Relicts, refugia and reticulation :a study of population history, hybrids and phylogeny in the long-lived flowering tree genus Tilia

PhD ThesisTilia L. (lime or basswood) is a genus of large trees that are widely distributed in the temperate regions of the Northern Hemisphere. Tilia is an under-investigated genus with unknown species relationships. Therefore, a phylogeny of the genus was reconstructed. This revealed disagreement of the phylogenetic placement of some species and also indicated extensive hybridization. To investigate this further, the most tractable and widely distributed species across Europe, T. cordata (Mill.) or small leaved lime and T. platyphyllos (Scop.) or large leaved lime, were selected for study. This study aims to increase the understanding of genetic diversity and hybridization between the two Tilia species. Also, to gain insight into postglacial recolonization in Tilia across Europe, the patterns of population genetic structure were investigated. In order to achieve the goals, 15 microsatellite markers were developed for detailed genetic analysis. These loci clearly discriminated the two Tilia species. Cross-amplification results indicated that twelve microsatellite markers amplified polymorphic loci in 24 species in the genus. A high level of polymorphism was observed in twenty-five populations of T. cordata and 15 populations of T. platyphyllos from natural woods across Europe. The level of genetic diversity in T. platyphyllos is higher than in T. cordata. Both microsatellite and morphological analysis revealed that natural hybridisation and introgression have occurred between T. cordata and T. platyphyllos in sympatric UK populations, which could be of importance for adaptation and other evolutionary processes. The partial congruence of molecular and morphological analysis suggests that molecular markers are more reliable than morphological analysis for detecting hybridization. The stronger genetic structure observed in T. platyphyllos than in T. cordata suggested that the migration and colonization in the northern areas of T. cordata occurred before those of T. platyphyllos. Microsatellite analysis suggested different possible colonization routes between the two Tilia species. However, T. cordata and T. platyphyllos seem to share the three main refugia in southern Europe (Iberia, Italy and the Balkans). In addition, T. cordata seems to have additional putative refugia in eastern areas. The haplotype network and some shared haplotypes of eight chloroplast regions indicate incomplete lineage sorting rather than recent hybridization.The Royal Thai Government

Development of expressed sequence tag resources for Vanda Mimi Palmer and data mining for EST-SSR

Vanda Mimi Palmer (VMP) is a highly sought as fragrant orchid hybrid in Malaysia. It is economically important in cosmetic and beauty industries and also a famous potted ornamental plant. To date, no work on fragrance-related genes of vandaceous orchids has been reported from other research groups although the analysis of floral fragrance or volatiles have been extensively studied. An expressed sequence tag (EST) resource was developed for VMP principally to mine any potential fragrance-related expressed sequence tag-simple sequence repeat (EST-SSR) for future development as markers in the identification of fragrant vandaceous orchids endemic to Malaysia. Clustering, annotation and assembling of the ESTs identified 1,196 unigenes which defined 966 singletons and 230 contigs. The VMP dbEST was functionally classified by gene ontology (GO) into three groups:molecular functions (51.2%), cellular components (16.4%)and biological processes (24.6%) while the remaining 7.8% showed no hits with GO identifier. A total of 112 EST-SSR(9.4%) was mined on which at least five units of di-, tri-,tetra-, penta-, or hexa-nucleotide repeats were predicted.The di-nucleotide motif repeats appeared to be the most frequent repeats among the detected SSRs with the AT/TA types as the most abundant among the dimerics, while AAG/TTC, AGA/TCT-type were the most frequent trimerics. The mined EST-SSR is believed to be useful in the development of EST-SSR markers that is applicable in the screening and characterization of fragrance-related transcripts in closely related species

Characterization of Microsatellite Loci in <i>Tilia platyphyllos</i> (Malvaceae) and Cross-Amplification in Related Species

Premise of the study: Microsatellite markers in the genus Tilia were developed to investigate the genetic variation in T. platyphyllos and its relationship with T. cordata. Methods and Results: Fifteen microsatellite markers were developed using a microsatellite enrichment protocol. Most loci show a high level of polymorphism in two T. platyphyllos populations from France. The number of alleles ranged from one to 15, with a mean of 8.96. The mean observed and expected heterozygosities were 0.71 and 0.70, respectively. Cross-amplification results indicated that 12 out of 15 loci amplified polymorphic loci in 23 species in the genus. Conclusions: These markers will be useful tools for investigating the phylogeography and hybridization of Tilia species