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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.

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. Introgression of the short arm of rye chromosome one into common wheat increases root biomass and drought tolerance, but the underlying genetic basis is unknown. Here, the authors report that dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate the differences of wheat root architecture.

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.

Good understanding of the genes controlling root development is required to engineer root systems better adapted to different soil types. In wheat (Triticum aestivum L.), the 1RS.1BL wheat–rye (Secale cereale L.) translocation has been associated with improved drought tolerance and a large root system. However, an isogenic line carrying an interstitial segment from wheat chromosome arm 1BS in the distal region of the 1RS arm (1RSRW) showed reduced grain yield and shorter roots both in the field and in hydroponic cultures relative to isogenic lines with the complete 1RS arm. In this study, we used exome capture to characterize 1RSRW and its parental lines T‐9 and 1B+40. We show that 1RSRW has a 7.0 Mb duplicated 1RS region and a 4.8 Mb 1BS insertion colinear with the 1RS duplication, resulting in triplicated genes. Lines homozygous for 1RSRW have short seminal roots, while lines heterozygous for this chromosome have roots of intermediate length. By contrast, near‐isogenic lines carrying only the 1BS distal region or the 1RS‐1BS duplication have long seminal roots similar to 1RS, suggesting a limited effect of the 1BS genes. These results suggest that the dosage of duplicated 1RS genes is critical for seminal root length. An induced deletion encompassing 38 orthologous wheat and rye duplicated genes restored root length and confirmed the importance of gene dosage in the short‐root phenotype. We explored the expression profiles and functional annotation of these genes and discuss their potential as candidate genes for the regulation of seminal root length in wheat.

Key messageChromosome regions affecting grain yield, grain yield components and plant water status were identified and validated in fall-sown spring wheats grown under full and limited irrigation.Increases in wheat production are required to feed a growing human population. To understand the genetic basis of grain yield in fall-sown spring wheats, we performed a genome-wide association study (GWAS) including 262 photoperiod-insensitive spring wheat accessions grown under full and limited irrigation treatments. Analysis of molecular variance showed that 4.1% of the total variation in the panel was partitioned among accessions originally developed under fall-sowing or spring-sowing conditions, 11.7% among breeding programs within sowing times and 84.2% among accessions within breeding programs. We first identified QTL for grain yield, yield components and plant water status that were significant in at least three environments in the GWAS, and then selected those that were also significant in at least two environments in a panel of eight biparental mapping populations. We identified and validated 14 QTL for grain yield, 15 for number of spikelets per spike, one for kernel number per spike, 11 for kernel weight and 9 for water status, which were not associated with differences in plant height or heading date. We detected significant correlations among traits and colocated QTL that were consistent with those correlations. Among those, grain yield and plant water status were negatively correlated in all environments, and six QTL for these traits were colocated or tightly linked (< 1 cM). QTL identified and validated in this study provide useful information for the improvement of fall-sown spring wheats under full and limited irrigation.

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 1RSWW) to develop and evaluate two sets of 1RS/1RSWW near isogenic lines (NILs). Field trials showed that standard 1RS lines had significantly higher yield and better canopy water status than the 1RSWW NILs in both well-watered and water-stressed environments. We intercrossed the 1RS and 1RSWW lines and generated two additional NILs, one carrying the distal (1RSRW) and the other carrying the proximal (1RSWR) wheat segment. Lines not carrying the distal wheat region (1RS and 1RSWR) showed significant improvements in grain yield and canopy water status compared to NILs carrying the distal wheat segment (1RSWW and 1RSRW), 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 1RSWW, 1RSWR and 1RSRW NILs have been deposited in the National Small Grains Collection.

The introgression of a small segment of wheat (Triticum aestivum L.) chromosome arm 1BS in the distal region of the rye (Secale cereale L.) 1RS.1BL arm translocation in wheat (henceforth 1RSRW) was previously associated with reduced grain yield, carbon isotope discrimination, and stomatal conductance, suggesting reduced access to soil moisture. Here we show that lines with the normal 1RS arm have longer roots than lines with the 1RSRW arm in both field and hydroponic experiments. In the 1RSRW lines, differences in seminal root length were associated with a developmentally regulated arrest of the root apical meristem (RAM). Approximately 10 d after germination, the seminal roots of the 1RSRW plants showed a gradual reduction in elongation rate, and stopped growing a week later. Seventeen days after germination, the roots of the 1RSRW plants showed altered gradients of reactive oxygen species and emergence of lateral roots close to the RAM, suggesting changes in the root meristem. The 1RSRW lines also showed reduced biomass (estimated by the normalized difference vegetation index) and grain yield relative to the 1RS lines, with larger differences under reduced or excessive irrigation than under normal irrigation. These results suggest that this genetic variation could be useful to modulate root architecture.

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.

Wheat is an important staple food crop with an annual production of ∼700 million tons, which accounts for roughly 20 % of calories and protein consumed by the human population. Yields must be increased to feed the rapidly growing world population, but water stress is a major constraint to increasing yields. One potential source of traits for improving water stress tolerance is from the introgression of chromatin from related species into wheat, often through chromosome arm translocations. One such alien chromosome arm introgression, the short arm of chromosome arm 1 from rye (1RS) has been used extensively in wheat breeding programs worldwide for its disease resistance and abiotic stress tolerance characteristics. One drawback of the 1RS translocation in wheat is that it reduces bread making quality. To remedy this, a recombinant 1RS chromosome arm was previously engineered with two interstitial wheat segments (henceforth 1RSWW) to remove the Sec-1 locus from rye and introduce the Glu-B3/Gli-B1 locus from wheat. The 1RSWW chromosome arm was introgressed into several wheat varieties containing the 1RS translocation, and field studies showed that grain yield was substantially decreased when the 1RSWW chromosome arm was present, particularly under drought stress conditions. The two interstitial wheat segments were separated and further field testing showed that the presence of the distal wheat segment (1RSRW) was associated with decreased yield, while the presence of the proximal wheat segment (1RSWR) had no effect. Physiological characterization showed that 1RS and 1RSWR plants had lower water use efficiency than the low yielding 1RSWW and 1RS RW plants. There was no difference in photosynthetic rate between the two groups, suggesting that the 1RS and 1RSWR plants had better access to stored soil moisture, presumably because of differences in the root system. Flow sorted telosomic 1RS and 1RSRW chromosome arms were sequenced and it was found that the interstitial wheat segment in the 1RSRW chromosome arm was not a replacement of rye chromatin, as originally hypothesized, but was an insertion of wheat chromatin into the 1RS arm. In the inserted wheat region 28 genes were identified, two of which were prioritized for further investigation based on their high expression in roots and low expression in other tissues. Both of the genes were involved in jasmonic acid biosynthesis, which has been shown to inhibit root growth. Using two large Targeting Induced Local Lesions IN Genomes (TILLING) populations in tetraploid and hexaploid wheat, mutations were identified in 25 of the 28 genes, with three genes having no mutations detected. Of the 25 genes with mutations, a total of 50 truncation mutations were identified in 15 of the 25 genes. There were missense mutations in 22 of the 25 genes with 991 missense mutations in total. No truncation mutations were detected in the two high priority candidate genes, but one of them contained 63 missense mutations, 42 of which were predicted by the SIFT (Sorting Intolerant From Tolerant) program to have a severe impact on protein function, with three missense mutations in active sites. For the other candidate, ten missense mutations were detected, seven of which were predicted to have a severe impact on protein function. These identified mutations will be tested to confirm whether or not they are involved in the drought stress response.