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

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Addis Ababa University


Chickpea (Cicer arietinum L.) is one of the most economically important food legumes cultivated in different parts of the world. Ethiopia is the largest producer, consumer and exporter of chickpea in Africa. However, several biotic and abiotic stresses restrict its potential productivity. Among the biotic stresses, fungal diseases are the major yield limiting factors throughout chickpea producing countries. Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (Foc), is one of the most dominant and destructive pathogen threatening chickpea production in Ethiopia. Breeding for host plant resistance is the most cost efficient and eco-friendly strategy to control the disease. Nevertheless, chickpea breeding for Fusarium wilt resistance is regularly challenged with high pathogenic variability and limited availability of good resistance sources. So far only few efforts have been made to investigate the genetic diversity and geographic distribution of Foc pathogen in Ethiopia for designing effective breeding and integrated disease management strategies. For this disease, no report is available that encompass the breadth of major and minor chickpea producing areas of the country. In this study four sets of experiments were executed with the main objectives to: investigate the intensity and association of Fusarium wilt/root rot disease of chickpea under diverse biophysical factors in Ethiopia; identify new resistance sources and map a novel wilt resistant quantitative trait loci (QTL) in chickpea; analyses the genomic diversity, pathogenic variability and geographic distribution of Foc pathogen in the country; and develop rapid and reliable disease diagnostic assay for accurate disease diagnosis. In 2015 and 2016 cropping seasons, geo-referenced field surveys were conducted covering a total of 62 major chickpea growing districts located in 19 diverse agro-ecological zones of Ethiopia, and a total of 217 diseased plant samples were collected for pathogen identification and genomics study. Among these, from 51 representative farmers’ fields, three 1 x 1 meter quadrat were surveyed along a diagonal transect to investigate the intensity and association of Fusarium wilt/root rot disease of chickpea under diverse biophysical factors in Ethiopia. Data on major biophysical factors were recorded, and pathogen was isolated based on the established morphological and cultural characteristics. For identification of new Fusarium wilt resistance sources, a total of 315 wild introgression lines and 47 recombinant inbred lines (RILs) were evaluated for Fusarium wilt resistance in sick plot at Debre Zeit Agricultural Research Center. To map Fusarium wilt resistance QTL in chickpea, total of 108 F2 hybrids were generated by crossing Fusarium wilt resistant variety Dera and Fusarium wilt susceptible genotype JG 62, and genotyping-by-sequencing identified 1,659 single nucleotide polymorphisms (SNPs) that distinguish the two parental lines. A total of 166 representative Fusarium isolates collected from different part of the country were sequenced using whole genome sequencing (WGS) with Illumina HiSeq 4000 platform to investigate the genomic diversity, pathogenic variability and geographic distribution of Foc pathogen. For rapid and accurate detection of Foc pathogen directly from symptomatic chickpea plants, broad specificity PCR primers were designed based on the alignment of selected Benchmarking Universal Single Copy Orthologs (BUSCO) genes present and highly conserved in the genomes of a set of 66 Fusarium isolates. Moreover, a cultureindependent broad-range18S amplicon survey was conducted to characterize chickpea-associated eukaryotic communities. The result indicated that Fusarium wilt disease was widely distributed in all growing areas of the country. Across all surveyed sites, Foc was the predominant species encountered among fungi cultured from plant tissue, representing 69.4 % of total isolates. Diseases pressure was significantly (P < 0.05) associated with heavy black soils, Desi type chickpea, early planting, flowering and plant maturity. The highest mean percent diseases incidence per m2 (45.65%) was recorded in the Amhara region, West Gojam zone, where heavy clay soils predominate. Wild introgression lines and advanced recombinant inbred lines showed significant genetic diversity for Fusarium wilt resistance and yield related traits that can be exploited to improve the agronomic value of the chickpea crop. In the present study 20 Fusarium wilt resistant RILs with high yield and desirable agro morphological traits were identified. For Fusarium wilt resistance QTL mapping, a total 836 high quality SNP markers were assigned to six genetic linkage groups, each corresponding to separate chromosomes, with a total map size of 274.9 cM and 3.12 cM average distance between mapped markers. Major QTL explaining 55.28 % of the observed phenotypic variation was identified on chromosome 4 at 44.29 cM with a logarithm of odds (LOD) score of 13.8. Interestingly, Nei’s genetic diversity analysis based on 196, 495 SNPs split test isolates into 20 distinct clusters irrespective of their regions of origin and geographical location. Among these,16 distinct clusters were Fusarium oxysporium ciceris (Foc) isolates. Phylogenetic analysis based on 1,052 highly conserved BUSCO genes also divided test isolates into six distinct Fusarium species, and 16 sub-groups (Foc isolates). Consistent with these results, pairwise average nucleotide identity (ANI) analysis based on 3,695 highly conserved BUSCO genes split test isolates in to six distinct Fusarium species, using 95 % ANI (ANI95) as the lower species boundary. Besides, dendrogram built based on virulence data split Foc isolates into four distinct virulence groups confirming the existence of high pathogenic variability between Foc isolates in Ethiopia irrespective of their geographical origin. Mantel correlation estimate showed very weak correlation between geographical distance and genetic distances of Fusarium isolates in Ethiopia with P = 0.280 and R2 = 0.0006. The results the PCR diagnostic assay showed that, on test with Fusarium specific PCR primer (EOG09331-PTT), 97.5 % of diseased plants with typical symptoms (39 out of 40 plants) gave uniformly strong amplification with the identity of amplicons confirmed by Sanger sequencing. However, some symptomatic plants yielded inconsistent results as PCR based disease diagnosis using organism-specific DNA amplification is unable to assess the presence of all other microbes that might better inform diagnosis. To address these issues, microbial community composition were surveyed using 18S amplicon sequencing. The result nominated Phytophthora medicaginis as alternative pathogens in some fields where Fusarium wilt was suspected. Such analyses represent a potentially powerful alternative to traditional plant disease diagnostics. Without the constraints of culturability and the bias of endpoint PCR, amplicon sequencing can provide powerful insights into disease dynamics. In conclusion, the novel major QTL and associated genetic markers identified in the present study offer molecular tools for breeding wilt resistant against Ethiopian Foc isolates. This study indicated the presence of high genetic diversity and pathogenetic variability between Fusarium isolates in Ethiopia. Therefore, designing effective country wide breeding and integrated disease management strategies against Foc pathogens is key to break the recurrent disease cycle in the country. The results of the present study provide detailed information and appropriate framework to develop effective breeding and integrated disease management strategies to combat Fusarium wilt disease of chickpea in Ethiopia.



ANI, BUSCO Gene, Chickpea, Fusarium, Genotyping by Sequencing, PCR, Snps, 18S