Whole Genome Sequencing and Expression Analysis of ToxA in Bipolaris Sorokiniana Provides Discernment of Pathogenicity Causing Spot Blotch of Wheat

Background: Spot blotch disease of wheat caused by Bipolaris sorokiniana Boerma (Sacc.) is an emerging problem in South Asian countries. In this study, whole genome of highly virulent isolate of Bipolaris sorokiniana (BS112) was sequenced, pathogenicity related gene(s) were identified and role of ToxA gene in spot blotch disease development was established.Results: Bipolaris sorokiniana isolate BS112 infecting wheat was sequenced using hybrid assembly approach. The assembly size of the genome was 35.64Mb (GenBank accession number RCTM00000000) with GC content of 50.2%, providing coverage of 97.6% on reference ND90Pr genome. Average gene density predicted was 250-300 genes/Mb. A total of 235 scaffolds were obtained using pyScaf assembler with N50 of 16,54,800 bp. In addition, 152 transcription factors involved in various biological processes were identified and a total of 682 secretory proteins were predicted using secretome analysis. ToxA gene (535bp) was analyzed and identified in the genome of B. sorokiniana which revealed 100% homology with ToxA gene of Pyrenophora tritici repentis. Further, ToxA gene was amplified, sequenced and validated in the 39 isolates of B. sorokiniana which confirmed the presence of ToxA gene in all the isolates of B. sorokiniana. All these ToxA sequences were submitted in NCBI database (MN601358-MN601396). As ToxA gene interacts with Tsn1 gene of host, 13 wheat genotypes were evaluated for the Tsn1 gene and five genotypes (38.4%) were found to be Tsn1 positive with more severe necrotic lesions compared to Tsn1 negative wheat genotypes. In vitro expression analysis of ToxA gene in B. sorokiniana isolate (BS112) using qPCR revealed maximum upregulation (14.67 fold) at 1st day after inoculation (DAI). Further, in planta expression analysis of ToxA gene in Tsn1 positive and Tsn1 negative genotypes, Agra local and Chiriya 7 respectively was also conducted. Results revealed maximum expression (7.89 fold) of ToxA gene in Tsn1 positive genotype, Agra local at 5th DAI compared to Tsn1 negative genotype Chiriya 7 which showed minimum expression (0.048 fold) at 5th DAI. Conclusions: Full genome of B. sorokiniana was sequenced; secreted proteins and virulence genes were identified in the genome. ToxA gene was validated in thirty nine isolates of B. sorokiniana. In planta ToxA-Tsn1 interaction studies established that spot blotch disease is more severe in Tsn1 positive genotypes. This genomic resource will provide a new insight into better understanding and management of spot blotch disease and B. sorokiniana of wheat.

Genomic Structure and Evolution of Multigene Families: “Flowers” on the Human Genome

We report the results of an extensive investigation of genomic structures in the human genome, with a particular focus on relatively large repeats (>50 kb) in adjacent chromosomal regions. We named such structures “Flowers” because the pattern observed on dot plots resembles a flower. We detected a total of 291 Flowers in the human genome. They were predominantly located in euchromatic regions. Flowers are gene-rich compared to the average gene density of the genome. Genes involved in systems receiving environmental information, such as immunity and detoxification, were overrepresented in Flowers. Within a Flower, the mean number of duplication units was approximately four. The maximum and minimum identities between homologs in a Flower showed different distributions; the maximum identity was often concentrated to 100% identity, while the minimum identity was evenly distributed in the range of 78% to 100%. Using a gene conversion detection test, we found frequent and/or recent gene conversion events within the tested Flowers. Interestingly, many of those converted regions contained protein-coding genes. Computer simulation studies suggest that one role of such frequent gene conversions is the elongation of the life span of gene families in a Flower by the resurrection of pseudogenes.

A Large Rearrangement Involving Genes and Low-Copy DNA Interrupts the Microcollinearity Between Rice and Barley at theRph7Locus

Grass genomes differ greatly in chromosome number, ploidy level, and size. Despite these differences, very good conservation of the marker order (collinearity) was found at the genetic map level between the different grass genomes. Collinearity is particularly good between rice chromosome 1 and the group 3 chromosomes in the Triticeae. We have used this collinearity to saturate the leaf rust resistance locus Rph7 on chromosome 3HS in barley with ESTs originating from rice chromosome 1S. Chromosome walking allowed the establishment of a contig of 212 kb spanning the Rph7 resistance gene. Sequencing of the contig showed an average gene density of one gene/20 kb with islands of higher density. Comparison with the orthologous rice sequence revealed the complete conservation of five members of the HGA gene family whereas intergenic regions differ greatly in size and composition. In rice, the five genes are closely associated whereas in barley intergenic regions are >38-fold larger. The size difference is due mainly to the presence of six additional genes as well as noncoding low-copy sequences. Our data suggest that a major rearrangement occurred in this region since the Triticeae and rice lineage diverged.