Application of SNP (Single Nucleotide Polymorphism) marker related to horse body length and chest circumference characters

Abstract

The invention relates to an application of an SNP (Single Nucleotide Polymorphism) marker related to a horse body length and chest circumference character. The application specifically comprises the following applications: (1) identifying or assisting in identifying the horse body length or chest circumference character; (2) preparing a product for identifying or assisting in identifying the body length or chest circumference character of the horse; (3) breeding horses with slender figures and large chest circumference; (4) preparing and breeding products of horses with slender figures and large chest circumference; the SNP marker is located at the 76th bp, the 788th bp and the 230th bp of DNA of the No.15 chromosome of a horse genome, an allele is A or G, and rs ID is rs1136176196. By detecting the genotype of the SNP marker provided by the invention to assist in breeding of breeding horses, AA genotype individuals can be directionally selected and reserved in the foal stage, and the AA genotype individuals are used as racing horses or racing horse breeding horses for culture after a quick-step race of a 1000 / 5000 m race.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and specifically relates to the application of SNP markers related to the traits of horse body length and chest girth. Background Technology

[0002] China has one of the world's longest histories of horse breeding. However, with social development and the widespread adoption of mechanization, the role of horses has gradually been replaced by machinery. The horse industry has shifted from daily applications such as military and agriculture to recreational applications such as equestrian sports and horse racing, as well as production applications such as dairy and meat. Xinjiang Uygur Autonomous Region has the largest horse population in my country, boasting abundant horse breeds, including four local breeds represented by the Kazakh horse and two developed breeds represented by the Ili horse. In recent years, equestrian sports have developed rapidly in Xinjiang Uygur Autonomous Region, with increasingly rich professional competitions. The horse industry is also gradually developing towards specialization, large-scale operation, and intensification. Currently, most of the sports horses in many equestrian clubs across the country come from Xinjiang Uygur Autonomous Region.

[0003] Bortala Mongol Autonomous Prefecture (hereinafter referred to as Bortala Prefecture) is an important border region in western my country, located in the center of the "Altai-Tianshan" horse industry belt. It is adjacent to traditional horse-breeding areas such as Altay, Tacheng, and Yining, boasting a long history of horse breeding and a rich horse culture, possessing a uniquely advantageous foundation for the development of the horse industry. In the past, Bortala Prefecture primarily bred Kazakh horses, which are known for their strength and tolerance to roughage, but their competitive ability lagged significantly behind that of superior domestic and international breeds, failing to meet the demands of modern competitive horse racing. In recent years, Bortala Prefecture has improved the Kazakh horse breed by introducing Thoroughbreds and Ili horses, among other superior domestic and international breeds. The resulting hybrid horses have shown significant improvements in body structure, body size, and competitive performance.

[0004] A horse's body conformation significantly impacts its quality and performance, being a key factor in determining its racing performance. Studies show a significant correlation between height, length, chest girth, and racing results. For instance, in a 1000m trot, higher height, longer length, larger chest girth, and smaller cannon bone girth, chest girth percentage, and cannon bone girth percentage all correlate with shorter race times. Similarly, in a 5000m trot, higher height, larger chest girth, longer length, and smaller cannon bone girth percentage all correlate with shorter race times. Scientific breeding and genetic improvement can cultivate horse breeds with superior conformation, enhancing overall horse performance, better meeting market demands, and increasing economic benefits. With the rapid development of molecular biology techniques, molecular genetic markers and marker-assisted selection are increasingly widely researched and applied in livestock breeding, paving a new path for fundamentally improving the genetic quality of horses and breeding high-performance breeds. Therefore, identifying SNP markers related to equine body size traits and applying them to the breeding of large-sized, high-performance horses will significantly improve breeding efficiency and accuracy, and accelerate the process of equine genetic improvement. Summary of the Invention

[0005] This invention identifies a single-NP marker that is significantly associated with the traits of horse body length and chest girth. By detecting the genotype of this SNP marker, the selection of racehorses or stallions can be assisted, allowing for the targeted selection of individuals with the target genotype during the foal stage for breeding as reserve racehorses or stallions. Based on this, this invention provides applications of SNP markers associated with the traits of horse body length and chest girth.

[0006] In a first aspect, the present invention provides the application of SNP markers related to horse body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0007] In a second aspect, the present invention provides the use of a reagent for detecting SNP markers associated with equine body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0008] Thirdly, the present invention provides the application of primer sets for amplifying SNP markers associated with equine body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

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

[0010] Fourthly, the present invention provides a method for identifying or assisting in the identification of body length or chest girth traits in horses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. Predicting body length traits in adult individuals based on genotype: If the genotype of the SNP marker associated with horse body length in the genome of the test individual is AA, then it is predicted that the test individual will grow up to be a horse with a slender body and a large chest. If the genotype of the SNP marker associated with horse body length in the genome of the test individual is AG, then it is predicted that the test individual will grow up to be a short-bodied horse with a small chest circumference. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0011] Fifthly, the present invention provides a method for breeding racehorses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are of the AG or GG genotype, the test individual will be eliminated. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0012] Sixthly, the present invention provides a method for breeding racehorses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are AG or GG genotypes, the test individual will be eliminated. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0013] In some embodiments of the present invention, the method for detecting the genotype of SNP markers related to horse body length and chest girth traits in the genome of the test individual is any one of whole genome sequencing, PCR, gene chip, and SNaPshot.

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

[0015] In some embodiments of the present invention, the test subject 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) The present invention screened a SNP marker related to the body length trait of horses in Bortala Prefecture. The SNP marker showed significant differences in body length among horses with different genotypes. The average body length of individuals with the AA genotype was 156.4 cm, and the average body length of individuals with the AG genotype was 148.3 cm. The AA genotype was the dominant genotype, and the corresponding individuals had a slender body shape.

[0017] (2) This SNP marker can also take into account the selection of chest girth traits in horses in the Bortala region. Horses with different genotypes of this SNP marker have significant differences in chest girth. The average chest girth of individuals with the AA genotype is 179.74 cm, and the average chest girth of individuals with the AG genotype is 172.83 cm. The AA genotype is the dominant genotype, and the corresponding individuals also have the large chest girth trait.

[0018] (3) By detecting the SNP markers provided by this invention to assist in the breeding of stallions, the body length and chest circumference of horses can be effectively increased by selecting AA genotype stallions at the foal stage, thereby enabling early selection of stallions, accelerating the genetic improvement and breeding process of stallions in Bortala Prefecture, and obtaining specialized horse breeds with slender bodies and large chest circumferences.

[0019] (4) By detecting the genotype of the SNP marker provided by the present invention to assist in the breeding of stallions, individuals with the AA genotype can be selected in a targeted manner at the foal stage to be trained as reserve racehorses for 1000 / 5000 m trotting races. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The selective pressure on the chromosomal region containing the rs1136176196 locus within the population.

[0022] Figure 2 The chromosomal region containing the rs1136176196 locus experiences selective pressure among extreme individuals. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] This invention screened out a SNP marker that is significantly associated with the traits of body length and chest girth in horses. The SNP marker is located at 76,788,230 bp on chromosome 15 of the horse genome, with alleles of A or G and rs ID of rs1136176196.

[0025] In this article, the term "genotype" refers to the allele combination of SNPs in an organism's somatic cells; "AA genotype" refers to SNPs with alleles A and A; "GG genotype" refers to SNPs with alleles G and G; and "AG genotype" refers to SNPs with alleles A and G.

[0026] In this article, the term "chest circumference" refers to the chest circumference trait in livestock body size traits, measured by the length around the shoulder blade vertically at the posterior edge. In this article, the terms "small chest circumference" and "large chest circumference" are used to describe the relative size of chest circumference in adult horses across all genotypes of this linked SNP marker.

[0027] In this article, the term "body length" refers to the straight-line distance from the front edge of the shoulder to the rear edge of the rump; the terms "slender body" and "short body" are used to describe the relative size of body length in adult horses across all genotypes of this linked SNP marker.

[0028] In this article, the terms “site”, “SNP site”, and “SNP marker” have the same meaning.

[0029] In this article, the term "subject individual" is defined as the biological individual from which the test sample originates.

[0030] In this article, the term "foal" is defined as an immature horse individual whose size continues to develop and change before reaching adulthood.

[0031] This invention revealed significant differences in the distribution of the three genotypes of this SNP marker among different horse breeds and individuals with extreme body lengths. In first-generation crossbreds, high-generation crossbreds, and Thoroughbreds, the AA genotype was the dominant genotype. In Kazakh horses, the AG genotype was the dominant genotype, while in Ili horses, the frequencies of the three genotypes were similar. In the extremely long body length group, the frequency of the AA genotype was 100%, significantly higher than the 57.1% in the extremely short body length group.

[0032] In the Bortala region, the AA genotype of this SNP marker corresponds to individuals with a slender body and large chest circumference, with an average body length of 156.4 cm and a chest circumference of 179.74 cm; the AG genotype corresponds to individuals with a short body length and small chest circumference, with an average body length of 148.3 cm and a chest circumference of 172.83 cm.

[0033] This invention can determine the genotype of the SNP marker by directly detecting it using whole-genome sequencing or by preparing a reagent for detecting the SNP marker. This allows for its application in any of the following scenarios: (1) predicting horse body length or chest girth traits; (2) selecting racehorses with slender bodies and large chest circumferences during the foal stage, and then selecting trotting racehorses for 1000 / 5000 m races; (3) selecting racehorse stallions during the foal stage for breeding horses with body length or chest girth traits, as well as improving horse breeds related to body length and chest girth traits, especially improving the germplasm resources of horses in the Bortala region.

[0034] Based on this, the present invention provides the application of SNP markers related to horse body length and chest girth traits, their detection reagents, and amplification primer sets in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rsID rs1136176196.

[0035] In some embodiments of the present invention, the product includes one of a reagent kit, a chip, or an instrument.

[0036] This invention provides a method for identifying or assisting in the identification of body length or chest girth traits in horses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. Predicting body length traits of test individuals as they grow up based on genotype.

[0037] This method can be directly used for breeding decisions (such as selecting reserve racehorses), for scientific research (analyzing the strength of the association between genotype and phenotype), or to assist veterinarians or breeders in developing personalized management plans (such as providing high-nutrient feed for foals predicted to have a "long-bodied" build). For example, the test individual is a foal. If the genotype of the SNP marker associated with horse body length in the test individual's genome is AA, then the test individual is predicted to grow into a long-bodied horse with a large chest; if the genotype of the SNP marker associated with horse body length in the test individual's genome is AG, then the test individual is predicted to grow into a short-bodied horse with a small chest.

[0038] This invention provides a method for breeding racehorses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are AG or GG genotypes, the test individual will be eliminated.

[0039] The body length trait of foals is not yet fully formed, making it difficult to predict their adult body length trait. If racehorses are selected based on phenotype after adulthood, their potential may not be fully realized due to a lack of targeted training during their foaling period. Conversely, if all horses are trained for racing during their foaling period, and those that fail to meet the standards are culled in adulthood, it will result in a significant waste of training resources. This invention performs genotyping on foals before they reach 24 months of age, allowing for the early culling of inferior individuals, reducing later feeding costs, and increasing the proportion of high-quality racehorses.

[0040] In some implementation methods, the test subjects are bred and managed as reserve racehorses, including: providing targeted training (such as endurance and speed training) and nutritional support (high-protein feed and micronutrient supplementation) to AA genotype foals; tracking and recording the foals' body length and racing performance (such as speed and endurance indicators) after adulthood, and promptly eliminating those that do not meet the requirements.

[0041] This invention also provides a method for breeding racehorses, comprising: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are AG or GG genotypes, the test individual will be eliminated.

[0042] Phenotypic selection of stallions typically occurs when horses are 3-4 years old, prolonging the breeding cycle and reducing selection efficiency. Furthermore, body length and chest girth phenotypes are easily influenced by environmental factors such as feeding conditions and training levels, potentially failing to accurately reflect the stallion's genetic potential and leading to selection results that deviate from actual genetic advantages. This invention can determine whether a test individual is suitable for breeding and managing as a racehorse stallion by detecting the genotype of SNP markers within a few days of birth.

[0043] In some embodiments of the present invention, the method for detecting the genotype of SNP markers related to horse body length and chest girth traits in the genome of the test individual is any one of whole genome sequencing, PCR, gene chip, and SNaPshot.

[0044] In some embodiments of the present invention, the test subject is a foal. Preferably, the age of the test subject is no more than 24 months; more preferably, the age of the test subject is no more than 12 months; further, the age of the test subject is no more than 1 month.

[0045] In some embodiments of the present invention, the test subject or its parent is a horse from the Bortala region.

[0046] The technical solution of the present invention will be described in detail below through specific embodiments: Example This invention utilizes genomic population evolution and selection analysis to identify genetic markers associated with body size traits in local horse breeds. These markers can be used to improve the accuracy of body length selection for horses in the Bortala Mongol Autonomous Prefecture. The specific steps are as follows: I. Materials and Methods 1.1 Blood collection and body size measurement of horses in Bortala Prefecture Study subjects: Fifty-six adult individuals were randomly selected from Bortala Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, including 30 first-generation crossbred horses, 12 advanced-generation crossbred horses, and 14 Kazakh horses. First-generation crossbred horses were obtained by crossing Thoroughbreds, Ili horses, and other bred breeds as sires with Kazakh horses from Bortala Prefecture as dams. Advanced-generation crossbred horses were obtained by crossing first-generation crossbred horses as dams with the aforementioned sires over multiple generations.

[0047] All blood samples were collected via the jugular vein, anticoagulated with EDTA, and stored at -20 °C for 10× genome sequencing. Simultaneously, the following measurements were taken of these horses: height (vertical height from the top of the forearm to the ground), body length (straight-line distance from the front edge of the shoulder to the rear edge of the rump), chest circumference (length around the posterior edge of the scapula vertically), and cannon bone circumference (length around the narrowest part of the upper third of the foreleg cannon). The body length percentage, chest circumference percentage, and cannon bone circumference percentage were calculated using the following formulas: Body length percentage (%) = Body length / Body height × 100%; Chest circumference percentage (%) = chest circumference / height × 100%; Tube circumference rate (%) = tube circumference / body height × 100%.

[0048] 1.2 Total DNA Extraction and Quality Inspection from Blood DNA extraction was performed using the Cowin CWE9600 fully automated nucleic acid extractor and the accompanying Cowin CWE9600 MagbeadBlood DNA Kit via magnetic bead extraction. 20 μL Proteinase K, 300 μL blood sample, 200 μL Buffer mL, 750 μL Buffer KL, 750 μL Buffer CW1, 750 μL Buffer GW2, 750 μL Buffer MW3, and 100 μL Buffer EB were added to the corresponding positions in a 96 DW deep-well plate. The magnetic bead holder was inserted into the 96 DW deep-well plate, and the Cowin Blood 300 program was run. After approximately 23 minutes, 310 μL of a thoroughly mixed isopropanol and magnetic bead mixture was added as prompted by the instrument, and the program was continued. The program finished after approximately 35 minutes, and the eluted product was transferred to a 1.5 mL centrifuge tube for cryogenic storage.

[0049] Take 2 μL of the elution product and spot it onto a 1.5% agarose gel. Electrophoresis is performed at 150 V for 25 min. The gel is then placed in a UV analyzer. If only one band is visible, the DNA sample has good integrity. The purity of the elution product is tested using a NanoDrop 2000 nucleic acid and protein analyzer. If the A260 / A280 ratio is in the range of 1.7 to 2.1 and the A260 / A230 ratio is in the range of 1.8 to 2.2, the DNA sample purity meets the requirements for library construction.

[0050] 1.3 Library Construction and Sequencing Genomic DNA with a total volume greater than 1 μg was randomly fragmented into approximately 300–350 bp fragments using the NadPrep® DNA fragmentation kit (for MGI) via a Covaris™ fragmentation instrument. After end repair, A-tailing, and ligation of sequencing adapters, DNA fragments of approximately 300–350 bp were selected using NadPrep® SP Beads. PCR amplification was performed, and the PCR products were purified again using NadPrep® SP Beads to obtain the final sequencing library. After library construction, preliminary quantification was performed using Qubit 2.0, followed by detection of the inserted fragments using Bioanalyzer® (Agilent). If the results met expectations, pooling was performed based on the library concentration and the target data volume requirements. Sequencing was then performed using a DNBSEQ-T7 sequencer with the PE150 sequencing strategy.

[0051] 1.4 Data Collection on Common Horse Breeds Resequencing data of 24 horses from two breeds, Thoroughbred and Ili, were downloaded from the NCBI database (see Table 1) as exogenous reference populations. These two breeds are representative breeding breeds worldwide and in the Xinjiang Uygur Autonomous Region, respectively. Reads in fastq format were extracted from the SRA files using SRAtools software.

[0052] Table 1. Information on resequencing data of well-known domestic and international horse breeds

[0053] 1.5 Data Filtering and Preprocessing FastQ is used to perform a series of quality control (QC) processes on the raw reads that are downloaded and downloaded. These processes include removing reads with adapters, removing paired reads with an N content exceeding 1% of the total number of bases in the read, and removing paired reads with a low-quality (Q ≤ 5) base content exceeding 50% of the total number of bases in the read.

[0054] After data filtering, clean reads were aligned to the reference genome (EquCab3.0) using BWA 0.7.17 (men) software. Sorting and indexing were performed using samtools 1.7. Deduplication of the Bam files was performed using GATK 4.1.8.0 software. Then, based on the Bam files, information such as sequencing depth and genome coverage of each sample was statistically analyzed to prepare for subsequent variant detection.

[0055] Based on the alignment results of clean reads with the reference genome, SNPs (single nucleotide polymorphisms) were called using GATK 4.1.8.0 software. SNPs were then rigorously 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 information was then annotated using SNPeff software on the filtered files.

[0056] 1.6 Inter-group Fst and θ π Ratio Population Evolution and Selection Analysis Principal component analysis (PCA) was performed on all individuals using Plink software, population structure analysis was performed using Admixture software, and a phylogenetic tree was constructed using VCF2DIS software. Based on these results, all individuals were grouped (see Table 2). After grouping, the Fst values ​​and θ values ​​of the modified population and other populations were calculated using VCFtools software. π Value, window size 100kb, step size 10kb. Calculate θ. π Ratio value, select Fst value and θ π The region with the highest Ratio value (top 1%) is selected as the candidate region.

[0057] 1.4 Fst and θ among extreme individuals π Ratio Population Evolution and Selection Analysis Individuals were sorted by body length, and 19 horses in the upper and lower quartiles were selected to construct a maximum body length group (body length > 150 cm) and a minimum body length group (body length < 140 cm). The Fst and θπ values ​​were calculated using vcftools software with a window size of 100 kb and a step size of 10 kb. The θπ Ratio was calculated, and the top 1% of regions with the highest Fst and θπ Ratio values ​​were selected as candidate regions related to body length traits.

[0058] 1.5 Association analysis with body size traits Intersections of candidate regions between populations and extreme individuals were calculated, and SNP markers and their genotypes were extracted from overlapping candidate regions. Genotype frequencies and allele frequencies at each locus were calculated using R software, and differential analysis was performed using the chi-square test. One-way ANOVA was used to compare differences in body size traits among individuals with different genotypes, and the LSD method was used for multiple comparisons of phenotypic values ​​of individuals with different genotypes.

[0059] II. Test Results 2.1 SNP markers associated with horse body length and chest girth traits One SNP marker associated with the body length trait in horses, rs1136176196, was identified. This locus is located at 76,788,230 bp on chromosome 15 of the horse. The two alleles at this locus are A / G, and their distribution varies significantly among different populations.

[0060] 2.2 Differentiation degree and frequency distribution of rs1136176196 locus among populations The Fst value and θ were used to measure the inter-group values ​​of 80 horses from 5 groups. π The Ratio value results show ( Figure 1 The chromosomal region containing the rs1136176196 locus is subject to selection in different populations. Fst = 0.10, θ π Ratio = 2.18), indicating that the polymorphism at this locus showed strong differentiation among different populations, suggesting that the polymorphism at this locus may be an important factor leading to phenotypic differences among different populations.

[0061] Chi-square test results showed that the distribution of each genotype of this SNP marker differed significantly in different populations (Table 2), with highly significant differences in the distribution of this locus between first-generation hybrid horses and Kazakh and Ili horses. p <0.01, the distribution of which differed significantly between first-generation hybrid horses and thoroughbreds. p <0.05, the difference was not significant between first-generation and high-generation crossbred horses ( p >0.05). Among first-generation crossbred horses, high-generation crossbred horses, and Thoroughbreds, the AA genotype is the dominant genotype. In Kazakh horses, the AG genotype is the dominant genotype. In Ili horses, the frequencies of the three genotypes are similar.

[0062] Table 2. Frequency of each genotype at the rs1136176196 locus in different populations.

[0063] 2.3 Differentiation degree and frequency distribution of rs1136176196 locus among extreme individuals The intergroup Fst value and θ were obtained from two extreme groups, totaling 19 horses. π Ratio value display ( Figure 2 Compared to the extremely small body length group, the chromosomal segment containing the rs1136176196 locus was selected in the extremely large body length group. Fst = 0.12, θ π Ratio =7.70), indicating that the polymorphism at this site showed strong differentiation among extreme individuals, suggesting that the polymorphism at this site may be an important factor leading to differences in individual body length.

[0064] The chi-square test results showed that the distribution of this SNP marker differed significantly between the maximum body length group and the minimum body length group. p <0.01). As shown in Table 3, the AA genotype accounted for 100% in the maximum body length group, while it accounted for only 57.1% in the minimum body length group.

[0065] Table 3. Frequency of different genotypes at the rs1136176196 locus in extreme individuals.

[0066] 2.4 Association analysis between rs1136176196 locus and body size trait The statistical results of seven physical characteristics (body height, body length, chest circumference, cannon circumference, body length percentage, chest circumference percentage, and cannon circumference percentage) of 30 first-generation crossbred horses and 12 high-generation crossbred horses are shown in Table 4.

[0067] Table 4 Statistical results of body size traits

[0068] Association analysis revealed the effects of different genotypes at the rs1136176196 locus on the body size traits of seven horses, as shown in Table 5. The AA and AG genotypes at the rs1136176196 locus significantly affected body length and chest girth in horses from the Bortala Mongol Autonomous Prefecture. p <0.05), with no significant effect on height, cannon bone circumference, body length percentage, chest circumference percentage, or cannon bone circumference percentage. Individuals with the AA genotype had greater average body length and chest circumference than those with the AG genotype, with average increases of 5.40% and 4.00%, respectively.

[0069] Table 5 Association analysis between rs1136176196 locus and body size trait (MEAN+SD)

[0070] Note: Different lowercase letters indicate different genotypes of the same trait at the same locus, and the differences are significant (P<0.05).

[0071] The above results show that the rs1136176196 mutation can affect the body length and chest girth traits of horses. In tall-body-length groups, the frequency of the AA genotype was 100%, significantly higher than its frequency in short-body-length groups. Furthermore, in the Bortala Mongol Autonomous Prefecture (BEA) horse population, individuals with the AA genotype had significantly larger body length and chest girth than those with the AG genotype. By genotyping this SNP marker, it is possible to predict the body length and chest girth traits of horses, allowing for early selection of horses with slender builds and large chests. SNP marker-assisted selection can also facilitate targeted breeding of trotting racehorses for 1000 / 5000 m races and improve the body length and chest girth traits of horses in the BEA region.

[0072] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0073] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0074] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the 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 invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of SNP markers related to horse body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

2. Application of reagents for detecting SNP markers associated with horse body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

3. Application of primer sets for amplifying SNP markers associated with equine body length and chest girth traits in any of (1) to (4): (1) To identify or assist in the identification of the body length or chest girth of horses; (2) To prepare products for identification or to assist in the identification of horse body length or chest girth traits; (3) Select and breed horses with slender bodies and large chests; (4) To prepare products of horses with slender body shape and large chest circumference; The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

4. The application according to any one of claims 1 to 3, characterized in that: The product includes one of the following: a reagent kit, a chip, or an instrument.

5. A method for identifying or assisting in the identification of body length or chest girth traits in horses, characterized in that, include: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. Predicting body length traits in adult individuals based on genotype: If the genotype of the SNP marker associated with horse body length in the genome of the test individual is AA, then it is predicted that the test individual will grow up to be a horse with a slender body and a large chest. If the genotype of the SNP marker associated with horse body length in the genome of the test individual is AG, then it is predicted that the test individual will grow up to be a short-bodied horse with a small chest circumference. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

6. A method for breeding racehorses, characterized in that, include: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are AG or GG genotypes, the test individual will be eliminated. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

7. A method for breeding racehorses, characterized in that, include: Genotypes of SNP markers associated with horse body length and chest girth traits were detected in the genomes of the test individuals. If the genotype of the SNP marker related to horse body length in the genome of the test individual is AA, then the test individual will be bred and managed as a reserve racehorse. If the SNP markers related to horse body length in the genome of the test individual are AG or GG genotypes, the test individual will be eliminated. The SNP marker is located at 76,788,230 bp on chromosome 15 of the equine genome, with alleles A or G and rs ID rs1136176196.

8. The method according to any one of claims 5 to 7, characterized in that: The method for detecting the genotype of linked SNP markers associated with the chest girth trait in the genome of the test individuals can be any one of whole genome sequencing, PCR, gene chip, or SNaPshot.

9. The method according to any one of claims 5 to 7, characterized in that: The subjects were foals.

10. The method according to claim 9, characterized in that: The test subjects or their parents were horses from the Bortala region.

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