Indiscriminate genetic manipulation to improve athletic ability is a major threat human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited ensure fairness. Therefore, development of detect indiscriminate are urgently needed. Here, we developed highly sensitive method horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, EPO transgene was cloned into plasmid for...

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports horseracing industries. Effective methods capable of detecting monitoring doping are urgently needed. Although several PCR-based that detect transgenes have been developed, many them focus only on single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed detection method targets multiple transgenes. We targeted 12 performance...

Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness digital real-time using primers probes that matched (P-true) or incompletely (P-false) template DNA. Fluorescence intensity was significantly reduced when substituted were used compared probe both assays. Digital...

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates two distinct ways heterochromatin. First, Pob3 mediates onto heterochromatin through dimerization tandem PH domains. Without Pob3, partially reduced, exhibiting a silencing defect impaired H2A/H2B organization. Second, protein 1 (HP1)/Swi6 by physical interaction of Swi6 chromo-shadow domain (CSD)...

To ensure fair competition and sports integrity, gene doping is prohibited in horseracing equine sports. One method by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable multiplex detection. In this proof-of-concept study, we a highly sensitive using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) polymerase chain...

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin is not reduced, and a predisposition to melanoma. causal mutation was initially reported as duplication 4.6 kb intronic sequence Syntaxin 17. speed greying varies considerably among Grey horses. Here we demonstrate the presence two different alleles, G2 carrying tandem copies duplicated G3 three. latter by far most common allele, probably due strong selection for striking white phenotype. Our results...

The Thoroughbred breed was formed by crossing Oriental horse breeds and British native horses is currently used in horseracing worldwide. In this study, we constructed a single-nucleotide variant (SNV) database using data from 101 racehorses. Whole genome sequencing (WGS) revealed 11,570,312 602,756 SNVs autosomal (1-31) X chromosomes, respectively, yielding total of 12,173,068 SNVs. About 6.9% identified were rare variants observed only one allele horses. number detected individual ranged...

Considering the personality traits of racehorses (e.g., flightiness, anxiety, and affability) is considered essential to improve training efficiency decrease accident frequency, especially when retraining for a second career that may involve contact with inexperienced personnel after retiring from racing. Studies on human personality-related genes are frequently conducted; however, such studies rare in horses because consistent methodology evaluation lacking. Using recently published whole...

Gene doping is prohibited for fair competition in human and horse sports. One style of gene the administration an exogeneous gene, called a transgene, to postnatal humans horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR digital PCR, have been recently developed, it remains difficult reliably detect low-copy transgenes. In this study, we developed validated nested method specifically The consists (1)...

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect a method utilizing quantitative polymerase chain reaction (qPCR) with hydrolysis probe was developed to test whole blood plasma samples, thereby protecting fairness competition rights stakeholders sports. Therefore, we aimed develop sample storage methods suitable for A B samples gene doping tests using...

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene administer exogenous genetic materials, called transgenes, into the bodies postnatal humans horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR real-time have been developed for testing However, significance inhibitors has not well evaluated. In this study, we evaluated effects on using against doping. Digital amplification was significantly inhibited...

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted the genome. In this study, we identified 62 processed pseudogene candidates intron-less genes from whole-genome sequencing (WGS) data Thoroughbred horses using delly structural variation software. The were confirmed by PCR amplification products. A total 11 pseudogenes in genome all 23 analysed horses, whereas three with structures ATP11B, DPH3 RPL17...

Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction domestic animals but also livestock production by enhancement of desirable traits. The existence the technology, however, leaves it open to misuse performance-led sports such as horseracing other equestrian events. Recent advances equine gene editing, regarding generation gene-edited embryos using CRISPR/Cas9 technology somatic cell nuclear transfer, have highlighted need develop tools...

Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene genome editing detection methods based on whole resequencing (WGR) using genomic DNA extracted from blood. this study, aimed to develop a WGR method extracts hair roots. Hair roots are preferred substrate because their collection less invasive than blood collection. also easier store for long periods of time. Although almost all root samples stored years at room...

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner gene doping. In this study, we developed a test to detect editing based on amplicon sequencing using next-generation (NGS). We designed 1012 amplicons target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% targeted exons were sequenced sufficient coverage (depth > 50) for calling variants. targets artificial defined homozygous alternative...

Thoroughbreds are some of the most famous racehorses worldwide and currently animals high economic value. To understand genomic variability in Thoroughbreds, we identified genome-wide insertions deletions (INDELs) obtained their allele frequencies this study. INDELs were from whole-genome sequencing data 101 Thoroughbred by mapping sequence reads to horse reference genome. By integrating individual data, 1,453,349 113,047 autosomal (1-31) X chromosomes, respectively, while 18 on...

Summary Gene doping is prohibited in horseracing. In a previous study, we developed method for non‐targeted transgene detection using DELLY, which based on split‐read (SR) and paired‐end (PE) algorithms to detect structural variants, WGS data. this validated the sensitivity of DELLY artificially generated sequence data 12 target genes. With at least one intron was detected as deletion eight targeted genes 150 bp PE read data, whereas all were by 100 The higher reads than reads, despite lower...

In human and equestrian sporting events, one method of gene doping is the illegal use therapeutic oligonucleotides to alter expression. this study, we aimed identify via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model oligonucleotides, 22 bp-long phosphorothioated (PSOs) were used. By Clarity OTX kit for extracting short-length spectrum singly charged PSO with mean intensity 6.08 × 104 (standard deviation: 4.34 103 )...

Abstract The Greying with age phenotype involves loss of hair pigmentation whereas skin is not reduced and a predisposition to melanoma. causal mutation was initially reported as duplication 4.6 kb intronic sequence in Syntaxin 17 . speed greying varies considerably among Grey horses. Here we demonstrate the presence two different alleles, G2 carrying tandem copies duplicated G3 three. latter by far most common allele, probably due strong selection for striking white phenotype. Our results...

One method of gene doping in horseracing is administering exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs reference materials (RMs) and/or positive template controls (PTCs) are necessary simultaneous analysis multiple transgene targets. In this study, we designed a RM simultaneously targets via microfluidic quantitative PCR...

We evaluated the utility of single nucleotide polymorphism (SNP) markers for parentage testing in Breton (BR) and Percheron (PR) horses Japan using proposed International Society Animal Genetics (P-ISAG) 147 SNP panel 414 autosomal SNPs. Genomic DNA was extracted from 98 two breeds, BR (n = 47) PR 51), sequenced next-generation sequencing. The average minor allele frequencies P-ISAG were 0.306 0.301, respectively. combined probabilities exclusion (PEs) given parents one offspring: exclude a...

Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations wild domestic animals. In horse racing industries, they extremely identifying registering individuals doping control to ensure fair competition. this study, we constructed an individual panel horses by using insertion deletion (INDEL) markers. The included 39 INDEL markers selected from a whole-genome database. Genotyping 89 Thoroughbreds showed polymorphisms with...