Application of linkage SNP (Single Nucleotide Polymorphism) marker related to horse chest circumference character

Abstract

The invention relates to an application of a linkage SNP marker related to horse chest circumference characters. The application specifically comprises the following applications: (1) identifying or assisting in identifying the horse chest circumference characters; (2) preparing a product for identifying or assisting in identifying the chest circumference character of the horse; (3) breeding horses with large chest circumference; (4) preparing and breeding products of horses with large chest circumference; the linkage SNP markers comprise an SNP1, an SNP2 and an SNP3; the allele of the SNP1 is C or G at the 26th bp, the 679th bp and the 922th bp of the No.13 chromosome DNA of a horse genome, and the rs ID is rs396976187; the allele of the SNP2 is A or G at the 26th bp, the 680th bp and the 067th bp of the DNA of the 13th chromosome of the horse genome, and the rs ID is rs395890483; the SNP3 is located at 26th bp, 680th bp and 139th bp of DNA of a No.13 chromosome of a horse genome, an allele of the SNP3 is C or G, and rs ID is rs394326854. By detecting the genotypes of the three SNP markers, the breeding of a racing horse or a racing horse breeding horse can be assisted, and a target genotype individual is directionally selected and reserved at a foal stage and is used as a backup racing horse or a racing horse breeding horse for culture.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to the application of linked SNP markers associated with horse chest girth traits. Background Art

[0002] With rapid social development and the advancement of mechanization, the traditional role of horses in labor and transportation has gradually faded. The role of horses has begun to shift towards a modern horse industry that integrates culture, sports, leisure, and tourism. With the continuous development and improvement of my country's modern horse industry, people are increasingly interested in sports, leisure, and fitness. In recent years, domestic demand for horses for competition, speed racing, trotting, long-distance endurance racing, and recreation has continued to grow, and the country has placed the development of animal husbandry on a high priority as a livelihood industry.

[0003] Due to its proximity to traditional horse-breeding regions such as Altay, Tacheng, and Yining, Bortala Mongol Autonomous Prefecture (hereinafter referred to as Bortala Prefecture) has long bred Kazakh horses. While Kazakh horses boast strength and tolerance to roughage, their competitive abilities lag significantly behind those of leading domestic and international breeds, making them inadequate for modern competitive racing. In recent years, Bortala Prefecture has achieved some success in improving the local Kazakh horse breed by introducing thoroughbreds and other leading domestic and international breeds, such as the Ili horse. However, this traditional crossbreeding method, characterized by its long production cycles, slow progress, and relatively low efficiency, has resulted in a limited supply of high-quality breeding stock and low performance, a key constraint on the development of the local horse industry.

[0004] Body conformation is a key factor influencing equine racing performance. Competitive animals with an appropriate body conformation not only have excellent practical value but also serve as a crucial evaluation criterion in breeding. Studies have shown that body measurements such as height, length, and chest circumference are significantly correlated with racing performance, with chest circumference exhibiting the highest and most significant correlation with the 5000 m trotting race. Middle- and long-distance racing requires a certain level of endurance from horses, placing higher demands on their cardiorespiratory function than short-distance racers. A larger chest circumference and a deeper thorax increase chest volume, leading to a more developed heart and lungs and enhanced oxygen supply. This allows the horse to more steadily maintain or increase stride length during middle- and long-distance races, thus completing the race in a shorter time. Identifying and identifying key genes or loci influencing chest circumference traits in horses, and using molecular marker-assisted selection, could accelerate the targeted breeding of middle- and long-distance racing horses. Summary of the Invention

[0005] The present invention has screened three SNP markers significantly associated with the chest girth trait in horses. Genotyping these three SNP markers can assist in the selection and breeding of racehorses or stallions, allowing targeted selection of individuals with the target genotype during the foal stage for future racehorse or stallion breeding and management. Based on this, the present invention provides applications for linked SNP markers associated with the chest girth trait in horses.

[0006] In a first aspect, the present invention provides a use of a linked SNP marker associated with a horse chest girth trait in any one of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0007] In a second aspect, the present invention provides a reagent for detecting a linked SNP marker associated with a horse chest girth trait, for use in any one of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0008] In a third aspect, the present invention provides a primer set for amplifying a linked SNP marker associated with a horse chest girth trait, as used in any one of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0009] In some embodiments of the present invention, the product includes one of a kit, a chip, and an instrument.

[0010] In a fourth aspect, the present invention provides a method for identifying or assisting in identifying chest girth traits of a horse, comprising: Detect the genotype of linked SNP markers related to horse chest girth traits in the genome of the test individuals; Predict the chest circumference traits of the test individuals based on genotype: The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0011] In some embodiments of the present invention, the method for detecting the genotype of the linked SNP markers related to the horse chest girth trait in the genome of the test individual is any one of the whole genome sequencing method, PCR method, gene chip method, and SNaPshot method.

[0012] In a fifth aspect, the present invention provides a method for breeding horses, comprising: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is CCAAGG genotype or CCAGGC genotype, the test individual will be managed as a reserve racehorse; If the SNP marker associated with horse chest circumference in the genome of the test individual is CGAGGC genotype, the test individual will be eliminated; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0013] In a sixth aspect, the present invention provides a method for breeding racehorse stallions, comprising: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is the CCAAGG genotype or the CCAGGC genotype, the test individual will be bred and managed as a racehorse stallion; If the SNP marker related to horse chest circumference in the genome of the test individual is CGAGGC genotype, it will be eliminated directly; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0014] In some embodiments of the present invention, the subject is a foal.

[0015] In some embodiments of the present invention, the test individual or its parent is a horse from the Bortala region.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: 1. By detecting the genotypes of the three SNP markers provided by the present invention to assist in horse breeding, individuals with the CCAAGG genotype can be targeted and retained at the foal stage for breeding as reserve racehorses or stallions for middle and long distance trotting. Individuals with the CCAGGC genotype can also be targeted and retained for breeding as reserve racehorses for middle and long distance trotting.

[0017] 2. The two SNP markers screened by the present invention are tightly linked. In molecular breeding, the use of multiple mutually linked SNP markers has higher sensitivity and accuracy than a single SNP marker, thereby improving the accuracy of seed selection and further accelerating genetic progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 : Linkage relationship of the chromosome region where the three SNP markers are located.

[0020] Figure 2 : The selection pressure among populations in the chromosome region where the three SNP markers are located.

[0021] Figure 3 : The selection pressure among extreme individuals in the chromosome region where the three SNP markers are located. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0023] The present invention screened out three SNP markers that are significantly correlated with and highly linked to the chest girth trait of horses, namely SNP1, SNP2, and SNP3. Among them, SNP1 is located at 26,679,922 bp of DNA on chromosome 13 of the horse genome, with an allele of C or G, and an rs ID of rs396976187; SNP2 is located at 26,680,067 bp of DNA on chromosome 13 of the horse genome, with an allele of A or G, and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of DNA on chromosome 13 of the horse genome, with an allele of C or G, and an rs ID of rs394326854. Note: rs ID refers to the SNP number of the SNP in the horse genome version of EquCab3.0, for example, the rs ID is rs396976187.

[0024] In this article, the term "genotype" refers to the allele combination of SNP1, SNP2, and SNP3 in the cells of an organism; "SNP1CC genotype" refers to the alleles of SNP1 being C and C; "SNP2 AA genotype" refers to the alleles of SNP2 being A and A; the term "CCAAGG genotype" refers to the alleles of SNP1 being C and C, the alleles of SNP2 being A and A, and the alleles of SNP3 being G and G; "CGAGGC genotype" refers to the alleles of SNP1 being C and G, the alleles of SNP2 being A and G, and the alleles of SNP3 being G and C; and so on for other terms.

[0025] As used herein, the term "chest girth" refers to the chest girth trait in livestock body size traits, measured as the length perpendicularly around the posterior edge of the shoulder blade. The terms "small chest girth" and "large chest girth" are used herein to describe the relative size of chest girth in adult horses across all genotypes of this linked SNP marker.

[0026] Herein, the terms "site", "SNP site" and "SNP marker" have the same meaning.

[0027] As used herein, the term "subject" is defined as the biological individual from which the test sample originates.

[0028] For the purposes of this article, the term "foal" is defined as an immature equine individual whose body size continues to develop and change until adulthood.

[0029] The present invention found that the distribution differences of the three SNP markers between different breeds and extreme individuals were significant. In first-generation hybrid horses, high-generation hybrid horses, Ili horses, and thoroughbred horses, the SNP1 CC genotype, SNP2 AA genotype, and SNP3 GG genotype were dominant genotypes, and the heterozygous genotype was the dominant genotype in Kazakh horses. In the extremely large chest girth group, the SNP1 CC genotype, SNP2AA genotype, and SNP3 GG genotype were dominant genotypes, while in the extremely small chest girth group, the heterozygous genotype was the dominant genotype. The three dominant homozygous genotypes had a significant effect on the chest girth of horses, with average increases of 4.14%, 3.32%, and 3.32%, respectively. Among the genotypes of the three SNP markers, the CCAAGG genotype was the dominant combination, the CCAGGC genotype was the suboptimal combination, and the CGAGGC genotype was the worst combination.

[0030] In addition, the above three SNP markers are in a tightly linked state, and joint detection has higher sensitivity and accuracy than single SNP marker detection, thereby improving the accuracy of seed selection and further accelerating genetic progress.

[0031] Based on this, the present invention provides a linked SNP marker associated with horse chest girth traits, a detection reagent thereof, and an amplification primer set for use in any one of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

[0032] In some embodiments of the present invention, the product includes one of a kit, a chip, and an instrument.

[0033] The present invention provides a method for identifying or assisting in identifying the chest girth trait of a horse, comprising: Detect the genotype of linked SNP markers related to horse chest girth traits in the genome of the test individuals; Predict the chest circumference of the subjects when they grow up based on their genotype.

[0034] This method can be used directly in breeding decisions (e.g., screening reserve racehorses), in scientific research (analyzing the strength of associations between genotype and phenotype), or to assist veterinarians or breeders in developing personalized management plans (e.g., providing high-nutrient feed to foals predicted to have large chest circumference). For example, the test subject is a foal. If the genotype of the SNP marker associated with chest circumference in the test subject's genome is CCAAGG, the test subject is predicted to grow up to be a horse with large chest circumference; if the genotype of the SNP marker associated with chest circumference in the test subject's genome is CGAGGC, the test subject is predicted to grow up to be a horse with small chest circumference.

[0035] The present invention also provides a method for breeding racehorses, comprising: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is CCAAGG genotype or CCAGGC genotype, the test individual will be managed as a reserve racehorse; If the SNP marker associated with horse chest circumference in the genome of the test individual is a CGAGGC genotype, the test individual will be eliminated.

[0036] The chest circumference of foals before sexual maturity (typically 3 months to 2 years old) is not yet fully developed, making it difficult to predict their chest circumference in adulthood. Selecting racehorses based on phenotype after adulthood prevents them from reaching their full potential due to a lack of targeted training during their early years. Furthermore, training all horses as racehorses during their early years and then eliminating unqualified ones after adulthood results in a significant waste of training resources. This invention, which performs genotyping testing on foals before 24 months of age, allows for the early elimination of inferior individuals, reducing subsequent rearing costs and increasing the proportion of high-quality racehorses.

[0037] In some embodiments, the test individuals are bred and managed as reserve racehorses, including: providing targeted training (such as endurance and speed training) and nutritional support (high-protein feed, trace element supplementation) to foals with the CCAAGG genotype or CCAGGC genotype; tracking and recording the chest circumference and competition performance (such as speed and endurance indicators) of the foals after they become adults, and promptly eliminating unqualified ones.

[0038] In some embodiments of the present invention, the foal is a horse from the Bozhou area.

[0039] The present invention also provides a method for breeding racehorse stallions, comprising: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is the CCAAGG genotype or the CCAGGC genotype, the test individual will be bred and managed as a racehorse stallion; If the SNP marker associated with horse chest circumference in the genome of the test individual is the CGAGGC genotype, it will be eliminated directly.

[0040] Phenotypic selection of stallions is typically performed when the horses are 3-4 years old, which prolongs the breeding cycle and reduces selection efficiency. Furthermore, phenotypes (such as body shape and endurance) are easily affected by environmental factors such as rearing conditions and training level, and may not accurately reflect the stallion's genetic potential, causing selection results to deviate from actual genetic advantage. Observing only chest girth based on phenotype can easily lead to the omission of potentially high-quality stallions. The present invention can determine the suitability of individual racehorse breeders by testing the genotype of SNP markers within a few days of birth.

[0041] In some embodiments of the present invention, the method for detecting the genotype of the SNP marker related to the horse body length trait in the genome of the test individual is any one of the whole genome sequencing method, PCR method, gene chip method, and SNaPshot method.

[0042] In some embodiments of the present invention, the subject is a foal. Preferably, the subject is no older than 24 months; more preferably, the subject is no older than 12 months; further, the subject is no older than 1 month.

[0043] In some embodiments of the present invention, the test individual or its parent is a horse from the Bortala region.

[0044] The technical solution of the present invention is described in detail below through specific embodiments: Example This study uses genomic population evolution and selection analysis to identify genetic markers associated with chest girth in local horse breeds. These markers can be used to improve the accuracy of chest girth selection in horses from the Bortala region. The specific steps are as follows: 1.1 Blood collection and body measurement of horses in Bortala Prefecture Study subjects: 56 adult horses were randomly selected from Bortala Mongol Autonomous Prefecture, Xinjiang, including 30 first-generation hybrid horses, 12 high-generation hybrid horses, and 14 Kazakh horses. First-generation hybrid horses were first-generation hybrids derived from crossing a bred horse (e.g., a Thoroughbred, Ili, or similar breed) with a Kazakh horse from Bortala Prefecture as the dam. High-generation hybrid horses were derived from crossing a first-generation hybrid horse with the aforementioned sires for multiple generations.

[0045] All blood samples were collected from the jugular vein, anticoagulated with EDTA, and stored at -20°C for 10× genome sequencing. The horses' height (vertical height from the top of the carapace to the ground), body length (straight-line distance from the front edge of the shoulder to the back edge of the hip), chest circumference (vertical length around the back edge of the shoulder blade), and canal circumference (horizontally around the thinnest point of the upper third of the canal of the foreleg) were measured. The body length ratio, chest circumference ratio, and canal circumference ratio were calculated using the following formulas: body length ratio (%) = body length / body height × 100%; chest circumference ratio (%) = chest circumference / body height × 100%; canal circumference ratio (%) = canal circumference / body height × 100%.

[0046] 1.2 Blood total DNA extraction and quality control DNA was extracted using the Cowin CWE9600 fully automated nucleic acid extraction instrument and the accompanying Cowin CWE9600 Magbead Blood DNA Kit using magnetic beads. To the corresponding positions of a 96-well deep-well plate, 20 μL of Proteinase K, 300 μL of blood sample, 200 μL of Buffer mL, 750 μL of Buffer KL, 750 μL of Buffer CW1, 750 μL of Buffer GW2, 750 μL of Buffer MW3, and 100 μL of Buffer EB were added. The magnetic rod holder was inserted into the 96-well deep-well plate and the Cowin Blood 300 program was run. After approximately 23 minutes, 310 μL of the thoroughly mixed isopropanol and magnetic bead mixture was added according to the instrument prompt and the program was continued. The program concluded after approximately 35 minutes, and the eluted product was transferred to a 1.5 mL centrifuge tube for cryopreservation.

[0047] Take 2 μL of the eluted product and apply it to a 1.5% agarose gel. Perform electrophoresis at 150 V for 25 min. Place the gel in a UV analyzer. If only one band is observed, the DNA sample is considered to have good integrity. Use a NanoDrop 2000 Nucleic Acid Protein Analyzer to test the purity of the eluted product. If the A260 / A280 ratio is within the range of 1.7-2.1 and the A260 / A230 ratio is within the range of 1.8-2.2, the DNA sample purity meets the requirements for library construction.

[0048] 1.3 Library construction and sequencing Genomic DNA (>1 μg) was randomly fragmented into fragments of approximately 300–350 bp using the NadPrep® DNA universal library construction kit (for MGI) using a Covaris™ fragmentor. After end-repair, A-tailing, and ligation with sequencing adapters, the 300–350 bp fragments were screened using NadPrep® SP Beads. PCR amplification was performed, and the PCR product was purified again using NadPrep® SP Beads to generate a sequencing library. After library construction, initial quantification was performed using Qubit 2.0, followed by detection of inserts using a Bioanalyzer® (Agilent). If the inserts met expectations, pooling was performed based on the library concentration and target data volume. Sequencing was then performed using a DNBSEQ-T7 sequencer using a PE150 sequencing strategy.

[0049] 1.4 Data Collection of Common Horse Breeds Resequencing data from 24 horses (Thoroughbred and Ili horses) were downloaded from the NCBI database (Table 1). These two horses serve as exogenous reference populations. These two horses are representative breeds worldwide and in Xinjiang, respectively. Fastq reads were extracted from the SRA files using SRAtools software.

[0050] Table 1 Resequencing data information of well-known domestic and foreign bred horse breeds

[0051] 1.5 Data filtering and pre-processing A series of quality controls (QC) were performed on the downloaded raw data using FastQ, including removing reads with adapters, removing paired reads with N content exceeding 1% of the total number of bases in the read, and removing paired reads with low-quality (Q ≤ 5) bases exceeding 50% of the total number of bases in the read.

[0052] After data filtering, clean reads were aligned to the reference genome (EquCab3.0) using BWA 0.7.17 (men) software, sorted and indexed using samtools 1.7, and the Bam file was deduplicated using GATK 4.1.8.0 software. The sequencing depth, genome coverage, and other information of each sample were then statistically analyzed based on the Bam file to prepare for subsequent variant detection.

[0053] Based on the alignment of clean reads to the reference genome, SNPs (single nucleotide polymorphisms) were called using GATK 4.1.8.0. SNPs were then strictly filtered using the following criteria: "QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0." Functional and positional annotations were performed on the filtered files using SNPeff software.

[0054] 1.6 Inter-population Fst and θ π Ratio population evolution and selection analysis All individuals were subjected to PCA principal component analysis using Plink software, population structure analysis using Admixture software, and phylogenetic tree construction using VCF2DIS software. Based on the above results, all individuals were grouped (see Table 2). After grouping, the Fst value and θ of the improved population and other populations were calculated using VCFtools software. π Calculate θ with a window of 100kb and a step size of 10kb. π Ratio value, select Fst value and θ π The regions with the top 1% Ratio values ​​are selected as candidate regions.

[0055] 1.7 Fst and θ between extreme individuals π Ratio population evolution and selection analysis Sequenced individuals were sorted by chest girth, and 19 horses in the upper and lower quartiles were selected to construct a maximum chest girth group (chest girth > 185 cm) and a minimum chest girth group (chest girth < 174 cm). Fst and θπ values ​​were calculated using vcftools software with a 100 kb window and 10 kb step size. θπ Ratio values ​​were calculated, and regions with the top 1% of Fst and θπ Ratio values ​​were selected as candidate regions associated with chest girth.

[0056] 1.8 Association analysis with body size traits The intersection of candidate regions between populations and extreme individuals was determined, and SNP loci and their genotypes were extracted from the overlapping candidate regions. The genotype and allele frequencies of each locus were calculated using R software, and linkage disequilibrium maps were plotted. Differences were analyzed using the chi-square test. Differences in body size traits between individuals with different genotypes were compared using one-way analysis of variance, and the LSD method was used for multiple comparisons of phenotypic values ​​of traits within individuals with different genotypes.

[0057] 2. Test results 2.1 SNP markers associated with horse chest girth traits Three linked SNP loci associated with horse chest girth traits were screened, namely rs396976187, rs395890483, and rs394326854, located at 26,679,922 bp, 26,680,067 bp, and 26,680,139 bp on chromosome 13, respectively ( Figure 1 ), the alleles at the three loci are C / G, A / G and C / G respectively.

[0058] 2.2 Differentiation degree and frequency distribution of each site among groups Fst values ​​and θ between groups π Ratio value display ( Figure 2 ), the chromosome segments where the three sites are located are selected among different populations ( Fst = 0.07, θ π Ratio = 1.23), indicating that the polymorphism of this site was strongly differentiated among different populations, suggesting that the polymorphism at this site may be an important factor leading to phenotypic differences among different populations.

[0059] The chi-square test results showed that the distribution of each genotype of this SNP site in different populations was significantly different (Table 2), among which the distribution difference of rs396976187 site in first-generation hybrid horses and Kazakh horses and Yili horses was extremely significant ( p <0.01), and the distribution was significantly different from that in thoroughbred horses ( p <0.05), and the distribution difference between them and high-generation hybrid horses was not significant ( p >0.05); the distribution differences of rs395890483 and rs394326854 in first-generation hybrid horses and Kazakh horses, Yili horses, and purebred horses were extremely significant ( p <0.01), and the distribution difference between the two groups was not significant ( p >0.05). In first-generation hybrid horses, high-generation hybrid horses, Ili horses, and purebred horses, CC and AA types were the dominant genotypes at the three SNP loci, while AG type was the dominant genotype in Kazakh horses.

[0060] Table 2 The frequency of each genotype at the 3 loci in different populations

[0061] 2.3 Differentiation degree and frequency distribution among extreme individuals at each site Fst values ​​and θ between extreme individuals π Ratio value display ( Figure 3 ), compared with the very small chest circumference group, the chromosome segment where the rs397436663 locus is located is selected in the very large chest circumference group ( Fst = 0.08, θ π Ratio = 2.86), indicating that the polymorphism of this site was strongly differentiated among extreme individuals, suggesting that the polymorphism at this site may be an important variation leading to individual differences in chest circumference.

[0062] The chi-square test results showed that the distribution of this SNP site in the extremely large chest circumference group and the extremely small chest circumference group was significantly different ( p <0.01). As shown in Table 3, the dominant genotypes of the three loci in the extremely large chest circumference group were CC, AA, and GG, respectively, while the dominant genotype in the extremely small chest circumference group was the heterozygous genotype.

[0063] Table 3 Frequency of each genotype at 3 loci in extreme individuals

[0064] 2.4 Association analysis between the three sites and body size traits The statistical results of seven body size traits (body height, body length, chest circumference, tube circumference, body length ratio, chest circumference ratio and tube circumference ratio) measured in 30 first-generation hybrid horses and 12 high-generation hybrid horses are shown in Table 4.

[0065] Table 4 Statistical results of body size traits

[0066] Association analysis showed the effects of different genotypes at the three loci on seven body size traits, and the results are shown in Table 5. Different genotypes at the three loci had a significant effect on horse chest circumference ( p <0.05), with no significant effect on height, length, tube circumference, body length ratio, chest circumference ratio, or tube circumference ratio. The mean chest circumference and chest circumference ratio of individuals with the dominant genotypes (CC, AA, and GG) were greater than those of the heterozygous genotype, with mean increases of 4.14%, 3.32%, and 3.32%, respectively.

[0067] Table 5 Association analysis between three loci and body size traits (MEAN ± SD)

[0068] Note: Different lowercase letters indicate significant differences between different genotypes of the same trait at the same locus (P<0.05).

[0069] The three loci constituted three genotype combinations in all individuals. Association analysis showed the effects of different genotype combinations on seven body size traits. The results are shown in Table 5. The genotype combination of the three loci had a significant effect on the chest circumference of horses ( p<0.05), with no significant effect on height, length, tube circumference, body length ratio, chest circumference ratio, or tube circumference ratio. The CCAAGG combination was the dominant combination, with a larger average chest circumference than the CGAGGC combination. The CCAGGC combination did not differ significantly from the other two combinations.

[0070] Table 6 Association analysis between genotype combinations of the three loci and body size traits (MEAN ± SD)

[0071] Note: Different lowercase letters indicate significant differences between different genotypes of the same trait at the same locus (P<0.05).

[0072] The above research results show that three SNP sites, rs396976187, rs395890483, and rs394326854, can affect the chest girth traits of horses and are closely linked. In the large chest girth population, the dominant genotypes at the three sites are CC, AA, and GG, respectively. The chest girth traits of horses in the Bortala region show significant differences between the different genotypes and genotype combinations of the three sites, with the optimal genotype combination being the CCAAGG genotype. By typing the three linked SNP sites, it is possible to predict the chest girth traits of foals as they grow up, select horses with large chest girths at an early stage, improve the chest girth traits of horses in the Bortala region, and achieve targeted breeding of horses for middle and long-distance competitions.

[0073] In the description of this specification, the description with reference to the terms "one embodiment / method", "some embodiments / methods", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in an appropriate manner in any one or more embodiments / methods or examples. In addition, those skilled in the art may combine and combine different embodiments / methods or examples described in this specification and the features of different embodiments / methods or examples, unless they are contradictory.

[0074] It should be noted that, in the present invention, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises", "includes" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a..." does not exclude the presence of other identical elements in the process, method, article or device comprising the element. In the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly specified.

[0075] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of linked SNP markers associated with horse chest girth traits in any of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

2. Use of a reagent for detecting linked SNP markers associated with chest girth traits in horses in any of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

3. Use of a primer set for amplifying a linked SNP marker associated with a horse chest girth trait in any one of (1) to (4): (1) Identify or assist in identifying the chest girth traits of horses; (2) Preparation of products for identifying or assisting in identifying chest girth traits of horses; (3) Breeding horses with large chest circumference; (4) Preparation of products for breeding horses with large chest girth; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

4. The use according to any one of claims 1 to 3, characterized in that: The product includes one of a kit, a chip, and an instrument.

5. A method for identifying or assisting in identifying chest girth traits of horses, characterized in that: include: Detect the genotype of linked SNP markers related to horse chest girth traits in the genome of the test individuals; Predict the chest circumference traits of the subjects based on genotype; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

6. A method for breeding racehorses, characterized in that: include: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is CCAAGG genotype or CCAGGC genotype, the test individual will be managed as a reserve racehorse; If the SNP marker associated with horse chest circumference in the genome of the test individual is CGAGGC genotype, the test individual will be eliminated; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

7. A method for breeding racing stallions, characterized in that: include: Detect linked SNP markers related to horse chest girth traits in the genome of the test individuals; If the SNP marker associated with horse chest circumference in the genome of the test individual is the CCAAGG genotype or the CCAGGC genotype, the test individual will be bred and managed as a racehorse stallion; If the SNP marker related to horse chest circumference in the genome of the test individual is CGAGGC genotype, it will be eliminated directly; The linked SNP markers include SNP1, SNP2, and SNP3; SNP1 is located at 26,679,922 bp on chromosome 13 of the horse genome, with alleles of C or G and rs ID rs396976187; SNP2 is located at 26,680,067 bp on chromosome 13 of the horse genome, with an allele of either A or G and an rs ID of rs395890483; SNP3 is located at 26,680,139 bp of chromosome 13 of the horse genome, with an allele of C or G and an rs ID of rs394326854.

8. The method according to any one of claims 5 to 7, characterized in that: The method for detecting the genotype of the linked SNP marker associated with the horse chest girth trait in the genome of the test individual is any one of the whole genome sequencing method, PCR method, gene chip method, and SNaPshot method.

9. The method according to any one of claims 5 to 7, characterized in that: The test subject is a foal.

10. The method according to claim 9, characterized in that: The test individuals or their parents are horses from Bozhou area.

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