Pursuit for est microsatellites in a tetraploid model from de novo transcriptome sequencing

Abstract

methodology provides abundant de novo assembled short contigs and holds great promise to mine numerous additional EST-SSR-containing markers for possible use in genetics population studies of tetraploid taxa within the genus Centaurium.

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species

“…The specificity of the microsatellite markers enabled us to analyze plant material collected directly from nature, without amplifying foreign (e.g., fungal or insect) DNA, which may give false variations when non-specific markers are used. In the previous study [36], we confirmed the transferability of the eight genic microsatellite markers, initially developed for C. erythraea , to seven other taxa, and, among them, the two studied here. Still, since EST-SSR markers are basically developed from C. erythraea transcriptome, it is not surprising that they showed higher variability in this species (Table 1) in comparison to the other two studied taxa [40,41].…”

“…To estimate genetic variation in natural populations of both parental species and the interspecific hybrid, the EST-SSR multilocus genotyping was performed and reported for the first time for the populations within the genus Centaurium . For this purpose, we employed newly designed genic microsatellite markers [36].…”

“…Primer pairs (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) for amplification of eight EST-SSR loci (M4, M5, M7, M8, M10, M12, M13 and M17), previously published by Banjanac et al [36], were employed to study the genetic variation within the sample set. PCR amplifications were conducted according to the same study as well.…”

“…Fragment analysis of PCR products was performed using the Lab-on-a-Chip technology (DNA 1000 LabChip) on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). PCR products’ peaks on electropherograms were aligned using Agilent 2100 Expert software (Agilent Technologies, Santa Clara, CA, USA) as described in Banjanac et al [36]. Alleles were scored manually.…”

“…Their interspecific hexaploid hybrids, named C. pannonicum , grow in stable populations in a few locations in the northern Serbia and were, however, found to be hexaploid [32]. With a hint that this might lead to speciation of this hybridogenic taxon, we aimed at analyzing the genetic basis of both parental taxa and the hybrid populations, implementing newly reported EST-SSR markers for the genus Centaurium [36] and analyzing the non-coding regions of the plastid DNA. As a contribution to perceiving the overall phenotypic picture of this interspecific hybridization case, which was started earlier by the phytochemical profiling [32], the divergence between the hybrid and the parental individuals is additionally investigated by a thorough analysis of the key morphological characteristics.…”