Systematic prediction of EMS-induced mutations in a sorghum mutant population.

The precise detection of causal DNA mutations (deoxyribonucleic acid) is very crucial for forward genetic studies. Several sources of errors contribute to false-positive detections by current variant-calling algorithms, which impact associating phenotypes with genotypes. To improve the accuracy of mutation detection, we implemented a binning method for the accurate detection of likely ethyl methanesulfonate (EMS)-induced mutations in a sequenced mutant population. We also implemented a clustering algorithm for detecting likely false negatives with high accuracy. Sorghum bicolor is a very valuable crop species with tremendous potential for uncovering novel gene functions associated with highly desirable agronomical traits. We demonstrate the precision of the described approach in the detection of likely EMS-induced mutations from the publicly available low-cost sequencing of the M3 generation from 600 sorghum BTx623 mutants. The approach detected 3,274,606 single nucleotide polymorphisms (SNPs), of which 96% (3,141,908) were G/C to A/T DNA substitutions, as expected by EMS-mutagenesis mode of action. We demonstrated the general applicability of the described method and showed a high concordance, 94% (3,074,759) SNPs overlap between SAMtools-based and GATK-based variant-calling algorithms. Our clustering algorithm uncovered evidence for an additional 223,048 likely false-negative shared EMS-induced mutations. The final 3,497,654 SNPs represent an 87% increase in SNPs detected from the previous analysis of the mutant population, with an average of one SNP per 125 kb in the sorghum genome. Annotation of the final SNPs revealed 10,263 high-impact and 136,639 moderate-impact SNPs, including 7217 stop-gained mutations, which averages 12 stop-gained mutations per mutant, and four high- or medium-impact SNPs per sorghum gene. We have implemented a public search database for this new genetic resource of 30,285 distinct sorghum genes containing medium- or high-impact EMS-induced mutations. Seedstock for a select 486 of the 600 described mutants are publicly available in the Germplasm Resources Information Network (GRIN) database.

Natural variation at sympathy for the ligule controls penetrance of the semidominant Liguleless narrow-R mutation in Zea mays.

Leaf architecture determines plant structural integrity, light harvesting, and economic considerations such as plant density. Ligules, junctions at the leaf sheath and blade in grasses, protect stalks from environmental stresses and, in conjunction with auricles, controls leaf angle. Previous studies in mutants have recessive liguleless mutants (lg1 and lg2) and dominant mutations in knotted1-like homeobox genes (Lg3-O, Lg4, and Kn1) involved in ligule development. Recently, a new semidominant liguleless mutant, Liguleless narrow (Lgn-R), has been characterized in maize that affects ligule and auricle development and results in a narrow leaf phenotype. We show that quantitative genetic variation affects penetrance of Lgn-R. To examine the genetic architecture underlying Lgn-R expressivity, crosses between Lgn-R/+ mutants in a B73 background and intermated B73 x Mo17 recombinant inbred lines were evaluated in multiple years and locations. A single main-effect quantitative trait locus (QTL) on chromosome 1 (sympathy for the ligule; sol) was discovered with a Mo17-contributed allele that suppressed Lgn-R mutant phenotypes. This QTL has a genetic-interaction with a locus on chromosome 7 (lucifer; lcf) for which the B73-contributed allele increases the ability of the sol(Mo17) allele to suppress Lgn-R. Neither of the genetic intervals likely to contain sol or lcf overlap with any current liguleless genes nor with previously identified genome-wide association QTL connected to leaf architecture. Analysis of phenotypes across environments further identified a genotype by enviroment interaction determining the strength of the sol x lcf interaction.

Forward genetics by genome sequencing reveals that rapid cyanide release deters insect herbivory of Sorghum bicolor.

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.