremely significantly lower than those of individuals with the CT genotype and the TT genotype. That is, the CC genotype is an excellent genotype, which is beneficial to enhancing the growth rate of pigs. Therefore, by detecting the genotype of this SNP molecular marker, marker-assisted selection can be carried out simultaneously for the two traits of the age at 100 kg body weight and the age at 120 kg body weight, effectively identifying whether the individual to be tested is a breeding pig with a significantly fast growth rate, so as to conduct early selection in the population; it provides a detection technical means for early selection and improving breeding efficiency, and also provides a reliable target site for the genetic improvement of pig growth traits. By selecting breeding pigs with the CC genotype, the growth rate of the population of pigs can be gradually increased, which helps to improve the economic benefits of pig breeding enterprises.

[0094] 7. Development of SNP molecular marker detection primers

[0095] Specific detection primers were designed for the nucleotide site C / T at position 58393394 on chromosome 14 for PCR detection of this SNP site. The targeted positions of the detection primers are shown underlined in Table 1, and the nucleotide sequences are as follows:

[0096] F: 5’-TGAAGGAAAGCGGATACCCA-3’ (see SEQ ID NO.2);

[0097] R: 5’-ATTAACCAATGTGCCACGACA-3’ (see SEQ ID NO.3).

[0098] The nucleotide sequence of the amplification product is as follows: (see SEQ ID NO.4), where Y = C or T.

[0100] According to the whole-genome DNA sequencing results, genomic DNAs of individuals with SNP sites of CC, CT, and TT genotypes were respectively selected as DNA templates to verify the specificity of the primers, the detection effect, and the authenticity of the molecular marker sites.

[0101] For PCR amplification of the target fragment, the reaction system was 50 μL in volume, including: 25.0 μL of 2×GS taq PCRMix (purchased from TaKara, catalog number R300A), 2 μL of the upstream primer, 2 μL of the downstream primer, 2 μL of genomic DNA, and 19.0 μL of deionized water (ddH2O). The reaction procedure included: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, extension at 72°C for 10 s, for a total of 35 cycles; running at 72°C for 5 min; and storing the amplified reaction solution at 4°C for standby.

[0102] The PCR amplification products were detected by agarose gel electrophoresis, including the following steps: Weigh agarose at a mass concentration of 20 g / L, add it to 1×TAE buffer, heat and dissolve it to prepare an agarose solution; add 5 μL of EB solution to every 100 mL of agarose, mix well, and after cooling, pour it onto the electrophoresis plate, insert the comb plate, let it solidify at room temperature to form a gel, then place it in 1×TAE buffer, and gently pull out the comb plate vertically upward; Mix 5 μL of the PCR amplification product with 1 μL of the loading buffer, add it to the gel sample well, and at the same time add a DNA molecular weight standard to one of the sample wells, connect the power supply, and perform electrophoresis for 15 - 30 min under the condition of 2 V / cm - 5 V / cm for detection. After electrophoresis, take out the agarose gel, place it on a gel imager or an ultraviolet transilluminator for imaging. Archive the electrophoresis results as an electronic file or take a photo with a photographic system.

[0103] Method for preparing loading buffer: Weigh 250 mg of bromophenol blue, add 10 mL of water, and dissolve it at room temperature for 12 h; weigh 250 mg of xylene cyanol FF, add 10 mL of water to dissolve; weigh 50 g of sucrose, add 30 mL of water to dissolve. Mix the above three solutions, make up the volume to 100 mL with water, and store at 4 °C.

[0104] Judge the size of the amplified band according to the DNA molecular weight standard. The results of agarose gel detection are as Figure 5 shown. It can be seen that the size of the amplified fragment is 448 bp, presenting a single target band, which proves that the detection primer has good specificity.

[0105] Cut the above gel electrophoresis band, use a gel extraction kit (purchased from TIANGEN, product number DP209) to extract the amplified product, and perform sequencing. The comparison results of the sequencing are shown in Figure 6 (In the figure, Target represents the 10-bp nucleic acid sequence where the mutation site is located, Model represents the 1000-bp nucleic acid sequences upstream and downstream of the mutation site, CC, CT, and TT represent genotypes, and F and R respectively represent the forward and reverse sequencing results of the corresponding genotypes), and the peak map results are shown in Figure 7 . The results show that there are C and / or T bases at the nucleotide site 58393394 on chromosome 14, thereby confirming the existence of the SNP molecular marker. When the mutation site is C, it is the CC genotype; when the mutation site is C and T, it is the CT genotype; when the mutation site is T, it is the TT genotype.

[0106] Therefore, the SNP molecular marker provided by the present invention and the PCR detection primer developed based on this molecular marker can be used for the identification or breeding of pig growth traits, and can realize the conversion of the phenotypic judgment into the genotype identification, and further quickly identify or screen breeding pigs with excellent growth traits from the genotype level.

[0107] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker related to pig growth traits, and its detection primer or kit for use in pig growth trait identification or breeding, characterized in that: The SNP molecular marker is located at the 1001th base of the nucleotide sequence shown in SEQ ID NO.1, Y is selected from C or T, and the SNP molecular marker has CC, CT and TT genotypes; The growth traits are reaching 100 kg body weight at day 1 and / or reaching 120 kg body weight at day 1, wherein the growth traits of pigs with CC genotype reaching 100 kg body weight at day 1 and / or reaching 120 kg body weight at day 1 are significantly lower than those of pigs with CT genotype and TT genotype.

2. The use of the SNP molecular marker associated with pig growth traits, its detection primer or kit in pig growth trait identification or breeding according to claim 1, characterized in that: The detection primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

3. The use of the SNP molecular marker associated with pig growth traits, its detection primer or kit in pig growth trait identification or breeding according to claim 1, characterized in that: The kit comprises the detection primers, which include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

4. The use of the SNP molecular marker associated with pig growth traits, the detection primers or the kit thereof in pig growth trait identification or breeding according to claim 3, characterized in that: The kit also includes a PCR amplification reagent, which includes Taq DNA polymerase, dNTP and a buffer reagent.

5. A detection primer for detecting SNP molecular markers related to pig growth traits, characterized in that: The detection primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

6. A kit for detecting SNP molecular markers associated with pig growth traits, characterized in that: The kit comprises the detection primer as claimed in claim 5.

7. The kit for detecting SNP molecular markers associated with pig growth traits according to claim 6, characterized in that: The kit also includes a PCR amplification reagent, which includes Taq DNA polymerase, dNTP and a buffer reagent.

8. A method for identifying growth traits of pigs, characterized in that: The following steps are involved: Extracting genomic DNA from the individual pigs to be tested; Amplifying the genomic DNA by polymerase chain reaction using the detection primers shown in SEQ ID NO.2-3 to obtain an amplified product; Detecting the genotype of the 301st base of the amplified product, wherein the 301st base has CC, CT and TT genotypes, and identifying the growth traits of the individual pig to be tested according to the genotype; Among them, the growth traits are reaching 100 kg body weight at day old and / or reaching 120 kg body weight at day old, and the test pig individuals with CC genotype reaching 100 kg body weight at day old and / or reaching 120 kg body weight at day old are significantly lower than the test pig individuals with CT genotype and TT genotype.

9. The method for identifying pig growth traits according to claim 8, characterized in that: The amplification reaction system was 50 μL, including: 2×GS taq PCR Mix 25.0 μL, upstream primer 2 μL, downstream primer 2 μL, genomic DNA 2 μL, and deionized water 19.0 μL; The amplification reaction program included: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, and extension at 72°C for 10 s, for a total of 35 cycles; and running at 72°C for 5 min.

10. A method for genetically improving growth traits of pigs, characterized in that: The following steps are involved: Extracting genomic DNA from the individual pigs to be tested; Amplifying the genomic DNA by polymerase chain reaction (PCR) technique using the detection primers shown in SEQ ID NO.2-3 to obtain an amplified product; The genotype of the 301st base of the amplified product is detected. The 301st base has CC, CT and TT genotypes, and the pig individual to be tested with CC genotype is retained as a breeding pig.

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