The current scenario and future perspectives of transgenic oilseed mustard by CRISPR-Cas9.

PURPOSE: Production of a designer crop having added attributes is the primary goal of all plant biotechnologists. Specifically, development of a crop with a simple biotechnological approach and at a rapid pace is most desirable. Genetic engineering enables us to displace genes among species. The newly incorporated foreign gene(s) in the host genome can create a new trait(s) by regulating the genotypes and/or phenotypes. The advent of the CRISPR-Cas9 tools has enabled the modification of a plant genome easily by introducing mutation or replacing genomic fragment. Oilseed mustard varieties (e.g., Brassica juncea, Brassica nigra, Brassica napus, and Brassica carinata) are one such plants, which have been transformed with different genes isolated from the wide range of species. Current reports proved that the yield and value of oilseed mustard has been tremendously improved by the introduction of stably inherited new traits such as insect and herbicide resistance. However, the genetic transformation of oilseed mustard remains incompetent due to lack of potential plant transformation systems. To solve numerous complications involved in genetically modified oilseed mustard crop varieties regeneration procedures, scientific research is being conducted to rectify the unwanted complications. Thus, this study provides a broader overview of the present status of new traits introduced in each mentioned varieties of oilseed mustard plant by different genetical engineering tools, especially CRISPR-Cas9, which will be useful to improve the transformation system of oilseed mustard crop plants. METHODS: This review presents recent improvements made in oilseed mustard genetic engineering methodologies by using CRISPR-Cas9 tools, present status of new traits introduced in oilseed mustard plant varieties. RESULTS: The review highlighted that the transgenic oilseed mustard production is a challenging process and the transgenic varieties of oilseed mustard provide a powerful tool for enhanced mustard yield. Over expression studies and silencing of desired genes provide functional importance of genes involved in mustard growth and development under different biotic and abiotic stress conditions. Thus, it can be expected that in near future CRISPR can contribute enormously in improving the mustard plant's architecture and develop stress resilient oilseed mustard plant species.

A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes.

Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species-B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single-molecule real-time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short-read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping-by-sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome-scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.

Mining of Cloned Disease Resistance Gene Homologs (CDRHs) in Brassica Species and Arabidopsis thaliana.

Various diseases severely affect Brassica crops, leading to significant global yield losses and a reduction in crop quality. In this study, we used the complete protein sequences of 49 cloned resistance genes (R genes) that confer resistance to fungal and bacterial diseases known to impact species in the Brassicaceae family. Homology searches were carried out across Brassica napus, B. rapa, B. oleracea, B. nigra, B. juncea, B. carinata and Arabidopsis thaliana genomes. In total, 660 cloned disease R gene homologs (CDRHs) were identified across the seven species, including 431 resistance gene analogs (RGAs) (248 nucleotide binding site-leucine rich repeats (NLRs), 150 receptor-like protein kinases (RLKs) and 33 receptor-like proteins (RLPs)) and 229 non-RGAs. Based on the position and distribution of specific homologs in each of the species, we observed a total of 87 CDRH clusters composed of 36 NLR, 16 RLK and 3 RLP homogeneous clusters and 32 heterogeneous clusters. The CDRHs detected consistently across the seven species are candidates that can be investigated for broad-spectrum resistance, potentially providing resistance to multiple pathogens. The R genes identified in this study provide a novel resource for the future functional analysis and gene cloning of Brassicaceae R genes towards crop improvement.