Mapping Of Novel Quantitative Trait Loci (qtl) For Fusarium Wilt Resistance In Chickpea (cicer Arietinum L.) And Analysis Of The Genomic Diversity Of Fusarium Oxysporum F. Sp. Ciceris In Ethiopia
Chickpea (Cicer arietinum L.) is one of the most economically important foodrnlegumes cultivated in different parts of the world. Ethiopia is the largest producer,rnconsumer and exporter of chickpea in Africa. However, several biotic and abioticrnstresses restrict its potential productivity. Among the biotic stresses, fungal diseasesrnare the major yield limiting factors throughout chickpea producing countries.rnFusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (Foc), is one of the mostrndominant and destructive pathogen threatening chickpea production in Ethiopia.rnBreeding for host plant resistance is the most cost efficient and eco-friendly strategyrnto control the disease. Nevertheless, chickpea breeding for Fusarium wilt resistance isrnregularly challenged with high pathogenic variability and limited availability of goodrnresistance sources. So far only few efforts have been made to investigate the geneticrndiversity and geographic distribution of Foc pathogen in Ethiopia for designingrneffective breeding and integrated disease management strategies. For this disease, nornreport is available that encompass the breadth of major and minor chickpea producingrnareas of the country. In this study four sets of experiments were executed with thernmain objectives to: investigate the intensity and association of Fusarium wilt/root rotrndisease of chickpea under diverse biophysical factors in Ethiopia; identify newrnresistance sources and map a novel wilt resistant quantitative trait loci (QTL) inrnchickpea; analyses the genomic diversity, pathogenic variability and geographicrndistribution of Foc pathogen in the country; and develop rapid and reliable diseaserndiagnostic assay for accurate disease diagnosis.rnIn 2015 and 2016 cropping seasons, geo-referenced field surveys werernconducted covering a total of 62 major chickpea growing districts located in 19rndiverse agro-ecological zones of Ethiopia, and a total of 217 diseased plant samplesrnrnwere collected for pathogen identification and genomics study. Among these, from 51rnrepresentative farmers’ fields, three 1 x 1 meter quadrat were surveyed along arndiagonal transect to investigate the intensity and association of Fusarium wilt/root rotrndisease of chickpea under diverse biophysical factors in Ethiopia. Data on majorrnbiophysical factors were recorded, and pathogen was isolated based on the establishedrnmorphological and cultural characteristics. For identification of new Fusarium wiltrnresistance sources, a total of 315 wild introgression lines and 47 recombinant inbredrnlines (RILs) were evaluated for Fusarium wilt resistance in sick plot at Debre ZeitrnAgricultural Research Center. To map Fusarium wilt resistance QTL in chickpea,rntotal of 108 F2 hybrids were generated by crossing Fusarium wilt resistant varietyrnDera and Fusarium wilt susceptible genotype JG 62, and genotyping-by-sequencingrnidentified 1,659 single nucleotide polymorphisms (SNPs) that distinguish the twornparental lines. A total of 166 representative Fusarium isolates collected from differentrnpart of the country were sequenced using whole genome sequencing (WGS) withrnIllumina HiSeq 4000 platform to investigate the genomic diversity, pathogenicrnvariability and geographic distribution of Foc pathogen. For rapid and accuraterndetection of Foc pathogen directly from symptomatic chickpea plants, broadrnspecificity PCR primers were designed based on the alignment of selectedrnBenchmarking Universal Single Copy Orthologs (BUSCO) genes present and highlyrnconserved in the genomes of a set of 66 Fusarium isolates. Moreover, a cultureindependentrnbroad-range18S amplicon survey was conducted to characterizernchickpea-associated eukaryotic communities.rnThe result indicated that Fusarium wilt disease was widely distributed in allrngrowing areas of the country. Across all surveyed sites, Foc was the predominantrnspecies encountered among fungi cultured from plant tissue, representing 69.4 % ofrntotal isolates. Diseases pressure was significantly (P < 0.05) associated with heavyrnblack soils, Desi type chickpea, early planting, flowering and plant maturity. Thernhighest mean percent diseases incidence per m2 (45.65%) was recorded in the Amhararnregion, West Gojam zone, where heavy clay soils predominate. Wild introgressionrnlines and advanced recombinant inbred lines showed significant genetic diversity forrnFusarium wilt resistance and yield related traits that can be exploited to improve thernagronomic value of the chickpea crop. In the present study 20 Fusarium wilt resistantrnRILs with high yield and desirable agro morphological traits were identified. ForrnFusarium wilt resistance QTL mapping, a total 836 high quality SNP markers werernrnassigned to six genetic linkage groups, each corresponding to separate chromosomes,rnwith a total map size of 274.9 cM and 3.12 cM average distance between mappedrnmarkers. Major QTL explaining 55.28 % of the observed phenotypic variation wasrnidentified on chromosome 4 at 44.29 cM with a logarithm of odds (LOD) score ofrn13.8. Interestingly, Nei’s genetic diversity analysis based on 196, 495 SNPs split testrnisolates into 20 distinct clusters irrespective of their regions of origin andrngeographical location. Among these,16 distinct clusters were Fusarium oxysporiumrnciceris (Foc) isolates. Phylogenetic analysis based on 1,052 highly conserved BUSCOrngenes also divided test isolates into six distinct Fusarium species, and 16 sub-groupsrn(Foc isolates). Consistent with these results, pairwise average nucleotide identityrn(ANI) analysis based on 3,695 highly conserved BUSCO genes split test isolates in tornsix distinct Fusarium species, using 95 % ANI (ANI95) as the lower species boundary.rnBesides, dendrogram built based on virulence data split Foc isolates into four distinctrnvirulence groups confirming the existence of high pathogenic variability between Focrnisolates in Ethiopia irrespective of their geographical origin. Mantel correlationrnestimate showed very weak correlation between geographical distance and geneticrndistances of Fusarium isolates in Ethiopia with P = 0.280 and R2 = 0.0006. Thernresults the PCR diagnostic assay showed that, on test with Fusarium specific PCRrnprimer (EOG09331-PTT), 97.5 % of diseased plants with typical symptoms (39 out ofrn40 plants) gave uniformly strong amplification with the identity of ampliconsrnconfirmed by Sanger sequencing. However, some symptomatic plants yieldedrninconsistent results as PCR based disease diagnosis using organism-specific DNArnamplification is unable to assess the presence of all other microbes that might betterrninform diagnosis. To address these issues, microbial community composition werernsurveyed using 18S amplicon sequencing. The result nominated Phytophthorarnmedicaginis as alternative pathogens in some fields where Fusarium wilt wasrnsuspected. Such analyses represent a potentially powerful alternative to traditionalrnplant disease diagnostics. Without the constraints of culturability and the bias ofrnendpoint PCR, amplicon sequencing can provide powerful insights into diseaserndynamics.rnIn conclusion, the novel major QTL and associated genetic markers identifiedrnin the present study offer molecular tools for breeding wilt resistant against EthiopianrnFoc isolates. This study indicated the presence of high genetic diversity andrnpathogenetic variability between Fusarium isolates in Ethiopia. Therefore, designingrnrneffective country wide breeding and integrated disease management strategies againstrnFoc pathogens is key to break the recurrent disease cycle in the country. The results ofrnthe present study provide detailed information and appropriate framework to developrneffective breeding and integrated disease management strategies to combat Fusariumrnwilt disease of chickpea in Ethiopia.