Abstract
Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.
Highlights
Jatropha curcas originates from Central America and currently is distributed throughout all tropical and subtropical regions
To screen for novel mutations in genes of interest and to understand in silico gene function prediction on a whole-genome scale, Normalized Genotyping by Sequencing (nGBS) and ddGBs based on Illumina sequencing were used on 82 mutants and 14 wild-type accessions of J. curcas
Obtained data showed that allelic variations and in silico analysis of gene functions could be rapidly identified in mutant populations using nGBS and digested GBS (ddGBS)
Summary
Jatropha curcas (family Euphorbiaceae) originates from Central America and currently is distributed throughout all tropical and subtropical regions. It could be one of the most valuable crops, as a biofuel plant and due to the high protein amount in its seeds, which make it attractive as animal feed and organic manure. Still, detailed information of the genetic landscape and gene function is a premise to (a) uncover why some accessions of J. curcas produce toxins and others not, (b) why – with their narrow genetic variation – J. curcas can grow in such diverse environmental conditions, (c) know, how biochemistry contributes to increases in oil yield and quality, (d) understand the processes of growth and development
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