Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis.

The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)-based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col-0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single-tube/single-capillary format. In addition, for 110 framework markers matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col-0 and C24. Subsets of 10-62 of the established markers discriminate between various combinations of the accessions Col-0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col-0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) x Col-0 cross.

Intra- and interspecific molecular polymorphism of thrips species.

Molecular polymorphism of six species of Thysanoptera of both sexes, collected from different locations and host plants in Hungary was studied by using RAPD-PCR technique. The specimens were classified according to sampling sites (Gödöllö, Nagykovácsi and Valkó), host plants (Lathyrus tuberosus, Medicago sativa, Taraxacum officinale, Trifolium pratense), sexes, and larvae in case of Aeolothrips intermedius. On the basis of the total of 103 fragments generated by 15 RAPD primers the genetic distances were calculated by cluster analysis using simple matching method. The dendrogram resulted in two main groups: Aeolothripidae (Aeolothrips intermedius) and Thripidae (Frankliniella intonsa, Kakothrips robustus, Odontothrips confusus, Thrips dilatatus and T. tabaci). Within the family Thripidae two subgroups were observed including (i) F. intonsa, T. dilatatus and T. tabaci, and (ii) K. robustus and O. confusus. Two population-specific and one sex-linked fragments were identified by the RAPD primers, OPQ 14, NO11 and OPA08, respectively.

Evaluation of genetic diversity of poplar genotypes by RAPD and AP-PCR analysis.

RAPD (randomly amplified polymorphic DNA) and AP-PCR (arbitrarily primed PCR) were utilized to establish the genetic diversity of 19 Populus genotypes. A set of 40 primers of random sequence was tested, of which 35 exhibited polymorphism. Eighteen primers generated 162 easily detectable bands between 250 and 2,500 base pairs in size, sufficient to distinguish between the genotypes. Similarity measures, cluster and multidimensional scaling analysis were performed to evaluate the RAPD and AP-PCR data. Our study demonstrated that in most instances similarity in the RAPD and AP-PCR banding patterns reflected the relationship due to origin. Nineteen primers gave a species or hybrid-specific pattern. One primer generated a specific pattern in P. euramericana. Ten primers produced specific fragments in VIF (P. alba), 4 primers in KOR (P. pyramidialis x P. berolinensis) and 4 primers in UNA and RAS (P. trichocarpa x P. deltoides). The results of this study demonstrated that RAPD or AP-PCR can be used to distinguish between poplar genotypes.