Development and multiplexing of microsatellite markers using pyrosequencing in the clonal plant Comarum palustre (Rosaceae).

Microsatellites represent one of the most commonly used genetic markers for population genetic studies. Traditionally, their development is quite time consuming, requiring construction of a genomic library enriched for repeated motifs. Using pyrosequencing, a fast and cost-effective new generation sequencing technique, we produced 24,340,862 bases in 63,860 short fragment reads, including 1170 dinucleotide motifs with a minimum of six repeats and 1383 trinucleotide motifs with a minimum of four repeats for the Marsh Cinquefoil, Comarum palustre L., an endangered marsh pioneer species. We selected 58 loci with SSR (Short Sequence Repeat) segments (at least 10 repeats) for a preliminary screening. Out of them, we screened 29 loci on a capillary sequencer after ligation in a vector and PCR using T7 forward primer labelled with FAM fluorescent dye and the specific unlabeled reverse primers. This procedure allowed us to screen large number of candidate loci with the same labelled primer and unlabelled specific primers. Finally, we characterized 20 polymorphic microsatellite markers, nine dinucleotides and 11 trinucleotides. We used these markers to assess genetic diversity and clonal structure in two Belgian populations. All loci showed a maximum of two alleles per individual, suggesting that they are from a diploid genome. One genet was detected in a newly extending population while 53 different genets in a long-term ecologically managed population. The number of alleles per locus ranged from 6 to 14 in this old population with an expected heterozygosity, ranging from 0.5964 to 0.8278. These preliminary results show a genet size up to 7.2 m.

Consequences of near and far between-population crosses for offspring fitness in a rare herb.

Crosses between plants from different populations may result in heterosis or outbreeding depression. However, despite its importance for conservation, little is known about the spatial scale over which these effects may arise. To investigate the consequences of between-population crosses at two distinct spatial scales, we conducted reciprocal crosses between four populations from two regions in the rare perennial herb Aster amellus. We assessed seed set and offspring fitness in a common garden experiment. Overall, between-population crosses within regions (10 km) resulted in 8% lower seed set than within-population crosses, while between-region crosses (70 km) resulted in 17% higher seed set than within-population crosses. Moreover, offspring from between-population crosses produced 18% more flower heads than offspring from within-population crosses. We conclude that hybridisation between A. amellus plants from different populations did not lead to immediate outbreeding depression and, thus, could represent a valid conservation option to increase genetic diversity. Moreover, our results suggest that the distance between populations affects the outputs of between-population crosses and therefore needs to be taken into account when promoting gene flow between populations.