A new 15 autosomal STRs multiplex for domestic horse genotyping

Builes, J.J.; Castro, Jaime; Velilla, C.; Bastidas, Ledesma; Afanador, C.H.; Manrique, A.; Aguirre, D.; Mendoza, L.; Bravo, M.L.J.

doi:10.1016/j.fsigss.2013.10.049

Cited by 2 publications

(4 citation statements)

References 4 publications

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“…In the past few years, many horse STR multiplex typing systems have been established , however, none of these systems were specifically developed for donkeys. Development of a multiplex STR typing system for donkeys is essential for the identification of individual donkeys, paternity testing and genetic analysis of populations.…”

Section: Discussionmentioning

confidence: 99%

“…Variation in the lengths of STRs can be detected using multiplex polymerase chain reaction (PCR) with fluorescently labelled primers that efficiently amplify polymorphic STRs. Multiplex STR typing systems have been established to identify individuals, and for paternity testing, in various mammals including horse , dog , boar , wolf , tiger and deer .…”

Section: Introductionmentioning

confidence: 99%

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A novel 13‐plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus)

Dang

Shang

Zhang

et al. 2019

Equine Veterinary Journal

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Summary Background Previous studies investigating donkey parentage and genetic diversity used horse‐specific multiplex systems. However, several mis‐allele and null‐allele issues were found with some of the horse primers when used in donkeys. In 2017, the International Society for Animal Genetics (ISAG) recommended 13 dinucleotide short tandem repeats (STRs) (AHT4, ASB23, HMS2, HMS3, HMS6, HMS7, HMS18, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343) as a core panel that should be used to identify individuals and to test for parentage in donkeys. To date, no single multiplex STR typing system containing all 13 donkey STRs recommended by the ISAG has been reported. Objectives To establish a novel and donkey‐specific multiplex STR typing system containing all 13 recommended STRs. Study design Assay development and validation in field population. Methods Primers for seven of the STRs were redesigned and conditions for polymerase chain reaction (PCR) were optimised. We analysed the allele sequences, sensitivity, species‐specificity and stutter ratios of this new system. Results A 13‐plex STR typing system for donkey was established. A full profile could be generated from a single PCR reaction using as little as 5 ng of DNA template with the 13 pairs of primers labelled with fluorescent dyes. An allele ladder, containing 101 alleles from the 13 STRs, was generated. No full genotype profile was generated with these primers if DNA from humans, or 11 other commonly encountered animals, was used. Genotypes could be generated for the horse and horse‐donkey hybrids (mule and hinny). Stutter ratios and population genetic parameters were calculated based on samples from 150 donkeys. The combined probabilities of paternity exclusion for this system were 0.988907326 (CPEduo) and 0.999665018 (CPEtrio). Main limitations This system cannot detect sex. Conclusions Our results indicate that our donkey‐specific 13‐plex STR typing system is sensitive, species‐specific and robust for individual identification, paternity testing and population genetic analysis in donkeys, and has potential forensic applications.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A novel 13‐plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus)

Dang

Shang

Zhang

et al. 2019

Equine Veterinary Journal

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“…The efficiency of STR genotyping can be greatly enhanced when applying a multiplex polymerase chain reaction (multiplex PCR) with fluorescent dye-labelled primers. STR multiplex PCR systems have been established in some animal species, including the dog [16][17][18], hen harrier [19], pig [20] and horse [21]. In 2003, Pero Dimsoski developed a 17-plex STR PCR kit for horses that contains the 12 most commonly used loci mentioned above [22].…”

Section: Introductionmentioning

confidence: 99%

“…However, this test did not contain the four most commonly used loci (AHT5, ASB17, ASB23 and HTG10), possibly due to unsatisfactory genotyping results for these loci [23]. One other horse 15-plex PCR system, that includes 15 new tetra-nucleotide STR loci, was developed but has not been widely used in practice [21]. In previous studies, the relatively high expense of PCR reagents or kits (Applied Biosystems AmpliTaq Gold Polymerase, Qiagen Multiplex PCR Kit, or Qiagen HotStar Taq Master Mix) has limited the growth of multiplex PCR applications for horse STRs, and none of them included a sex chromosome STR to determine horse gender.…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a novel 13‐plex PCR system for commonly used short tandem repeats in horses (Equus caballus)

Shang

Zhang

Zhao

et al. 2018

Equine Veterinary Journal

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Summary Background Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time. Objectives We aimed to develop a novel 13‐plex STR typing system to resolve the above issues. Study design Experimental study. Methods Twelve autosomal and most commonly used di‐nucleotide STRs (AHT4, AHT5, ASB2, ASB17, ASB23, HMS2, HMS3, HMS6, HMS7, HTG4, HTG10 and VHL20), and a Y‐chromosomal STR (YJ10) were included. We redesigned the primers of eight STRs to establish a novel multiplex PCR system and tested this system for species specificity, sensitivity and repeatability. Results Full profiles were easily generated in one fast PCR reaction using a low‐cost polymerase, as little as 1 ng of horse DNA template and 13 pairs of primers labelled with fluorescent dyes. No full profile was generated from DNA templates of humans or other commonly encountered animals. We also established an allelic ladder that contained 110 alleles based on 200 horses from 12 breeds and calculated standard population genetic parameters based on 150 Thoroughbreds. Stutter analysis showed that the averages of the stutter ratios were distinctly lower than those of lower allele ratios and the combined probability of paternity exclusion for this system were 0.994659935 (CPEduo) and 0.999854032 (CPEtrio). Main limitations A nonspecific and relatively low peak at 316 bp was frequently observed in locus HMS2, which is a nonexistent allele in all horses and should be ignored. Conclusions Our results indicate that this 13‐plex STR genotyping system is sensitive, species‐specific, cost‐effective and robust for applications in the horse industry and forensic investigation.

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A new 15 autosomal STRs multiplex for domestic horse genotyping

Cited by 2 publications

References 4 publications

A novel 13‐plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus)

A novel 13‐plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus)

Development and validation of a novel 13‐plex PCR system for commonly used short tandem repeats in horses (Equus caballus)

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