Premium
Repetitive flanking sequences challenge microsatellite marker development: a case study in the lepidopteran Melanargia galathea
Author(s) -
Schmid Max,
Csencsics Daniela,
Gugerli Felix
Publication year - 2016
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12547
Subject(s) - biology , microsatellite , primer (cosmetics) , genetics , multiplex , evolutionary biology , allele , chemistry , organic chemistry , gene
Microsatellite DNA families ( MDF ) are stretches of DNA that share similar or identical sequences beside nuclear simple‐sequence repeat ( nSSR ) motifs, potentially causing problems during nSSR marker development. Primers positioned within MDF s can bind several times within the genome and might result in multiple banding patterns. It is therefore common practice to exclude MDF loci in the course of marker development. Here, we propose an approach to deal with multiple primer‐binding sites by purposefully positioning primers within the detected repetitive element. We developed a new protocol to determine the family type and the primer position in relation to MDF s using the software packages repark and repeatmasker together with an in‐house R script. We re‐evaluated newly developed nSSR markers for the lepidopteran Marbled White ( Melanargia galathea ) and explored the implications of our results with regard to published data sets of the butterfly Euphydryas aurinia, the grasshopper Stethophyma grossum, the conifer Pinus cembra and the crucifer Arabis alpina . For M. galathea , we show that it is not only possible to develop reliable nSSR markers for MDF loci, but even to benefit from their presence in some cases: We used one unlabelled primer, successfully binding within an MDF , for two different loci in a multiplex PCR , combining this family primer with uniquely binding and fluorescently labelled primers outside of MDF s, respectively. As MDF s are abundant in many taxa, we propose to consider these during nSSR marker development in taxa concerned. Our new approach might help in reducing the number of tested primers during nSSR marker development.