RT Dissertation/Thesis
T1 Resistance gene analogues as a tool for basic and applied resistance genetics exemplified by sugarcane mosaic virus resistance in maize (Zea mays L.)
A1 Quint,Marcel
WP 2004/02/24
AB With the recent cloning of a number of plant disease resistance genes (R genes) it became apparent that R genes share certain homologies in conserved amino acid domains. PCR amplification of genomic DNA using degenerate primers on the basis of these conserved amino acid domains identified sequences with homologies to plant disease R genes - resistance gene analogues (RGAs). RGAs exist in large numbers in plant genomes and provide new possibilities for the investigation of resistance genetics in general and also for the analysis of certain plant disease resistances. 
   	The overall objective of this thesis was to evaluate the use of RGAs for plant breeding for the example of sugarcane mosaic virus (SCMV) resistance in maize. SCMV is one of the most important virus diseases of maize and causes serious yield losses in susceptible cultivars. Owing to the non-persistent manner of transmission, control of aphid vectors by chemical means is not effective and therefore, cultivation of resistant maize varieties is the most efficient method of virus control. Previous studies on the inheritance of oligogenic SCMV resistance located two major quantitative trait loci (QTLs) - Scmv1 and Scmv2 - on chromosomes 6S and 3L, respectively. The objectives of this study were to (1) give an overview on the current status of breeding for virus resistance in maize, (2) identify and genetically map candidate genes for Scmv1 and Scmv2, (3) use potential sequence homologies of linked RGAs for targeted increase of the number of candidate genes in the target regions, (4) convert closely linked amplified fragment length polymorphism (AFLP) markers into codominant, simple PCR-based markers as a tool for marker-assisted selection (MAS) and map-based cloning, (5) evaluate RGAs for the development of molecular markers, MAS, and map-based cloning, and (6) investigate the consequences of duplicate markers for the construction of linkage maps and their implications for MAS and map-based cloning.
   	Three previously published RGAs, pic13, pic21, and pic19 were cloned from six maize inbred lines, converted to cleaved amplified polymorphic sequence (CAPS) markers, and mapped in relation to SCMV R genes (Scmv1, Scmv2) in maize. Pairwise sequence alignments among the six inbreds revealed a frequency of one single nucleotide polymorphism (SNP) per 33 bp for the three RGAs, indicating a high degree of polymorphism and a high probability of success in converting RGAs into codominant CAPS markers compared to other sequences. Therefore, RGAs meet important requirements for the development of molecular markers, i.e., a high degree of polymorphism and availability in great numbers throughout the genome. In contrast to this, the degree of polymorphism for AFLPs closely linked to Scmv1 an Scmv2 was significantly lower in the same six inbred lines compared to RGAs. Only two of eight AFLP markers could be converted into one CAPS and one indel (insertion/deletion) marker. By genetic mapping, pic21 was shown to be different from Scmv2, whereas pic19 and pic13 could be mapped as single-copy markers to the target regions and are candidates for Scmv1 and Scmv2, respectively, due to genetic mapping and consistent restriction patterns of ancestral lines. 
   	Subsequently, pic19 was used as candidate for Scmv1 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 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% at the nucleotide level. The high sequence similarity and the multi-locus character of the previously single-copy mapped RGA pic19 show potential problems for the use of RGAs as molecular markers. The existence of ghost markers analogous to ghost QTL was suggested to be a result of simultaneous mapping of several homologous gene family members which cannot be distinguished at the level of PCR.
   	The idea of ghost loci derived by potentially duplicated sequences such as expressed sequence tags (ESTs), AFLPs, or simple sequence repeats (SSRs) was the subject of a theoretical and computer simulation study. Simultaneous amplification of homologous sequences results in an excess of heterozygotes causing distorted segregation ratios. We were able to theoretically prove the existence of such ghost markers resulting in changes of the correct marker orders. If these fictive ghost markers are part of a genetic map which is the subject of MAS or map-based cloning this may have fatal effects like locating a target gene into an incorrect marker interval. This incorrect locus order caused by duplicate marker loci can negatively affect the assignment of target genes to chromosome regions in a map-based cloning experiment, hinder indirect selection for a favourable allele at a QTL, and decrease the efficiency of reducing the chromosome segment attached to the target gene in marker-assisted backcrossing.
	In conclusion, this thesis demonstrates the use of RGAs for plant breeding and resistance genetics in general. RGAs provide a good source for the development of simple PCR-based markers. Furthermore, RGAs are an excellent tool for MAS, the identification of candidate genes and effective increase of such candidates in target regions using sequence homologies between RGAs. The duplicate nature of RGAs revealed potential problems for genetic mapping of potentially duplicated sequences which are widespread in eukaryote genomes and existent for several types of molecular markers. For resistance genetics in general, investigation of RGAs is important for the understanding of R gene organisation and evolutionary genetics of plant disease resistance.
K1 Epidemiologie
K1 Genetik
PP Hohenheim
PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
UL http://opus.uni-hohenheim.de/volltexte/2004/52