ABSTRACT
The first hurdle in developing microsatellite markers, cloning, has been overcome by next-generation sequencing. The second hurdle is testing to differentiate polymorphic from nonpolymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still performed manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Of 48 tested two markers failed to amplify, but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multilocus markers largely overlapped. Of the remainder, half were replicate markers (i.e. multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6-20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy therefore represents a major step forward in the development of highly polymorphic microsatellite markers.
Subject(s)
Computational Biology/methods , Genetic Variation , Genotyping Techniques/methods , Microsatellite Repeats , Repetitive Sequences, Nucleic Acid , Transcriptome , Molecular Sequence Data , Rosa/classification , Rosa/genetics , Sequence Analysis, DNAABSTRACT
Ten polymorphic microsatellite markers have been developed for Gonystylus bancanus (Ramin), a protected tree species of peat swamp forests in Malaysia and Indonesia. Eight markers were also shown to be polymorphic in other Gonystylus species. The markers will enable assessing the amount of genetic variation within and among populations and the degree of population differentiation, such that donor populations can be selected for reforestation projects. They may be used for tracing and tracking of wood in the production chain, so that legal trade in this Convention on International Trade in Endangered Species of Wild Fauna and Flora-protected timber species, derived from specifically described origins, can be distinguished from illegally logged timber.
