Transcriptome analysis of Bipolaris sorokiniana - Hordeum vulgare provides insights into mechanisms of host-pathogen interaction.

Spot blotch disease incited by Bipolaris sorokiniana severely affects the cultivation of barley. The resistance to B. sorokiniana is quantitative in nature and its interaction with the host is highly complex which necessitates in-depth molecular analysis. Thus, the study aimed to conduct the transcriptome analysis to decipher the mechanisms and pathways involved in interactions between barley and B. sorokiniana in both the resistant (EC0328964) and susceptible (EC0578292) genotypes using the RNA Seq approach. In the resistant genotype, 6,283 genes of Hordeum vulgare were differentially expressed out of which 5,567 genes were upregulated and 716 genes were downregulated. 1,158 genes of Hordeum vulgare were differentially expressed in the susceptible genotype, out of which 654 genes were upregulated and 504 genes were downregulated. Several defense-related genes like resistant gene analogs (RGAs), disease resistance protein RPM1, pathogenesis-related protein PRB1-2-like, pathogenesis-related protein 1, thaumatin-like protein PWIR2 and defensin Tm-AMP-D1.2 were highly expressed exclusively in resistant genotype only. The pathways involved in the metabolism and biosynthesis of secondary metabolites were the most prominently represented pathways in both the resistant and susceptible genotypes. However, pathways involved in MAPK signaling, plant-pathogen interaction, and plant hormone signal transduction were highly enriched in resistant genotype. Further, a higher number of pathogenicity genes of B. sorokiniana was found in response to the susceptible genotype. The pathways encoding for metabolism, biosynthesis of secondary metabolites, ABC transporters, and ubiquitin-mediated proteolysis were highly expressed in susceptible genotype in response to the pathogen. 14 and 11 genes of B. sorokiniana were identified as candidate effectors from susceptible and resistant host backgrounds, respectively. This investigation will offer valuable insights in unraveling the complex mechanisms involved in barley- B. sorokiniana interaction.

Draft genome sequencing of Tilletia caries inciting common bunt of wheat provides pathogenicity-related genes.

Common bunt of wheat caused by Tilletia caries is an important disease worldwide. The T. caries TC1_MSG genome was sequenced using the Illumina HiSeq 2500 and Nanopore ONT platforms. The Nanopore library was prepared using the ligation sequencing kit SQK-LSK110 to generate approximately 24 GB for sequencing. The assembly size of 38.18 Mb was generated with a GC content of 56.10%. The whole genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JALUTQ000000000. Forty-six contigs were obtained with N50 of 1,798,756 bp. In total, 10,698 genes were predicted in the assembled genome. Out of 10,698 genes, 10,255 genes were predicted significantly in the genome. The repeat sequences made up approximately 1.57% of the genome. Molecular function, cellular components, and biological processes for predicted genes were mapped into the genome. In addition, repeat elements in the genome were assessed. In all, 0.89% of retroelements were observed, followed by long terminal repeat elements (0.86%) in the genome. In simple sequence repeat (SSR) analysis, 8,582 SSRs were found in the genome assembly. The trinucleotide SSR type (3,703) was the most abundant. Few putative secretory signal peptides and pathogenicity-related genes were predicted. The genomic information of T. caries will be valuable in understanding the pathogenesis mechanism as well as developing new methods for the management of the common bunt disease of wheat.

Transcriptome Analysis of Wheat-Tilletia indica Interaction Provides Defense and Pathogenesis-Related Genes.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host−pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat−T. indica interaction for managing Karnal bunt disease of wheat.

Genome-Wide Association Mapping of Virulence Genes in Wheat Karnal Bunt Fungus Tilletia indica Using Double Digest Restriction-Site Associated DNA-Genotyping by Sequencing Approach.

Tilletia indica is a quarantine fungal pathogen that poses a serious biosecurity threat to wheat-exporting countries. Acquiring genetic data for the pathogenicity characters of T. indica is still a challenge for wheat breeders and geneticists. In the current study, double digest restriction-site associated-DNA genotyping by sequencing was carried out for 39 T. indica isolates collected from different locations in India. The generated libraries upon sequencing were with 3,346,759 raw reads on average, and 151 x 2 nucleotides read length. The obtained bases per read ranged from 87 Mb in Ti 25 to 1,708 Mb in Ti 39, with 505 Mb on average per read. Trait association mapping was performed using 41,473 SNPs, infection phenotyping data, population structure, and Kinship matrix, to find single nucleotide polymorphisms (SNPs) linked to virulence genes. Population structure analysis divided the T. indica population in India into three subpopulations with genetic mixing in each subpopulation. However, the division was not in accordance with the degree of virulence. Trait association mapping revealed the presence of 13 SNPs associated with virulence. Using sequences analysis tools, one gene (g4132) near a significant SNP was predicted to be an effector, and its relative expression was assessed and found upregulated upon infection.

Marker-assisted transfer of PinaD1a gene to develop soft grain wheat cultivars.

Grain softness has been a major trait of interest in wheat because of its role in producing flour suitable for making high-quality biscuits, cookies, cakes and some other products. In the present study, marker-assisted backcross breeding scheme was deployed to develop advanced wheat lines with soft grains. The Australian soft-grained variety Barham was used as the donor parent to transfer the puroindoline grain softness gene Pina-D1a to the Indian variety, DBW14, which is hard grained and has PinaD1bPinbD1a genes. Foreground selection with allele-specific PCR-based primer for Pina-D1a (positive selection) was used to identify heterozygous BC1F1 plants. Background selection with 173 polymorphic SSR primers covering all the 21 chromosomes was also carried out, in the foreground-selected BC1F1 plants. BC1F2 plants were selected by ascertaining the presence of Pina-D1a (positive selection) and absence of Pina-D1b (negative selection). Using the approach of positive, negative and background selection with molecular markers, 15 BC1F2 and 31 BC2F1 plants were finally selected. The 15 BC1F2 plants were selfed and the 31 BC2F1 plants were further backcrossed and selfed to raise BC3F1 and BC2F2 progenies, respectively. A part of the BC2F2 seed of each of the 31 plants was analyzed for grain hardness index (GHI) with single-kernel characterization system. The GHI varied from 12.1 to 37.1 in the seeds borne on the 31 BC2F1 plants. The reasons for this variation and further course of action are discussed.