Although simple sequence repeat (SSR) markers have been developed for species in the closely related genera Fragaria L. (strawberry) and Rubus L. (raspberry and blackberry), the number of SSRs available is insufficient for genetic mapping. Our objective was to use and compare multiple approaches for developing additional SSRs for Fragaria and Rubus. The approaches included: the development of SSRs from GenBank sequences from species of varied relatedness to Fragaria and Rubus and identified with two different data-mining methods (BLAST and SSRIT); the evaluation of some previously published SSRs designed from related species; and the development of SSRs from a genomic library made from F. ×ananassa Duschene ex Rozier `Earliglow'. When an SSR was developed from a known gene sequence, the location of the repeat in the gene was determined to evaluate the effect on amplification and polymorphism detection. Cross-generic amplification between closely related Fragaria and Rubus as well as transference from species of varied relatedness to Fragaria and Rubus also was evaluated and indicated limited transference within the subfamily Rosoideae. However, development of SSRs for Fragaria and Rubus from Rosa L. (rose) and Rosaceae genera outside Rosoideae was not efficient enough to be practical for new map development. SSRIT was superior to BLAST for identifying GenBank sequences containing repeats. SSRs developed from repeats found in either the 5′UTR (80% polymorphic) or 3′UTR (85% polymorphic) were most likely to detect polymorphisms, compared with those developed from coding regions (30%). SSRs developed from the genomic library were only slightly superior to GenBank-derived SSRs in their ability to detect polymorphisms.
K.S. Lewers, S.M.N. Styan, S.C. Hokanson and N.V. Bassil
David R. Sandrock, Anita N. Azarenko, Ruth M. Martin and Nahla V. Bassil
The NRT1gene family encodes transport proteins with dual or low affinity for nitrate. The objectives of this experiment were to develop a system that could be used to compare the expression of the NRT1genes between species. This was accomplished by comparing sequences of NRT1homologues from various species and designing degenerate primers in regions of high homology. These primers were used to amplify a region of the NRT1gene from species of interest. A 635 bp PCR product was amplified from each species using the MD2-1 (5' ATGTTACCAAYWTGGGCMAC-3') and MD2-2 (5'-GCCAMWARCCARTAGAAAT-3') primers. The PCR products were cloned and sequenced. At the nucleotide level, CornussericeaL. `Kelseyi' and RhododendronL. `Unique' were 79.52% identical. Species-specific primers were designed and used for RT-PCR to compare NRT1expression in roots of hydroponically grown C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique'. The relative levels of NRT1expression, normalized using 18S rRNA as a standard, were ≈3.2 to 1.7 to 1.0 for C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique', respectively. This approach may eventually be used to examine nitrate uptake potential in different taxa of plants at different times during the growing season.