Development of molecular based tools for resistance breeding of sorghum to colletotrichum sublineolum
Sorghum is a major staple food accounting for over 65 % of the carbohydrate requirements and 30% of the daily calorie intake for over 300 million people in developing economies of Africa and Asia. Sorghum production world over is affected among others, by anthracnose caused by Colletotrichum sublineolum. The disease affects all above ground parts of the crop causing losses of up to 50% grain yield. Breeding for resistance remains the most economically viable option for long-term disease management. Resistance to the disease has been reported as qualitative and quantitative with quantitative forms being more durable but difficult to breed for. Development of a sound scheme for pyramiding resistance genes can best be developed after characterization of resistance loci and identification of molecular makers closely linked and co-segregating with it. One of the approaches to elucidate the active defence loci is by identification of candidate genes. Once identified, these resistance and defence response genes can be used to develop “functional markers” from polymorphic sites within gene coding sequences. This is the underpinning for this thesis research whose overall aim was to elucidate the molecular basis of resistance in sorghum to C. sublineolum by identifying and characterizing key genes of potential use to breeding for durable resistance. The specific objectives of the study were to 1. Identify and characterize differentially expressed genes in sorghum in response to infection with Colletotrichum sublineolum. 2. Develop functional PCR based molecular marker for anthracnose resistance breeding. 3. Investigate the genetics of anthracnose resistance within and among the sorghum races. The candidate gene approach based on cDNA-AFLP transcript profiling was used on two contrasting sorghum genotypes inoculated with Colletotrichum sublineolum. In total, approximately 3,000 transcript-derived fragments (TDFs) were monitored ranging from 50 to 600 bp in size. Unique, up- or down-regulated transcripts in the resistant BS04/05 genotype compared to susceptible MU07/193D sorghum line, sampled at 24, 48 and 72 hours post inoculation (hpi) were excised, sequenced and analyzed. In a final set of 126 sequenced genes, putative functions by homology database searches were assigned of which15 were identified as stress related. However, majority of the differentially expressed genes with known function were categorized under metabolism, transcription and signaling, suggesting metabolic reprogramming during the infection process. Seven of the plant-derived genes were selected for functional analysis using a Brome mosaic virus-based virus-induced gene silencing (VIGS) system followed by fungal inoculation and quantitative real-time PCR analysis. The candidate set comprised genes encoding resistance proteins (Cs1A, Cs2A), a lipid transfer protein (SbLTP1), a zinc finger-like transcription factor (SbZnTF1), a rice defensin-like homolog (SbDEFL1), a cell death related protein (SbCDL1), and an unknown gene harboring a casein kinase 2-like domain (SbCK2). The results demonstrate that down-regulation of Cs1A, Cs2A, SbLTP1 SbZnF1 and SbCD1 via VIGS, significantly compromised the resistance response while milder effects were observed with SbDEFL1 and SbCK2. Expanded genome analysis revealed that Cs1A and Cs2A genes are located in two different loci on chromosome 9 closely linked with duplicated genes Cs1B and Cs2B, respectively. The data suggest that Cs1A and Cs2A play major roles in this defense interaction and could be exploited for resistance breeding. Two molecular markers from candidate genes based on sequence variation within the coding regions were identified and found to co-segregate with anthracnose resistance. These markers are bound to simplify and streamline MAS for resistance breeding to anthracnose, as they are completely predictive of the functional nucleotide polymorphism (FNP) that differentiates resistant and susceptible alleles. Efficient breeding and selection of resistant varieties as a disease management strategy requires the knowledge and the understanding of the underlying genetics. Both qualitative and quantitative resistance to anthracnose has been previously reported. In this study, field and greenhouse evaluations of the different populations show that resistance to anthracnose among sorghum races is monogenic in nature being controlled by either dominant or recessive genes. Four sources of resistance were identified among different sorghum races an indication of unique sources of resistance among sorghum races. Resistance in races Durra and Caudatum is controlled by dominant genes and recessive in kafir while both dominant and recessive genes are present in race bicolor. The simple inheritance found among sorghum accessions irrespective of race affiliation, indicates that the introgression of resistance genes to anthracnose is relative straightforward and can be achieved by backcrossing. Collectively, results from this thesis provide useful information as diverse genes for resistance were identified and their functional relevancy confirmed. These genes would be used to develop durable resistant cultivars through pyramiding.