Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals.

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.

Development of microsatellite markers in Gonystylus bancanus (Ramin) useful for tracing and tracking of wood of this protected species.

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.

Genetic structure in populations of an ancient woodland sedge, Carex sylvatica Hudson, at a regional and local scale.

Wood sedge (Carex sylvatica) is a well-known ancient woodland species with a long-term persistent seed bank and a caespitose growth habit. All thirteen isolated Carex sylvatica populations in the Dutch Rhine floodplain (including the river branches Waal and IJssel) were mapped in detail and analysed for genetic variation at a large number of AFLP loci and one microsatellite locus. Across all populations, only 40 % of the sampled individuals (n=216) represented a unique genotype. A high number of the studied patches (spatial clusters of tussocks, 2-10 m in diameter) within populations contained only one or a few genotypes. Identical plants (tussocks) were also found 20-500 m apart and in one case even 1000 m apart. Observed heterozygosity levels (H(O)=0.029) were low, indicating low levels of gene flow, which is in agreement with the selfing nature of other caespitose sedges. Although the number of genotypes in populations is low, these genotypes are genetically very distinct and variation within populations accounted for 55% of the total variation. The absence of a correlation between genetic and geographic distances among populations, and the scattered distribution of genotypes among patches within woodlands, support our hypothesis of rare establishments and subsequent local dispersal within woodlands in this forest floor species, which may benefit from and partly depend on human land use and forest management activities.