Genetic analysis and functional genomic tool development to characterize resistance gene candidates in wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) is the most widely cultivated food crop in the world. Biotic factors such as insects and pathogens cause a significant loss in wheat yield worldwide. Deployment of resistance genes is usually the most effective, economical and environmentally sound method of managing pest-related losses. Linked molecular markers can help in accurate and targeted deployment of resistance genes.;Majority of the cloned plant resistance genes belong to the NBS-LRR class of resistance genes. Analyses of NB/LRR-containing sequences, PCR amplified from various crops, have suggested that only a small fraction of the R-gene-like genomic sequences may be functional, and this fraction is expected to be even smaller in crop plants with larger genomes. Therefore, resistance gene-like sequences amplified from the expressed fraction of the genomes may be better markers if not the resistance genes itself. In a previous study, 184 R-gene candidates (RGCs) were amplified from the expressed portion of the wheat genome of which 121 physically mapped to 18 regions containing 82 wheat resistance genes. With the overall goal to characterize the RGCs, the main objectives of this study were to test linkage of RGCs to known resistance genes and to develop reverse genetic tools for transient and stable RNAi to facilitate characterization of the RGCs.;Linkage relationship of 14 phenotypically characterized resistance genes was investigated with 47 RGCs mapping in their reported chromosomes/arms/regions. Chromosomal location of nine resistance genes, H13, H22, H24, H25, Lr9, Lr16, Yr10, Pm3a and Pm20, was determined relative to RGCs. Seven of the nine genes were physically mapped. In total, 26 RGCs were found linked (≤ 30 cM) with nine resistance genes. Nine RGCs showed close linkage (≤ 10cM) with seven resistance genes of which BF482366 is 3.8cM from H24, BE492937 and BF474204 are 4.7cM and 5.6cM from Yr10, respectively.;Virus-induced gene silencing (VIGS), using barley stripe mosaic virus based vector, was optimized for transient silencing of genes in important wheat tissues including leaf, root and developing seed. Monitored using green fluorescent protein (GFP), spread of VIGS vector was observed in inoculated leaf tissue, phloem and root cortex at 10 and 17 days post-inoculation, but was absent in apical meristems and reproductive tissues. Compared to control plants, an antisense construct of the wheat coronatine insensitive1 ( TaCOI1) gene reduced TaCOI1 transcripts by 60% in roots and 65% in foliage of cultivar Scarlet. The seed-specific granule bound starch synthase (GBSS) gene was successfully silenced with antisense and hairpin constructs which resulted in up to 81% reduction in amylose content. Antisense and hairpin constructs produced similar levels of gene silencing except in one case where hairpin construct showed a significantly higher level of silencing. Monitored by typical symptoms of PDS gene silencing, 8 to 11% of the progeny of the inoculated plants showed transmission of gene silencing. In the next generation, however, 53 to 61% of the selfed progeny, of the plants that showed silencing in previous generation, showed silencing.;Stable RNAi approach involving Agrobacterium-mediated transformation with hpRNA expressing construct was optimized in wheat. A clear and repeatable procedure was standardized whereby, starting with immature embryos, transformants can be produced at a frequency of up to 5.5% in eight to ten weeks. In one experiment, the optimized procedure produced 22 transgenic plants from 400 immature embryos. The GatewayRTM cloning technology based RNAi vector pHELLSGATE 8 was investigated for its use in high throughput construction of hpRNA constructs and for gene silencing. Nine sequences, ranging in length from 225--633bp, were successfully cloned in sense and antisense orientation to make hpRNA constructs. Irrespective of the length of the sequence, 50--67% of the constructs had sequences cloned in proper orientation. Plants transformed with pHELLSGATE 8-based RNAi construct showed 80--100% reduction in transcript level of targeted wheat expressed sequence tag BE405778.