Pramod Prasad

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution

Abstract Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina. The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000–32,000 sequence contigs (N50;7.4–9.2 kb), which accounted for ~86X–105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2–70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases

A comprehensive germplasm evaluation study of wheat accessions conserved in the Indian National Genebank was conducted to identify sources of rust and spot blotch resistance. Genebank accessions comprising three species of wheat–Triticum aestivum, T. durum and T. dicoccum were screened sequentially at multiple disease hotspots, during the 2011–14 crop seasons, carrying only resistant accessions to the next step of evaluation. Wheat accessions which were found to be resistant in the field were then assayed for seedling resistance and profiled using molecular markers. In the primary evaluation, 19,460 accessions were screened at Wellington (Tamil Nadu), a hotspot for wheat rusts. We identified 4925 accessions to be resistant and these were further evaluated at Gurdaspur (Punjab), a hotspot for stripe rust and at Cooch Behar (West Bengal), a hotspot for spot blotch. The second round evaluation identified 498 accessions potentially resistant to multiple rusts and 868 accessions potentially resistant to spot blotch. Evaluation of rust resistant accessions for seedling resistance against seven virulent pathotypes of three rusts under artificial epiphytotic conditions identified 137 accessions potentially resistant to multiple rusts. Molecular analysis to identify different combinations of genetic loci imparting resistance to leaf rust, stem rust, stripe rust and spot blotch using linked molecular markers, identified 45 wheat accessions containing known resistance genes against all three rusts as well as a QTL for spot blotch resistance. The resistant germplasm accessions, particularly against stripe rust, identified in this study can be excellent potential candidates to be employed for breeding resistance into the background of high yielding wheat cultivars through conventional or molecular breeding approaches, and are expected to contribute toward food security at national and global levels.

Investigating Disease Controlling Ability of Brassica Volatiles and Their Compatibility with Trichoderma harzianum

The present investigation aimed to test the in vitro toxicity of Brassica volatiles against soil-borne plant pathogens (Rhizoctonia solani Kuhn, Sclerotium rolfsii Sacc., Fusarium oxysporum f.sp. ciceris (Padwick) Matuo & K. Sato and Sclerotinia sclerotiorum (Lib.) de Bary) and biocontrol fungus Trichoderma harzianum Rifai. Moreover, the response of T. harzianum to toxic volatiles was also studied in terms of glucanase and chitinase gene expression. A strong fungistatic effect of B. alba treatment was recorded against all the test pathogens. Noticeably, S. sclerotiorum manifested least sensitivity among all the tested pathogens. All T. harzianum isolates were less sensitive as compared to the assayed pathogens. Based on the in vitro study involving 6 T. harzianum isolates, T55 and Th-R showed least, while T39 showed highest sensitivity to the volatiles. Interestingly, the patterns relating to the effect of volatiles on inoculum density of T. harzianum isolates were similar to their effect on fungus growth in vitro. More importantly, expression of chitinase and glucanase genes in different Trichoderma isolates was up-regulated, which could improve the biocontrol activity of T. harzianum. Therefore, here the authors envisage that combining biocontrol and biofumigation has the potential to provide sustainable and cost-effective strategies to manage soil-borne plant pathogens.

Efficiency of double haploid production in wheat through wide hybridization and embryo rescue

The effect of haploid induction in wheat F1s by Zea mays, Imperata cylindrica and growth environments was investigated. Doubled Haploid (DH) plant production via maize and I. cylindrica technique from field grown LWH x VL616 F1 plants was equally efficient. In second environment, four F1 hybrids viz. LWH x VL616, HPW266 x Yr15 (CN25087), HS490 x HI1563 and Local Red x WHD938 were grown in greenhouse conditions and pollinated with maize. Efficiency of DH production was considerably high from greenhouse grown F1s as compared to field grown F1s. We also report an efficient protocol for DH production in bread wheat.