Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Abstract

Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of next-generation sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, which encodes a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of (1) a sequenced genome, (2) a strongly penetrant and easily measurable phenotype, and (3) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the postgenomic era.

Natural Variation in Synthesis and Catabolism Genes Influences Dhurrin Content in Sorghum

“…In this study we identified a wide range of genetic variability for leaf dhurrin accumulation within the SC panel. Because dhurrin is known to play an active role as a plant herbivory defense compound (Krothapalli et al, 2013; Gleadow and Møller, 2014), as well as other potential roles such as an osmoprotectant and an N storage compound, it could be an evolutionary advantage for many sorghums to contain elevated levels of dhurrin. Interestingly, when we screened multiple breeding lines commonly used as parents in commercial breeding programs, most lines were nearly devoid of dhurrin or were well below average levels seen in the SC panel (data not presented).…”

“…As a plant metabolite, dhurrin is thought to play an important role during the growth and development of sorghum. Examples of dhurrin's role include defense against insect hervibory (Krothapalli et al, 2013), as a storage source of nitrogen, and as an osmoprotectant (Busk and Møller, 2002). Recently, Burke et al (2013) observed significant variation in leaf dhurrin content among sorghum cultivars differing in pre‐ and post‐flowering drought tolerance, providing evidence that the metabolite can act as an osmoprotectant when sorghum is exposed to limited water.…”

A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses

“…The development of TILLING (targeting induced local lesions in genomes) techniques, which can detect singlenucleotide mismatches in heteroduplexes of PCR amplicons, made EMS mutagenesis a workhorse technique for the study of recalcitrant metabolic and signaling processes in plants, animals, and microorganisms (Greene et al, 2003;Winkler et al, 2005;Gilchrist et al, 2006). In sorghum, a few mutated genes have been identified by TILLING based on individual amplicons Blomstedt et al, 2012;Krothapalli et al, 2013). Recent advances in high-throughput next-generation sequencing have made it possible to catalog EMS-induced mutations on a large scale (Thompson et al, 2013;Henry et al, 2014).…”

Discovery of a Dhurrin QTL in Sorghum: Co‐localization of Dhurrin Biosynthesis and a Novel Stay‐green QTL

“…Although HCN is known to be highly toxic and volatile, the metabolite dhurrin itself is sequestered within the vacuoles of plant cells and is non‐toxic (Busk and Møller, 2002). Dhurrin has also been proposed to function in plant defense against insect herbivory, and as a storage source of N with osmoprotectant properties (Burke et al, 2015; Busk and Møller, 2002; Krothapalli et al, 2013). Recently, Burke et al (2013) demonstrated a correlation between elevated leaf dhurrin levels before flowering and enhanced stay‐green.…”