Forward Genetics by Sequencing EMS Variation-Induced Inbred Lines

Abstract

In order to leverage novel sequencing techniques for cloning genes in eukaryotic organisms with complex genomes, the false positive rate of variant discovery must be controlled for by experimental design and informatics. We sequenced five lines from three pedigrees of ethyl methanesulfonate (EMS)-mutagenized Sorghum bicolor, including a pedigree segregating a recessive dwarf mutant. Comparing the sequences of the lines, we were able to identify and eliminate error-prone positions. One genomic region contained EMS mutant alleles in dwarfs that were homozygous reference sequences in wild-type siblings and heterozygous in segregating families. This region contained a single nonsynonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in the Sorghum ortholog encoding the gibberellic acid (GA) biosynthetic enzyme ent-kaurene oxidase. Application of exogenous GA rescued the mutant phenotype. Our method for mapping did not require outcrossing and introduced no segregation variance. This enables work when line crossing is complicated by life history, permitting gene discovery outside of genetic models. This inverts the historical approach of first using recombination to define a locus and then sequencing genes. Our formally identical approach first sequences all the genes and then seeks cosegregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extension of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.

Common metabolic networks contribute to carbon sink strength of sorghum internodes: implications for bioenergy improvement

“…In addition, sweet sorghum can accumulate considerable amount of starch in the internode [11] and has differential expression patterns of the cell wall-related genes compared to non-sweet genotypes [12], indicating that the distribution of carbon utilization within sweet sorghum internodes may be redirected to establish sink strength. Also, sorghum is an emerging bioenergy crop with multiple advantages: (i) a ~ 730-Mb diploid genome and several reference assemblies with great synteny to maize and sugarcane [13–16]; (ii) good tolerance to several abiotic stresses and desirable agronomical features, such as the stay-green trait [7, 17, 18]; (iii) rich genetic resources [19], such as several EMS resources [20–22]; (iv) ability to be transformed and genome-edited [23, 24]; (iv) potential in phytoremediation of soil pollution [25].…”

Mapping-by-Sequencing via MutMap Identifies a Mutation in ZmCLE7 Underlying Fasciation in a Newly Developed EMS Mutant Population in an Elite Tropical Maize Inbred

“…To solve these difficulties, several approaches combining next generation sequencing and mapping populations generated in the same genetic background have been developed. These include crossing to the unmutagenized parent in the Mutmap method in rice [27] and in maize [28], or crossing to a normal looking plant of a different M2 family (the so called "evil twin" in Sorghum [29]) or even not crossing at all, but only sequencing M3 plants as in Mutmap + (rice) [30], or sequencing individual M2 plants combined with zygosity analysis via progeny in maize [9]. These approaches utilized only the segregating mutagen-induced variants between the two parents or siblings to map, and hence they all shared the limitation of relying on the low number of mutagen-induced variants.…”

Whole-Genome Sequence Accuracy Is Improved by Replication in a Population of Mutagenized Sorghum

“…We identified and cataloged the relatively few genomic positions of the sorghum reference genome, consisting of 57,872 positions, that were most probably error-prone, and which produced 4,123,621 SNP calls of high statistical quality (File S2). Similar approaches for elucidating this experimentally identifiable set of replicate SNP positions in any species, which lack the expected EMS mutation spectrum, correspond to repeated sequences, and contain paralogs with the predicted SNP, should improve the accuracy of SNP calling ( Henry et al 2014 ; Addo-Quaye et al 2017 ). Similarly, filtering of the indel alleles retained <1% (17,760) as nonreplicate indels ( Figure S4 and Tables S7 and S8 in File S26 ).…”

“…Thus, it offers a potentially simpler system in which to discover mechanisms of drought tolerance, plant nutrition, weed biology, the evolution of C4 photosynthesis, and undertake biofuel research. Recent forward genetics studies using EMS mutagenesis have provided key insights into the function of genes involved in insect–herbivory defense, cell wall and lignin biosynthesis, protein digestibility, leaf vein density, and plant height ( Pedersen et al 2005 ; Peters et al 2009 ; Xin et al 2009 ; Blomstedt et al 2012 ; Koegel et al 2013 ; Krothapalli et al 2013 ; Petti et al 2013 , 2015 ; Wu et al 2013 ; Sattler et al 2014 ; Rizal et al 2015 ; Scully et al 2015 ; Addo-Quaye et al 2017 ). The recent characterization of mutations in pooled samples derived from a population of 256 EMS-mutagenized sorghum BTx623 lines is a major step toward improving resources for sorghum genetics ( Jiao et al 2016 ).…”