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GENETIC RESISTANCE TO FUNGAL PATHOGENS IN SORGHUM [SORGHUM BICOLOR (L.) MOENCH]
Sorghum [Sorghum bicolor (L.) Moench] is the fifth most widely grown cereal crop in the world that serves as a staple food for millions of people. Grain mold of sorghum, caused by a consortium of fungal pathogens, is a leading constraint to sorghum production. A second sorghum disease with significant economic impact is anthracnose caused by the ascomycete fungus Colletotrichum sublineolum (Cs). Grain mold causes yield reduction and is highly detrimental to food quality due to contamination by toxigenic fungi and mycotoxins while anthracnose results in significant yield reduction in susceptible cultivars. Genetic resistance is considered the only effective and sustainable way to control both diseases, but the genetic control of these diseases are not well understood. In this project, we implemented genetic, genomic and molecular approaches to identify loci and/or genes underlying resistance to the two diseases. The results presented in Chapters 2 to 5 provide new insights to the genetic and genomic architecture of resistance to grain mold and anthracnose. Chapter 1 provides background information and review of the literature on the pathology of the two diseases, the contrasting and shared mechanisms of genetic resistance and approaches to QTL and gene identification. Chapter 2 and Chapter 3 describe genome wide association studies (GWAS) conducted on sorghum landrace accessions from Ethiopia. Results of both sets of GWAS were recently published (Nida et al., 2019, Journal of Cereal Science 85, 295-304, https://doi.org/10.1016/j.jcs.2018.12.016; Nida et al., 2021, Theoretical Applied Genetics, https://doi.org/10.1007/s00122-020-03762-2). Chapter 4 describes global transcriptome profiles of early stage of the developing grain from resistant and susceptible sorghum genotypes which uncovered process that correlate with resistance or susceptibility to grain mold. Results of this study was also recently published (Nida et al., 2021, BMC Genomics 22, 295, https://doi.org/10.1186/s12864-021-07609-y. Finally, Chapter 5 summarizes two anthracnose resistance genes identified through whole genome resequencing and genetic mapping.
In Chapter 2, genomic regions associated with grain mold resistance were identified through GWAS conducted using sorghum landraces. A major grain mold resistance locus containing tightly linked and sequence related MYB transcription factor genes were identified based on association between SNPs and grain mold resistance scores of 1425 accessions. The locus contains YELLOW SEED1 (Y1, Sobic.001G398100), a likely non-functional pseudo gene (Y2, Sobic.001G398200), and YELLOW SEED3 (Y3, Sobic.001G397900). SNPs and other sequence polymorphisms that alter the Y1 and Y3 genes correlated with susceptibility to grain mold and provided a strong genetic evidence. Although Y1 has long been known as a regulator of kernel color and the biosynthesis of 3-deoxyanthocynidin phytoalexins, it was not annotated in the sorghum genome. The data suggest that the MYB genes and their grain and glume specific expressions determine responses to molding fungi.
Chapter 3 focuses on GWAS conducted on a subset of early to medium flowering accessions to identify grain mold resistance loci. In addition, because of the caveats associated with grain flavonoid mediated mold resistance, we specifically aimed to identify resistance loci independent of grain flavonoids. A multi-environment grain mold phenotypic data and 173,666 SNPs were used to conduct GWAS using 635 accessions and a subset of non-pigmented accessions, potentially producing no tannins and/or phenols. A novel sorghum KAFIRIN gene encoding a seed storage protein, and LATE EMBRYOGENESIS ABUNDANT 3 (LEA3) gene encoding a protein with differential accumulation in seeds were identified. The KAFIRIN and LEA3 loci were also grain mold resistance factors in accessions with non-pigmented grains. Moreover, the known SNP (S4_62316425) in TAN1 gene, a regulator of tannin accumulation in sorghum grain was significantly associated with grain mold resistance. These data suggest the critical role of loci harboring seed protein genes for resistance to sorghum grain mold.
In Chapter 4, global transcriptome profiles of developing grain of resistant and susceptible sorghum genotypes were studied. The developing kernels of grain mold resistant RTx2911 and susceptible RTx430 sorghum genotypes were inoculated with a mixture of fungal pathogens mimicking the species complexity of the diseases under natural infestation. Global transcriptome changes corresponding to multiple molecular and cellular processes, and biological functions including defense, secondary metabolism, and flavonoid biosynthesis were observed with differential regulation in the two genotypes. Genes encoding pattern recognition receptors (PRRs), regulators of growth and defense homeostasis, antimicrobial peptides, pathogenesis-related proteins, zein seed storage proteins, and phytoalexins showed increased expression correlating with resistance. The data suggest a pathogen inducible defense system in the developing grain of sorghum that involves the chitin PRR, MAPKs, key transcription factors, downstream components regulating immune gene expression and accumulation of defense molecules.
Finally, Chapter 5 deals with anthracnose resistance loci and subsequent genetic mapping and identification of two resistance genes. The sorghum line SAP135 was previously described for its broad-spectrum resistance to anthracnose. To identify the specific resistance gene, a mapping population was generated by crossing SAP135 with the susceptible line TAM428. Bulked-segregant analysis (BSA) combined with whole genome re-sequencing of resistant and susceptible pools (BSA-seq) of the mapping population defined a single major peak on chromosome 8 for resistance to the Cs strain Csgrg which was designated as ANTHRACNOSE RESISTANCE GENE 4 (ARG4). ARG4 was co-localized with a locus identified in a parallel but an independent mapping study conducted using the sorghum line P9830 against another Cs strain Csgl1. Fine mapping revealed that the resistance loci from the two populations delineated two tightly linked loci, the latter locus designated as ANTHRACNOSE RESISTANCE GENE 5 (ARG5). ARG4 (Sobic.008G166400) and ARG5 (Sobic.008G177900) encode canonical NBS-LRR proteins widely known intracellular immune receptors. Interestingly, SAP135 carries a functional ARG4 but lacks ARG5 whereas P9830 harbors a functional ARG5 and lacks ARG4 and both show sequence homology to wheat rust resistance genes. Csgrg and Csgl1 are both virulent on sorghum lines TAM428 and BTx623, thus both lines carry susceptible alleles of ARG4 and ARG5. Supplemental information for the unpublished chapters are presented in the appendix section of this thesis.