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To improve the efficiency of high-density genotype data storage and imputation in bread wheat (Triticum aestivum L.), we applied the Practical Haplotype Graph (PHG) tool. The Wheat PHG database was built using whole-exome capture sequencing data from a diverse set of 65 wheat accessions. Population haplotypes were inferred for the reference genome intervals defined by the boundaries of the high-quality gene models. Missing genotypes in the inference panels, composed of wheat cultivars or recombinant inbred lines genotyped by exome capture, genotyping-by-sequencing (GBS), or whole-genome skim-seq sequencing approaches, were imputed using the Wheat PHG database. Though imputation accuracy varied depending on the method of sequencing and coverage depth, we found 92% imputation accuracy with 0.01× sequence coverage, which was slightly lower than the accuracy obtained using the 0.5× sequence coverage (96.6%). Compared to Beagle, on average, PHG imputation was ∼3.5% (P-value < 2 × 10-14) more accurate, and showed 27% higher accuracy at imputing a rare haplotype introgressed from a wild relative into wheat. We found reduced accuracy of imputation with independent 2× GBS data (88.6%), which increases to 89.2% with the inclusion of parental haplotypes in the database. The accuracy reduction with GBS is likely associated with the small overlap between GBS markers and the exome capture dataset, which was used for constructing PHG. The highest imputation accuracy was obtained with exome capture for the wheat D genome, which also showed the highest levels of linkage disequilibrium and proportion of identity-by-descent regions among accessions in the PHG database. We demonstrate that genetic mapping based on genotypes imputed using PHG identifies SNPs with a broader range of effect sizes that together explain a higher proportion of genetic variance for heading date and meiotic crossover rate compared to previous studies.

To improve the efficiency of high-density genotype data storage and imputation in bread wheat (Triticum aestivum L.), we applied the Practical Haplotype Graph (PHG) tool. The wheat PHG database was built using whole-exome capture sequencing data from a diverse set of 65 wheat accessions. Population haplotypes were inferred for the reference genome intervals defined by the boundaries of the high-quality gene models. Missing genotypes in the inference panels, composed of wheat cultivars or recombinant inbred lines genotyped by exome capture, genotyping-by-sequencing (GBS), or whole-genome skim-seq sequencing approaches, were imputed using the wheat PHG database. Though imputation accuracy varied depending on the method of sequencing and coverage depth, we found 92% imputation accuracy with 0.01x sequence coverage, which was slightly lower than the accuracy obtained using the 0.5x sequence coverage (96.6%). Compared to Beagle, on average, PHG imputation was ∼3.5% (p-value < 2 x 10-14) more accurate, and showed 27% higher accuracy at imputing a rare haplotype introgressed from a wild relative into wheat. We found reduced accuracy of imputation with independent 2x GBS data (88.6%), which increases to 89.2% with the inclusion of parental haplotypes in the database. The accuracy reduction with GBS is likely associated with the small overlap between GBS markers and the exome capture dataset, which was used for constructing PHG. The highest imputation accuracy was obtained with exome capture for the wheat D genome, which also showed the highest levels of linkage disequlibrium and proportion of identity-by-descent regions among accessions in the PHG database. We demonstrate that genetic mapping based on genotypes imputed using PHG identifies SNPs with a broader range of effect sizes that together explain a higher proportion of genetic variance for heading date and meiotic crossover rate compared to previous studies.

Soil-borne wheat mosaic virus (SBWMV) causes a serious viral disease that can significantly reduce grain yield in winter wheat worldwide. Using resistant cultivars is the only feasible strategy to reduce the losses caused by SBWMV. To fine map the resistance gene Sbwm1, 205 wheat accessions was genotyped using wheat Infinium iSelect Beadchips with 90 K SNPs. Association analysis identified 35 SNPs in 12 wheat genes and one intergenic SNP in the Sbwm1 region that showed a significant association with SBWMV resistance. Those SNPs were converted into Kompetitive Allele-Specific Polymerase assays (KASP) and analyzed in two F6-derived recombinant inbred line (RIL) populations derived from the crosses between two resistant cultivars Wesley and Deliver and a susceptible line OK03825-5403-6. Linkage analysis mapped this gene on chromosome 5D at intervals of 5.1 cM and 3.4 cM in the two populations, respectively. The two flanking markers in both populations delimited the gene to a 620 kb region where 19 genes were annotated. Comparative analysis identified a syntenic region of 660 kb in Ae. tauschii with 18 annotated genes and a syntenic region in chromosome 1 of B. distachyon. The candidate region includes several disease resistance related genes and we identified a PTI1-like tyrosine-protein kinase 1 gene as a putative candidate gene for Sbwm1. The two flanking SNPs for Sbwm1 can effectively separate the resistant and susceptible lines in a new diversity panel of 159 wheat germplasm. The results from this study lay a solid foundation for the cloning, functional characterization and marker-assisted selection of Sbwm1.

Greenbug, Schizaphis graminum, is one of the important cereal aphid pests of sorghum in the United States and other parts of the world. Sorghum bicolor variety PI 607900 carries the Schizaphis graminum resistance (SgR1) gene that underlies plant resistance to greenbug biotype I (GBI). Now, the SgR1 has been determined as the major gene conferring greenbug resistance based on the strong association of its presence with the resistance phenotype in sorghum. In this study, we have successfully isolated the SgR1 gene using a map-based cloning approach, and subsequent molecular characterization revealed it encodes a leucine-rich repeat containing receptor-like protein (LRR-RLP). According to DNA sequence analysis, the SgR1 gene are conserved among GBI-resistance sorghum accessions but are variable within susceptible lines. Furthermore, an InDel (−965 nt) at its promoter region and a single-nucleotide polymorphism (SNP, 592 nt) in the CDS of the SgR1 were detected and they are well conserved within resistant genotypes. When the SgR1 gene was cloned and transferred into Arabidopsis plants, the SgR1 was activated in the transgenic Arabidopsis plants in response to attack by green peach aphids according to the results of the histochemical assay, and GUS activity was detected in situ in spots around the vasculature of the leaf where the phloem is located, suggesting its biological function in those transgenic Arabidopsis plants. Overall, this study confirms that the SgR1 gene coding for an LRR-RLP is the major resistance gene to greenbug, a destructive pest in sorghum and wheat. This represents the first greenbug resistance gene cloned so far and indicates that the simple-inherited GBI resistance gene can be used for sorghum improvement with genetic resistance to GBI via molecular breeding or cross-based conventional breeding technologies. [ABSTRACT FROM AUTHOR]

IDEAL PLANT ARCHITECTURE1 (IPA1) is a pivotal gene controlling plant architecture and grain yield. However, little is known about the effects of Triticum aestivum SQUAMOSA PROMOTER‐BINDING‐LIKE 14 (TaSPL14), an IPA1 ortholog in wheat, on balancing yield traits and its regulatory mechanism in wheat (T. aestivum L.). Here, we determined that the T. aestivum GRAIN WIDTH2 (TaGW2)‐TaSPL14 module influences the balance between tiller number and grain weight in wheat. Overexpression of TaSPL14 resulted in a reduced tiller number and increased grain weight, whereas its knockout had the opposite effect, indicating that TaSPL14 negatively regulates tillering while positively regulating grain weight. We further identified TaGW2 as a novel interacting protein of TaSPL14 and confirmed its ability to mediate the ubiquitination and degradation of TaSPL14. Based on our genetic evidence, TaGW2 acts as a positive regulator of tiller number, in addition to its known role as a negative regulator of grain weight, which is opposite to TaSPL14. Moreover, combinations of TaSPL14‐7A and TaGW2‐6A haplotypes exhibit significantly additive effects on tiller number and grain weight in wheat breeding. Our findings provide insight into how the TaGW2‐TaSPL14 module regulates the trade‐off between tiller number and grain weight and its potential application in improving wheat yield. [ABSTRACT FROM AUTHOR]

A precise regulation of flowering time is critical for plant reproductive success, and therefore, a better understanding of the natural variation in genes regulating the initiation of the reproductive phase is required to develop well-adapted varieties. In both monocot and dicot species, the FLOWERING LOCUS T (FT) is a central integrator of seasonal signals perceived by the leaves. The encoded mobile protein (florigen) is transmitted to the apical meristem where it induces flowering. The FT homolog in barley (Hordeum vulgare L.), designated HvFT1, was shown to correspond to the vernalization locus VRN-H3, and natural alleles for spring and winter growth habit were identified. In this study, we demonstrate that the HvFT1 allele present in the barley genetic stock (BGS213) associated with a dominant spring growth habit carries at least four identical copies of HvFT1, whereas most barley varieties have a single copy. Increased copy number is associated with earlier transcriptional up-regulation of HvFT1 and a spring growth habit. This allele is epistatic to winter alleles for VRN-H1 and VRN-H2. Among accessions with one HvFT1 copy, haplotype differences in the HvFT1 promoter and first intron are also associated with differences in flowering time, which are modulated by genetic background. These different HvFT1 alleles can be used to develop barley varieties adapted to different or changing environments. Our results, together with studies of other wheat and barley flowering genes, show that copy number variation plays an important role in the regulation of developmental processes in the temperate cereals.

In diploid wheat (Triticum monococcum), and likely in other Triticeae species, the VRN1 gene is essential for the initiation of the reproductive phase, and therefore, a detailed characterization of its regulatory regions is required to understand this process. A CArG-box (MADS-box-binding site) identified in the VRN1 promoter upstream from the transcription initiation site has been proposed as a critical regulatory element for the vernalization response. This hypothesis was supported by the genetic linkage between CArG-box natural deletions and dominant Vrn1 alleles for spring growth habit and by physical interactions with VRT2, a MADS-box protein proposed as a putative flowering repressor regulated by vernalization. Here, we describe a T. monococcum accession with a strong vernalization requirement and a 48-bp deletion encompassing the CArG-box in the VRN1 promoter. Genetic analyses of 2 segregating populations confirmed that this VRN1 allele is completely linked with a strong winter growth habit (vrn-A(m)1b). Transcript levels of the VRN1 allele with the 48-bp deletion were very low in unvernalized plants and increased during vernalization to levels similar to those detected in other wild-type vrn-A(m)1 alleles. Taken together, these results indicate that the CArG-box found upstream of the VRN1 transcription initiation site is not essential for the vernalization response.

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.

Summary: Wheat needs different durations of vernalization, which accelerates flowering by exposure to cold temperature, to ensure reproductive development at the optimum time, as that is critical for adaptability and high yield. TaVRN1 is the central flowering regulator in the vernalization pathway and encodes a MADS‐box transcription factor (TF) that usually works by forming hetero‐ or homo‐dimers. We previously identified that TaVRN1 bound to an MADS‐box TF TaSOC1 whose orthologues are flowering activators in other plants. The specific function of TaSOC1 and the biological implication of its interaction with TaVRN1 remained unknown. Here, we demonstrated that TaSOC1 was a flowering repressor in the vernalization and photoperiod pathways by overexpression and knockout assays. We confirmed the physical interaction between TaSOC1 and TaVRN1 in wheat protoplasts and in planta, and further validated their genetic interplay. A Flowering Promoting Factor 1‐like gene TaFPF1‐2B was identified as a common downstream target of TaSOC1 and TaVRN1 through transcriptome and chromatin immunoprecipitation analyses. TaSOC1 competed with TaVRT2, another MADS‐box flowering regulator, to bind to TaVRN1; their coding genes synergistically control TaFPF1‐2B expression and flowering initiation in response to photoperiod and low temperature. We identified major haplotypes of TaSOC1 and found that TaSOC1‐Hap1 conferred earlier flowering than TaSOC1‐Hap2 and had been subjected to positive selection in wheat breeding. We also revealed that wheat SOC1 family members were important domestication loci and expanded by tandem and segmental duplication events. These findings offer new insights into the regulatory mechanism underlying flowering control along with useful genetic resources for wheat improvement. [ABSTRACT FROM AUTHOR]

Wheat is usually classified as a long day (LD) plant because most varieties flower earlier when exposed to longer days. In addition to LD, winter wheats require a long exposure to low temperatures (vernalization) to become competent for flowering. Here we show that in some genotypes this vernalization requirement can be replaced by interrupting the LD treatment by 6 weeks of short day (SD), and that this replacement is associated with the SD down-regulation of the VRN2 flowering repressor. In addition, we found that SD down-regulation of VRN2 at room temperature is not followed by the up-regulation of the meristem identity gene VRN1 until plants are transferred to LD. This result contrasts with the VRN1 up-regulation observed after the VRN2 down-regulation by vernalization, suggesting the existence of a second VRN1 repressor. Analysis of natural VRN1 mutants indicated that a CArG-box located in the VRN1 promoter is the most likely regulatory site for the interaction with this second repressor. Up-regulation of VRN1 under SD in accessions carrying mutations in the CArG-box resulted in an earlier initiation of spike development, compared to other genotypes. However, even the genotypes with CArG box mutations required LD for a normal and timely spike development. The SD acceleration of flowering was observed in photoperiod sensitive winter varieties. Since vernalization requirement and photoperiod sensitivity are ancestral traits in Triticeae species we suggest that wheat was initially a SD-LD plant and that strong selection pressures during domestication and breeding resulted in the modification of this dual regulation. The down-regulation of the VRN2 repressor by SD is likely part of the mechanism associated with the SD-LD regulation of flowering in photoperiod sensitive winter wheat.

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits - winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele.

VRN-2 is a dominant repressor of flowering that plays a central role in the vernalization pathway of wheat. This gene is a zinc-finger/CCT domain transcription factor (ZCCT-1) down-regulated by vernalization. In barley, there are three related and linked ZCCT genes designated VRN-H1a, b, and c. A deletion of these three genes was observed in six putative spring induced mutants from winter barley variety 'Chikurin Ibaraki I'. However, analysis of additional molecular markers demonstrated that these deletions were originated by outcrossing with a spring barley variety carrying the natural ZCCT deletion. Hybridization of Southern blots from 84 barley varieties with a wheat ZCCT1 probe showed three or more fragments in 23 winter varieties but none in 60 out of the 61 vrn-H2 spring varieties. The vrn-H2-spring barley variety 'Fan' showed only the ZCCT-Hb gene, suggesting that this gene is not sufficient to determine winter growth habit in barley. We also show in this study, that the effect of VRN-H2 on flowering time completely disappears in the presence of the dominant Vrn-H1 allele in a Morex x Hordeum spontaneum segregating population. A different result was described before for the epistatic interactions between the orthologous genes in diploid wheat. We discuss the possible causes of these differences.

This article, published in the Plant Biotechnology Journal, discusses the discovery of a gene in wheat called GRAIN WIDTH2 (TaGW2) that has a dual effect on enhancing leaf rust resistance and increasing grain weight. Leaf rust, caused by the fungal pathogen Puccinia triticina Eriksson, is a major threat to global wheat production. The limited number of cloned resistance genes often have undesirable agronomic traits, so finding new resistance genes while balancing yield is important for breeding strategies. The researchers used CRISPR/Cas9 technology to edit TaGW2 and found that knockout plants had increased resistance to leaf rust and increased grain weight, offering a novel approach to enhancing wheat's resistance without sacrificing yield. [Extracted from the article]