Grupo de Investigación en Acuicultura, Instituto Universitario de Sanidad Animal y Seguridad Alimentaria, Instituto Canario de Ciencias Marinas, Universidad de Las Palmas de Gran Canaria, Carretera Trasmontaña s/n, Arucas, Las Palmas, Spain.
Anim Genet. 2013 Aug;44(5):533-46. doi: 10.1111/age.12037. Epub 2013 Apr 10.
The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.
为了满足所有用户安全可靠使用的需求，迫切需要开发一套可重复使用的标记物。为此，本研究开发了首个适用于真鲷的标准化双微卫星多重 PCR 标记物panel。根据遗传连锁图谱中 138 个特异性微卫星的遗传变异性、等位基因大小范围和基因分型可靠性，对其引物进行重新设计和评估。提出了一种鉴定和分类基因分型错误或潜在错误的方案，以评估每个标记的可靠性。根据评估结果，从最佳标记中选择 11 个设计了两个新的多重 PCR，分别命名为 SMsa1 和 SMsa2（SuperMultiplex Sparus aurata）。本研究使用了来自三个西班牙不同公司的三个亲鱼群体（59、47 和 98 个繁殖者）和每个群体的 80 个后代样本，对这些多重 PCR 在亲鱼鉴定中的应用进行了验证。使用 SMsa1 和/或 SMsa2 的排除法，可以将每个后代分配给一对亲鱼（成功率 100%）。在每个基因分型样本中，都使用了参考样本（Ref-sa），其 DNA 可根据请求提供，类似于 genemapper（v.3.7）软件的 bin set 基因分型试剂盒（kit-SMsa1 和 kit-SMsa2）。本研究开发的标准化多重 PCR 标记物 panel 将成为分析系谱或鉴定群体的有力而有效的工具，并将被提议作为该物种的国际标记物 panel。
