High Throughput Polymorphism Discovery and Genotyping in a Mutant Population of Jatropha curcas

Jatropha curcas, an economically important biofuel crop, can grow under unfavorable climatic and soil conditions that are unsuitable for food production. However, the lack of high genetic variability in J. curcas limits the success of breeding programs and makes it harder to exploit the full potential of Jatropha, thus requiring other strategies to increase the genetic basis using chemical induced mutations.  In order to screen rare mutations in genes of interest and to understand gene function on a whole-genome scale, Double Digest RADseq (ddRADseq) and normalized Genotyping-by-Sequencing (nGBS) based on Illumina sequencing were used on a mutant population generated from 100 seeds of J. curcas treated with Ethyl Methane Sulphonate.  Single nucleotide polymorphisms (SNPs) calling of the raw sequencing data was performed using the Illumina analysis pipeline. High quality reads were mapped to a reference sequence, using Burrows-Wheeler Aligner 0.7.5a. SNP discovery and genotyping of samples were performed and, finally, high confidence SNPs and indels were annotated and identified by applying appropriate filter conditions. Results showed that SNPs discovery was robust in all samples, and the method provided high sensitivity and specificity as well as higher numbers of SNPs from NGBS over ddRADseq. This could be explained by the relative amount of genome methylation.  Reverse genetic approaches allowed the detection of induced point mutations in mutagenized Jatropha populations, which can help to identify novel variation for Jatropha breeding. Finally, the combination of random mutagenesis with high throughput mutation discovery revealed a spectrum of stable point-alleles.