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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Date
2021-05-01
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Addis Ababa University
Abstract
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.
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Keywords
ANI, BUSCO Gene, Chickpea, Fusarium, Genotyping by Sequencing, PCR, Snps, 18S