Publikationen - Molekulare Signalverarbeitung

The resistance gene analogue (RGA) pic19 in maize, a candidate for sugarcane mosaic virus (SCMV) resistance gene (R gene) Scmv1, was used to screen a maize BAC library to identify homologous sequences in the maize genome and to investigate their genomic organisation. Fifteen positive BAC clones were identified and could be classified into five physically independent contigs consisting of overlapping clones. Genetic mapping clustered three contigs into the same genomic region as Scmv1 on chromosome 6S. The two remaining contigs mapped to the same region as a QTL for SCMV resistance on chromosome 1. Thus, RGAs mapping to a target region can be successfully used to identify further-linked candidate sequences. The pic19 homologous sequences of these clones revealed a sequence similarity of 94–98% on the nucleotide level. The high sequence similarity reveals potential problems for the use of RGAs as molecular markers. Their application in marker-assisted selection (MAS) and the construction of high-density genetic maps is complicated by the existence of closely linked homologues resulting in 'ghost' marker loci analogous to 'ghost' QTLs. Therefore, implementation of genomic library screening, including genetic mapping of potential homologues, seems necessary for the safe application of RGA markers in MAS and gene isolation.

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5′-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Quantitative trait loci (QTLs) and bulked segregant analyses (BSA) identified the major genes Scmv1 on chromosome 6 and Scmv2 on chromosome 3, conferring resistance against sugarcane mosaic virus (SCMV) in maize. Both chromosome regions were further enriched for SSR and AFLP markers by targeted bulked segregant analysis (tBSA) in order to identify and map only markers closely linked to either Scmv1 or Scmv2. For identification of markers closely linked to the target genes, symptomless individuals of advanced backcross generations BC5 to BC9 were employed. All AFLP markers, identified by tBSA using 400 EcoRI/MseI primer combinations, mapped within both targeted marker intervals. Fourteen SSR and six AFLP markers mapped to the Scmv1 region. Eleven SSR and 18 AFLP markers were located in the Scmv2 region. Whereas the linear order of SSR markers and the window size for the Scmv2 region fitted well with publicly available genetic maps, map distances and window size differed substantially for the Scmv1 region on chromosome 6. A possible explanation for the observed discrepancies is the presence of two closely linked resistance genes in the Scmv1 region.