Genome wide transcriptome profiling of Fusarium oxysporum f sp. ciceris conidial germination reveals new insights into infection-related genes.

Vascular wilt caused by Fusarium oxysporum f. sp. ciceris (Foc) is a serious disease of chickpea (Cicer arietinum L.) accounting for approximately 10-15% annual crop loss. The fungus invades the plant via roots, colonizes the xylem vessels and prevents the upward translocation of water and nutrients. Infection is initiated by conidia that invade the host tissue often by penetration of intact epidermal cells. Here, we report the characterization of the transcriptome of Foc sequenced using Illumina Hiseq technology during its conidial germination at different time points. Genome-wide expression profiling revealed that genes linked to fungal development are transcribed in successive ways. Analysis showed that Foc have large sets of germination-related genes and families of genes encoding secreted effectors, cell wall/pectin-degrading enzymes, metabolism related enzymes, transporters and peptidases. We found that metabolism related enzymes are up-regulated at early time point whereas most transporters and secondary metabolites important for tissue colonization and pathogenicity are up-regulated later as evident from the qRT-PCR. The study demonstrated that early conidial germination in Foc is accompanied by rapid shifts in gene expression that prepare the fungus for germ tube outgrowth, host cell invasion and pathogenesis. This work lays the foundation for facilitating further research towards understanding this host-pathogen interaction.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

BACKGROUND: Soil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world's second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula. RESULTS: Focusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp. CONCLUSIONS: We demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn't share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Development of DArT markers and assessment of diversity in Fusarium oxysporum f. sp. ciceris, wilt pathogen of chickpea (Cicer arietinum L.).

BACKGROUND: Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. The severity and yield losses of Fusarium wilt differ from place to place owing to existence of physiological races among isolates. Diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. The advantages of using molecular markers to understand the distribution of genetic diversity in Foc populations is well understood. The recent development of Diversity Arrays Technology (DArT) offers new possibilities to study the diversity in pathogen population. In this study, we developed DArT markers for Foc population, analysed the genetic diversity existing within and among Foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of Foc in India. RESULTS: We report the successful development of DArT markers for Foc and their utility in genotyping of Foc collections representing five chickpea growing agro-ecological zones of India. The DArT arrays revealed a total 1,813 polymorphic markers with an average genotyping call rate of 91.16% and a scoring reproducibility of 100%. Cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of Foc were partially classified based on geographical source. Diversity in Foc population was compared with the phenotypic variability and it was found that DArT markers were able to group the isolates consistent with its virulence group. A number of race-specific unique and rare alleles were also detected. CONCLUSION: The present study generated significant information in terms of pathogenic and genetic diversity of Foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. The DArT markers were proved to be a powerful diagnostic tool to study the genotypic diversity in Foc. The high number of DArT markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population.

Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies.

Molecular markers are the most powerful genomic tools to increase the efficiency and precision of breeding practices for crop improvement. Progress in the development of genomic resources in the leading legume crops of the semi-arid tropics (SAT), namely, chickpea (Cicer arietinum), pigeonpea (Cajanus cajan) and groundnut (Arachis hypogaea), as compared to other crop species like cereals, has been very slow. With the advances in next-generation sequencing (NGS) and high-throughput (HTP) genotyping methods, there is a shift in development of genomic resources including molecular markers in these crops. For instance, 2,000 to 3,000 novel simple sequence repeats (SSR) markers have been developed each for chickpea, pigeonpea and groundnut. Based on Sanger, 454/FLX and Illumina transcript reads, transcriptome assemblies have been developed for chickpea (44,845 transcript assembly contigs, or TACs) and pigeonpea (21,434 TACs). Illumina sequencing of some parental genotypes of mapping populations has resulted in the development of 120 million reads for chickpea and 128.9 million reads for pigeonpea. Alignment of these Illumina reads with respective transcriptome assemblies have provided more than 10,000 SNPs each in chickpea and pigeonpea. A variety of SNP genotyping platforms including GoldenGate, VeraCode and Competitive Allele Specific PCR (KASPar) assays have been developed in chickpea and pigeonpea. By using above resources, the first-generation or comprehensive genetic maps have been developed in the three legume speciesmentioned above. Analysis of phenotyping data together with genotyping data has provided candidate markers for drought-tolerance-related root traits in chickpea, resistance to foliar diseases in groundnut and sterility mosaic disease (SMD) and fertility restoration in pigeonpea. Together with these traitassociated markers along with those already available, molecular breeding programmes have been initiated for enhancing drought tolerance, resistance to fusarium wilt and ascochyta blight in chickpea and resistance to foliar diseases in groundnut. These trait-associated robust markers along with other genomic resources including genetic maps and genomic resources will certainly accelerate crop improvement programmes in the SAT legumes.

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

The first set of EST resource for gene discovery and marker development in pigeonpea (Cajanus cajan L.).

BACKGROUND: Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs). RESULTS: A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (or= 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. Occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assay. CONCLUSION: The pigeonpea EST dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. Sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. Validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding.

Biological Control of Late Leaf Spot of Peanut (Arachis hypogaea) with Chitinolytic Bacteria.

ABSTRACT Late leaf spot (LLS), caused by Phaeoisariopsis personata, is a foliar disease of groundnut or peanut (Arachis hypogaea) with high economic and global importance. Antifungal and chitinolytic Bacillus circulans GRS 243 and Serratia marcescens GPS 5, selected among a collection of 393 peanut-associated bacteria, were applied as a prophylactic foliar spray and tested for control of LLS. Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 resulted in improved biological control of LLS disease. Supplementation of bacterial cells with 1% (wt/vol) colloidal chitin reduced lesion frequency by 60% compared with application of bacterial cells alone, in the greenhouse. Chitinsupplemented application of GRS 243 and GPS 5 also resulted in improved and stable control of LLS in a repeated field experiment and increased the pod yields by 62 and 75%, respectively, compared with the control. Chitin-supplemented application of GPS 5 was tested in six onfarm trials, and the increase in pod yields was up to 48% in kharif (rainy season). A 55-kDa chitinase was purified from the cell-free culture filtrate of GPS 5 by affinity chromatography and gel filtration. Purified chitinase of S. marcescens GPS 5 (specific activity 120 units) inhibited the in vitro germination of P. personata conidia, lysed the conidia, and effectively controlled LLS in greenhouse tests, indicating the importance of chitinolysis in biological control of LLS disease by GPS 5.