Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Novel Chitinase Gene LOC_Os11g47510 from Indica Rice Tetep Provides Enhanced Resistance against Sheath Blight Pathogen Rhizoctonia solani in Rice.

Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

Functional Characterization of Novel Chitinase Genes Present in the Sheath Blight Resistance QTL: qSBR11-1 in Rice Line Tetep.

Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 h after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.