Gene polymorphisms for elucidating the genetic structure of the heavy-metal hyperaccumulating trait in Thlaspi caerulescens and their cross-genera amplification in Brassicaceae

Abstract

Genetic polymorphism was investigated in Thlaspi caerulescens J. & C. Presl at 15 gene regions, of which seven have been identified to putatively play a role in heavy-metal tolerance or hyperaccumulation. Single nucleotide and length polymorphisms were assessed at four cleaved amplified polymorphic sequences (CAPS) and 11 simple sequence repeat (microsatellite) loci, respectively. The utility of these loci for genetic studies in T. caerulescens was measured among seven natural populations (135 individuals). Fourteen loci rendered polymorphism, and the number of alleles per locus varied from 2 to 5 and 1 to 27 for CAPS and microsatellites, respectively. Up to 12 alleles per locus were detected in a population. The global observed heterozygosity per population varied between 0.01 and 0.31. Additionally, cross-species/genera amplification of loci was investigated on eight other Brassicaceae (five individuals per population). Overall, 70% of the cross-species/genera amplifications were successful, and among them, more than 40% provided intraspecific polymorphisms within a single population. This indicates that such markers may, as well, allow comparative population genetic or mapping studies between and within several Brassicaceae, particularly for genes involved in traits such as heavy-metal tolerance and/or hyperaccumulation.

The use of comparative genome analysis and syntenic relationships allows extrapolating the position of Zn tolerance QTL regions from Arabidopsis halleri into Arabidopsis thaliana

“…In particular, the higher density of markers in the QTL intervals provided by this work has drastically reduced the length of the QTL two-LOD support intervals from 24, 4 and 13 cM ) to 5, 3 and 12 cM for Zntol-1, Zntol-2 and Zntol-3, respectively. Moreover, most of these primers can probably be used to amplify other Brassicaceae species, as shown by Basic and Besnard (2006), who reported that, using primers anchored on gene coding sequences, 70% of cross-species PCR amplification between species representative of Brassicaceae was successful. Consequently, the primers designed in this study can be further used as a source of transferable gene markers for comparative mapping analyses among species in the Brassicaceae family.…”

“…Most of the primers amplifying these newly defined markers were designed in exons (maximizing cross-species amplification of the markers), to amplify fragments displaying a size between 600 and 850 base pairs (bp) and to scan for polymorphism between species, within at least one intron. The sequences of the primers Tc-up3, Tc-up2, Ah-IRT2 were designed in Thlaspi caerulescens by Basic and Besnard (2006). To obtain labelled PCR products detectable on the automated genotyper Li-Cor 4200 (Li-Cor-ScienceTec), the forward and reverse primer contained a 5′-tail of 19 bp (forward primer) or 20 bp (reverse primer) homologous to the universal consensus M13 primer sequence, followed by the locus-specific sequence (Oetting et al 1995).…”

“…a Primers were designed in Basic and Besnard (2006 Increasing the density of the linkage map, and QTL analysis…”

Plastid DNA variation in Prunus serotina var. serotina (Rosaceae), a North American tree invading Europe

“…The last cycle was followed by a 10-min extension at 72°C. Electrophoresis procedures were used as described by Basic and Besnard (2006).…”

“…Digestion of the PCR fragments was performed using the restriction enzyme TasI (Fermentas, St Leon-Rot, Germany) according to the manufacturer's recommendations. Electrophoresis procedures were used as described by Basic and Besnard (2006).…”