Anther Culture in Jatropha curcas L.: A Tree Species.

The obstacles to breeding programs in Jatropha are the long reproductive cycle with a juvenile phase that lasts several months, the highly heterozygous nature of the genome, the large canopy size, and self-incompatibility that is a long-term process which requires multiple cycles of self-pollination to achieve complete homozygosity. In vitro plant tissue culture-based tools such as haploids and doubled haploid techniques can increase the selection efficiency, resulting into selection of superior plants with complete homozygosity in one generation. It bypasses the complications of greenhouse field evaluation or off-season generation advancement, which takes about 8-10 generations in traditional breeding with the time line of 10-12 years. The haploids have in fact a single set of chromosomes, which undergoes duplication spontaneously during in vitro culture conditions, and are further converted into doubled haploid plants. This represents a major biotechnological tool to accelerate plant breeding. Here, we have established a reproducible, unique anther culture protocol in Jatropha curcas to develop haploid and doubled haploid plants.

Exploitation of Hi-C sequencing for improvement of genome assembly and in-vitro validation of differentially expressing genes in Jatropha curcas L.

Jatropha curcas is one of the major sources of renewable energy due to potential use of its oil as a biofuel. The genome of this crop is constituted by the high content of repetitive elements. We employed the Hi-C proximity ligation technique to re-scaffold our existing hybrid genome assembly of an elite genotype (RJC1) developed using Illumina and Pacbio technologies. We assembled 99.81% of non-truncated reads to achieve 266.80 Mbp of the genome with an N50 value of 1.58 Mb. Furthermore, we compared the efficiency of Hi-C-augmented genome assembly with the hybrid genome assembly and observed a ~ 50% reduction in scaffolds and a tenfold increase in the N50 value. The gene ontology analysis revealed the identification of terms for molecular function (45.52%), cellular component (33.47%), and biological function (20.99%). Comparative genomic analysis of 13-plant species showed the conservation of 414 lipid metabolizing genes identified in the KEGG pathway analysis. Differential gene expression (DGE) studies were conducted in the healthy and Jatropha mosaic virus-infected leaves via RNA-seq analysis and observed gene expression changes for 2185 genes. Out of these, we observed 546 genes having more than two-fold change of transcript level and among these 259 genes were down-regulated and 287 genes were up-regulated. To validate RNA-seq data, two DEGs were selected for gene expression analysis using qRT-PCR and the data was in correlation with in silico results. RNA-seq analysis further shows the identification of some of the candidate genes and may be useful to develop JMV resistant plants after functional validation. This Hi-C genome assembly provides a detailed accurate reference genome which could be utilized to improve Jatropha and other economically important Euphorbiaceae family members.