一种新型的 13 重 STR 分型系统，用于驴（Equus asinus）的个体识别和亲子关系鉴定。

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

机构信息

Institute of Equine Sciences, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.

Liaoning Province Engineering Center of Modern Agricultural Production Base, Shenyang, China.

出版信息

Equine Vet J. 2020 Mar;52(2):290-297. doi: 10.1111/evj.13158. Epub 2019 Sep 5.

DOI:10.1111/evj.13158

PMID:31398262

Abstract

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.

摘要

背景

之前研究驴亲缘关系和遗传多样性使用的是马专用多重体系。然而，当在驴中使用一些马引物时，发现了一些等位基因错配和缺失等位基因的问题。2017 年，国际动物遗传学会（ISAG）推荐了 13 个二核苷酸短串联重复序列（STRs）（AHT4、ASB23、HMS2、HMS3、HMS6、HMS7、HMS18、HTG7、HTG10、TKY297、TKY312、TKY337 和 TKY343）作为核心面板，用于鉴定个体和检测驴的亲子关系。迄今为止，还没有报道过包含 ISAG 推荐的所有 13 个驴 STR 的单一多重 STR 分型系统。

目的

建立一个新的、专门针对驴的包含所有 13 个推荐 STR 的多重 STR 分型系统。

研究设计

在野外种群中进行检测和验证。

方法

重新设计了其中 7 个 STR 的引物，并优化了聚合酶链反应（PCR）的条件。我们分析了这个新系统的等位基因序列、灵敏度、物种特异性和 stutter 比率。

结果

建立了一个 13 重 STR 分型系统用于驴。使用少至 5ng 的 DNA 模板，通过 13 对标记有荧光染料的引物进行单次 PCR 反应，即可生成完整的图谱。生成了一个包含 13 个 STR 的 101 个等位基因的等位基因梯。如果使用人类或 11 种其他常见动物的 DNA，则不会生成这些引物的完整基因型图谱。可以为马和马驴杂种（骡子和混血儿）生成基因型。根据 150 头驴的样本计算了 stutter 比率和群体遗传参数。该系统的双亲排除概率为 0.988907326（CPEduo）和 0.999665018（CPEtrio）。