Genetic Association of MSTN Gene Variant (18:66493737T>C) with Track Performance & Muscle Development in Pakistani Horses

The horse, revered for its diverse traits including racing prowess, gaitedness, and distinctive behavior, plays a pivotal role in various sports. Numerous studies have linked racing performance in horses to the MSTN gene across global populations. Objectives: To investigate the genetic variability of the 18:66493737T>C variant in Pakistani random-bred horses. Methods: ARMS-PCR was employed where 24 horses sourced from UVAS equine clinic were genotyped. Results: Revealing a population distribution of 54% homozygous wild-type (TT), 41% heterozygous (TC), and 4% homozygous mutant (CC) at this locus. The alternative allele frequency within elite performers and control horses stood at 0.36 and 0.12, respectively. Application of the Chi-Square association test using the PLINK data toolset yielded a highly -6 signi�cant p-value of 7.832×10 . Conclusions: This underscores signi�cant genetic variability at the locus in the Pakistani horse population, aligning with global patterns. Future studies are advocated, incorporating racing performance data and encompassing diverse indigenous horse breeds with substantial sample sizes. Identi�cation of subject markers can inform targeted breeding strategies, contributing to the enhancement and preservation of desirable traits across various horse breeds.

differentiation factor 8) is encoded by MSTN gene.This protein belongs to the TGF-β (transforming growth factor beta) family of growth and differentiation factors [4,5].This protein restricts the development and growth of muscle bers [6].In recent studies, researchers found that the MSTN is linked to racing performance, growth of skeletal muscle, and carcass quality traits in different species including livestock animals, dogs, and horses [7].Moreover, the Myostatin gene is a major determinant of racing performance because it also in uences the proportion of fast twitch and slow twitch muscle bers in

I N T R O D U C T I O N
Horse (Equus caballus) racing is one of the ancient sports that has gained popularity with time in the modern era [1].It has developed as a public recreation to utilize their leisure time.Moreover, horses are also used in different sports like polo, tent pegging, etc.This is all because of the horse's phenotypic traits of athleticism.This trait in horses is due to the size and development of the skeletal muscular structure.Both genetic and environmental factors may in uence racing traits in horses [2].The horse's speed performance is genetically associated with the MSTN gene [3].Myostatin protein, also known as GDF-8 (Growth Volume 4, Issue 1 (Jan-Mar 2024) horses [5,8,9].The MSTN gene in Equus caballus is located on chromosome 18, accession ID NM_001081817.1 in assembly EquCab2.0 (GCF_000002305.2).It contains three exons and two intronic regions with a combined length of 4979 nucleotides.In horses, single nucleotide p o l y m o r p h i s m ( S NP ) a t r e g i o n 1 8 : 6 6 4 9 3 7 3 7 T > C (rs397152648) had been identi ed as a genetic marker to analyze the racing performance [10].From the previous genetic studies, the wild-type genotype (T/T) is linked to horse's stamina and muscle development, heterozygous (T/C) genotype horses give better performance in middle distance races whereas homozygous mutant type (C/C) horses are top performers, fast-paced and short-distance sprinters [5,11].Furthermore, the studies also suggested that fast twitch glycolytic, type II-B muscle bers provide short bursts of power in horses.Hence, the C/C horses have best racing performance and to be considered as sprinters.Whereas, in T/T horses the proportion slow twitch oxidative, type I muscle bers are present which are required for showing better stamina performances [12].The remaining horses with T/C genotype have both the muscle bers in their body which shows mild racing performance [13].The objective of current study was to investigate the MSTN gene variants associated with the racing performance among random-bred Pakistani horse population.

Sample Collection and DNA Extraction
A heterogeneous horse breeds were chosen for investigating the association between the g.66493737T>C variant and its potential impact on racing performance and muscle development traits within the local equine population.Blood samples were meticulously obtained from 24 horses, with a distribution of 10 best performers and 14 controls.The sample of top performers was obtained from owners whose horses have consistently maintained top positions in past races, ensuring a representation of elite racing genetics within the study cohort.Collection was performed via jugular vein puncture using EDTA vacutainers, facilitated through consultations with horse attendants or owners.The collected samples were promptly stored at 4°C for subsequent analysis.Genomic DNA extraction from the horse blood samples was conducted using the GDSbio genomic DNA extraction kit, ensuring accuracy and reliability in the genetic analysis process.

M E T H O D S
Tm = Melting Temperature; bp = base pairs Subject Variant Ampli cation Through ARMS-PCR The SimpliAmp thermal cycler (Applied Biosystems) was utilized to amplify both wild and mutant type variants.Each sample underwent two separate PCR reactions, with each reaction employing either the normal or mutant type ARMS reverse primer in combination with the common forward primer.Concurrently, an internal control region was ampli ed using a standard set of primers from an adjacent locus in each tube.A reaction mixture totaling 25µL was prepared, comprising 1µL of 50ng extracted genomic DNA, 1µL of 10.0 mM concentration of each primer (N or M reverse primers, forward common primer, forward & reverse IC), 1 unit of Taq polymerase, 2.5mM MgCl2, 2.5mM dNTPs, 1X Taq buffer, and PCR grade water.The PCR protocol consisted of primer, along with reverse primers speci c to the normal allele (N) and the mutant allele (M).These primers were designed from the 3' -end to facilitate the ampli cation of both wild-type and mutant variants of the targeted sequence.To enhance the speci city and e ciency of the ARMS-PCR assay, a secondary mismatch was deliberately introduced at the 4th nucleotide position from the 3' end of the reverse primers designed for both wild-type and mutant alleles.Additionally, we designed two supple mentary primers, one forward and one reverse, to amplify a region adjacent to our targeted sequence.This region served as an internal control (IC) to validate the accuracy and reliability of the ampli cation process (table 1).

Statistical Association
Genotyping of g.66493737T>C locus was analyzed on our collected horse samples followed by Chi-Square association testing using PLINK data analysis toolset for genotypic and allelic frequencies along with their p-value calculation.

Variant Genotyping
A total of 24 horses were sampled, which includes (best performers controls n=14) Genotypic variability of g.66493737T>C variants was analyzed in Pakistani horse population.The ARMS-PCR results showed signi cant genotypic variability of g.66493737T>C locus which is associates with racing performance of horses as described in different studies [5].Out of total 24 horses, 13 possess T/T homozygous wild genotype which may be considered to be the best for long-distance racing (stayer category), 10 horses showed T/C heterozygous genotype which may be considered as the best performers in middle-distance races (up to 8-12 furlongs) and only one horse had C/C homozygous mutant genotype which may be considered as the best performer in short distance races (sprinters category).As C/C genotype down regulates the expression of MSTN which increase the growth of muscle bers.Consequently, more body-to mass ratio leads to better racing performance.Sampled horses are shown in figure 2B.

Association Analysis
The statistical association along with both genotypic and allelic frequencies were calculated.The allele frequency distribution showed that the T/T homozygous wild genotype is common among Pakistani random-bred horses.Our screening results showed 54.16% of horse population were homozygous wild (T/T), while 41.66% were heterozygous (T/C) and the remaining were homozygous mutant with (C/C) genotype which was from the from top performer.Furthermore, ARMS-PCR results showed that "T" allele was more frequent than "C".The Chi-statistics was applied using PLINK data analysis toolset to calculate the p-value which was 7.832×10-6.
o an initial denaturation step at 95 C for 5 minutes, followed o by 30 cycles of denaturation at 95 C for 30 seconds, o o annealing at 60 C for 30 seconds, and extension at 72 C for 30 seconds.Subsequently, nal extension step was o conducted at 72 C for 10 minutes ( gure 1).

Figure 1 :
Figure 1 : Thermal Cyclic Conditions of ARMS-PCR

Figure 2 :
Figure 2: (A) ARMS-PCR ampli cation of targeted locus with laboratory case number from 1-21 and genotype, N and M represent ampli cation result of normal and mutant allele, L represent the Thermo Scienti c GeneRuler 1 kb DNA Ladder, with internal control and ampli ed allele at 700bp and 300bp respectively (B) few sampled horses

Table 1 :
Primers Sequences and its Attributes

Table 2 :
Association Analysis of MSTN (rs397152648) in Pakistani Horse Population

Saif R et al., DOI:https://doi.org/10.54393/fbt.v4i01.89
66493737T>C signi cantly in uenced sport traits, suggesting its potential use in selection for improved sport performance in this breed[17].Another study explored MSTN gene variants in Polish horse breeds and their association with height at the withers in Arabians.Five SNPs, including g.66493737T>C, were identi ed, with signi cant effects of g.66495696T>C on height observed in Arabian foals.These ndings underscore the relevance of MSTN polymorphisms for morphological traits in Arabian Genetic Association of MSTN Gene Variant in Horses