No abstract

The Misaki horse is a Japanese native horse, known as the “feral horse of Cape Toi”. In this study, we acquired the genetic information to establish their studbook, and analyzed their genetic characteristics for conservation. We genotyped 32 microsatellites and a mitochondrial D-loop region in 77 Misaki horses (80.2% of the population). The average number of alleles, observed heterozygosity, and expected heterozygosity were 3.4, 0.509, and 0.497, respectively. A neighbor-joining phylogenetic tree of individuals was constructed. Moreover, the results suggested that Misaki horses experienced a bottleneck, but it was neither severe nor recent. In addition, three mitochondrial haplotypes were confirmed. Consequently, we clarified the genetic background of Misaki horses that have been resident at Cape Toi for a long time.

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A routine parentage test as part of a conservation program for Kiso horses identified a possible sex chromosome anomaly in a 7 months-old filly because of an aberrant result using LEX3, an X-linked marker. We then analyzed X-linked markers (LEX26, TKY38, and TKY270), Y-linked markers (Eca.YH12, Eca.YM2, Eca.YA16, and the sex-determining region Y gene), and an X/Y marker (Amelogenin gene). This analysis demonstrated that the filly had not inherited an X chromosome from her sire. A karyotyping analysis confirmed that the filly was 63,XO. As it was suspected that the horse would be sterile, we avoided using the horse as a broodmare; the information should also serve to prevent unnecessary conflict between owners transferring and receiving the horse.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix‐assisted laser desorption/ionisation‐time‐of‐flight mass spectrometry (MALDI‐TOF MS). As a model of therapeutic oligonucleotides, 22 bp‐long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short‐length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI‐TOF MS. In addition, a 17 bp sequence was determined using in‐source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.