Comprehensive reverse genetic resources, which have been key to understanding gene function in diploid model organisms, are missing in many polyploid crops. Young polyploid species such as wheat, which was domesticated less than 10,000 y ago, have high levels of sequence identity among subgenomes that mask the effects of recessive alleles. Such redundancy reduces the probability of selection of favorable mutations during natural or human selection, but also allows wheat to tolerate high densities of induced mutations. Here we exploited this property to sequence and catalog more than 10 million mutations in the protein-coding regions of 2,735 mutant lines of tetraploid and hexaploid wheat. We detected, on average, 2,705 and 5,351 mutations per tetraploid and hexaploid line, respectively, which resulted in 35–40 mutations per kb in each population. With these mutation densities, we identified an average of 23–24 missense and truncation alleles per gene, with at least one truncation or deleterious missense mutation in more than 90% of the captured wheat genes per population. This public collection of mutant seed stocks and sequence data enables rapid identification of mutations in the different copies of the wheat genes, which can be combined to uncover previously hidden variation. Polyploidy is a central phenomenon in plant evolution, and many crop species have undergone recent genome duplication events. Therefore, the general strategy and methods developed herein can benefit other polyploid crops.

Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single-nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either bidimensional or tridimensional pooling schemes. The method compared favorably with other molecular and computational approaches, providing high sensitivity and specificity.

Key message This study identifies a small distal region of the 1RS chromosome from rye that has a positive impact on wheat yield. Abstract The translocation of the short arm of rye (Secale cereale L.) chromosome one (1RS) onto wheat (Triticum aestivum L.) chromosome 1B (1RS.1BL) is used in wheat breeding programs worldwide due to its positive effect on yield, particularly under abiotic stress. Unfortunately, this translocation is associated with poor bread-making quality. To mitigate this problem, the 1RS arm was engineered by the removal and replacement of two interstitial rye segments with wheat chromatin: a distal segment to introduce the Glu-B3/Gli-B1 loci from wheat, and a proximal segment to remove the rye Sec-1 locus. We used this engineered 1RS chromosome (henceforth 1RS^WW) to develop and evaluate two sets of 1RS/1RS^WW near isogenic lines (NILs). Field trials showed that standard 1RS lines had significantly higher yield and better canopy water status than the 1RS^WW NILs in both well-watered and water-stressed environments. We intercrossed the 1RS and 1RS^WW lines and generated two additional NILs, one carrying the distal (1RS^RW) and the other carrying the proximal (1RS^WR) wheat segment. Lines not carrying the distal wheat region (1RS and 1RS^WR) showed significant improvements in grain yield and canopy water status compared to NILs carrying the distal wheat segment (1RS^WW and 1RS^RW), indicating that the 1RS region replaced by the distal wheat segment carries the beneficial allele(s). NILs without the distal wheat segment also showed higher carbon isotope discrimination and increased stomatal conductance, suggesting that these plants had improved access to water. The 1RS^WW, 1RS^WR and 1RS^RW NILs have been deposited in the National Small Grains Collection.

Forward genetic screens of induced mutant plant populations are powerful tools to identify genes underlying phenotypes of interest. Using traditional techniques, mapping causative mutations from forward screens is a lengthy, multi-step process, requiring the identification of a broad genetic region followed by candidate gene sequencing to characterize the causal variant. Mapping by whole genome sequencing accelerates the identification of causal mutations by simultaneously defining a mapping region and providing information on the induced genetic variants. In wheat, although the availability of a high-quality draft genome assembly facilitates mapping and mutation calling, whole genome resequencing remains prohibitively expensive due to its large genome. In the current study, we used exome sequencing as a complexity reduction strategy to detect mutations associated with a target phenotype. In a segregating wheat EMS population, we identified a clear peak region on chromosome arm 4BS associated with increased plant height. Although none of the significant SNPs seemed causative for the mutant phenotype, they were sufficient to identify a linked ~ 1.9 Mb deletion encompassing nine genes. These genes included Rht-B1, which is known to have a strong effect on plant height and is a strong candidate for the observed phenotype. We performed simulation experiments to determine the impacts of sequencing depth and bulk size and discuss the importance of considering each factor when designing mapping-by-sequencing experiments in wheat. This approach can accelerate the identification of candidate causal point mutations or linked deletions underlying important phenotypes.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

Genetic markers are essential when developing or working with genetically variable populations. Indel Group in Genomes (IGG) markers are primer pairs that amplify single-locus sequences that differ in size for two or more alleles. They are attractive for their ease of use for rapid genotyping and their codominant nature. Here, we describe a heuristic algorithm that uses a k-mer-based approach to search two or more genome sequences to locate polymorphic regions suitable for designing candidate IGG marker primers. As input to the IGG pipeline software, the user provides genome sequences and the desired amplicon sizes and size differences. Primer sequences flanking polymorphic insertions/deletions are produced as output. IGG marker files for three sets of genomes, Solanum lycopersicum/Solanum pennellii, Arabidopsis (Arabidopsis thaliana) Columbia-0/Landsberg erecta-0 accessions, and S. lycopersicum/S. pennellii/Solanum tuberosum (three-way polymorphic) are included.