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Development of the first standardised panel of two new microsatellite multiplex PCR s for gilthead seabream ( Sparus aurata L.)
Author(s) -
LeeMontero I.,
Navarro A.,
Borrell Y.,
GarcíaCeldrán M.,
Martín N.,
NegrínBáez D.,
Blanco G.,
Armero E.,
Berbel C.,
Zamorano M. J.,
Sánchez J. J.,
Estévez A.,
Ramis G.,
Manchado M.,
Afonso J. M.
Publication year - 2013
Publication title -
animal genetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.756
H-Index - 81
eISSN - 1365-2052
pISSN - 0268-9146
DOI - 10.1111/age.12037
Subject(s) - biology , multiplex , microsatellite , multiplex polymerase chain reaction , fishery , genetics , polymerase chain reaction , gene , allele
Summary The high number of multiplex PCR s 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 PCR s 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 PCR s from the best assessed markers were designed with 11 markers in each, named SM sa1 and SM sa2 (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 SM sa1 and/or SM sa2. 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‐ SM sa1 and kit‐ SM sa2). 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.