Primer-enzyme Combinations Research Articles

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) I. Wood Density

Abstract The identification of AFLP markers and their subsequent conversion to SCAR-markers linked to wood density of Norway Spruce (Picea abies L [Karst.]) is described for the first time. In AFLP-analyses, 102 different primer enzyme combinations were screened in a bulked segregant approach comparing individuals with high and low wood density. A total of 107 polymorphic AFLP fragments were obtained between the DNA-pools. Twenty-three markers were selected for further analyses to verify their linkage to wood density based on individuals used for pool constitution and additional unrelated clonal material. For 15 markers, a significant linkage to wood density was confirmed by a two-sided Fisher’s-exact test. Four markers were converted into SCAR markers and validated for plant material assayed for wood density by X-ray microdensitometry. For each marker a monomorphic band was obtained using sets of nested primers or restriction site-specific primers (RSS), which include the AFLP-restriction recognition sites. For two markers that are linked to high wood density, a separation from unlinked size homologous marker-alleles was realized by a PCR-restriction approach. Validation of these markers in different full-sib families confirmed their usability to separate the classes for low and high wood density of Picea abies.

Molecular characterisation of grapevine yellows associated phytoplasmas of the stolbur-group based on RFLP-analysis of non-ribosomal DNA

Grapevines, alternative host plants, and vectors from different viticultural areas of Germany were surveyed for the presence of stolbur-group phytoplasmas that are associated with grapevine yellows. Isolates from these field samples and periwinkle isolates of stolbur were characterised by RFLP-analysis of non-ribosomal DNA fragments using a combination of 6 polymorphic primer-enzyme combinations. Three of the 5 different restriction profiles that could be distinguished were found in grapevine and other field samples of plants and insects. A previously unknown isolate was detected in grapevine, vectors and the newly identified herbaceous host Calystegia sepium . The further analysis of the phylogenetic relationship between the 5 RFLP groups revealed a close relationship between two of the isolates associated with grapevine yellows which were also found in the two Convolvulaceae Convolvulus arvensis and Calystegia sepium . The third isolate which was detected in grapevine and stinging nettle Urtica dioica is more closely related to a periwinkle isolate obtained from Lavandula officinalis than to the other two grapevine yellows isolates. Based on the data of the field survey a specific association of the three isolates to different alternative host plants is discussed.