Sequencing 4.3 million mutations in wheat promoters to understand and modify gene expression.

Wheat is an important contributor to global food security, and further improvements are required to feed a growing human population. Functional genetics and genomics tools can help us to understand the function of different genes and to engineer beneficial changes. In this study, we used a promoter capture assay to sequence 2-kb regions upstream of all high-confidence annotated genes from 1,513 mutagenized plants from the tetraploid wheat variety Kronos. We identified 4.3 million induced mutations with an accuracy of 99.8%, resulting in a mutation density of 41.9 mutations per kb. We also remapped Kronos exome capture reads to Chinese Spring RefSeq v1.1, identified 4.7 million mutations, and predicted their effects on annotated genes. Using these predictions, we identified 59% more nonsynonymous substitutions and 49% more truncation mutations than in the original study. To show the biological value of the promoter dataset, we selected two mutations within the promoter of the VRN-A1 vernalization gene. Both mutations, located within transcription factor binding sites, significantly altered VRN-A1 expression, and one reduced the number of spikelets per spike. These publicly available sequenced mutant datasets provide rapid and inexpensive access to induced variation in the promoters and coding regions of most wheat genes. These mutations can be used to understand and modulate gene expression and phenotypes for both basic and commercial applications, where limited governmental regulations can facilitate deployment. These mutant collections, together with gene editing, provide valuable tools to accelerate functional genetic studies in this economically important crop.

Genome-Enabled Analysis of Population Dynamics and Virulence-Associated Loci in the Oat Crown Rust Fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genotypic and Resistance Profile Analysis of Two Oat Crown Rust Differential Sets Urge Coordination and Standardization.

Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Virulence Patterns of Oat Crown Rust in Australia - Season 2022.

Puccinia coronata f. sp. avenae (Pca) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterized using a collection of oat lines often utilized in rust surveys in the United States and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, which contrasts with the population in Western Australia (WA). Several oat lines/Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the United States oat differential set that display disease resistance to Pca in WA, with some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the United States. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the United States is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic Analysis and Physical Mapping of Oat Adult Plant Resistance Loci Against Puccinia coronata f. sp. avenae.

Six quantitative trait loci (QTLs) for adult plant resistance against oat crown rust (Puccinia coronata f. sp. avenae) were identified from mapping three recombinant inbred populations. Using genotyping-by-sequencing with markers called against the OT3098 v1 reference genome, the QTLs were mapped on six different chromosomes: Chr1D, Chr4D, Chr5A, Chr5D, Chr7A, and Chr7C. Composite interval mapping with marker cofactor selection showed that the phenotypic variance explained by all identified QTLs for coefficient of infection range from 12.2 to 46.9%, whereas heritability estimates ranged from 0.11 to 0.38. The significant regions were narrowed down to intervals of 3.9 to 25 cM, equivalent to physical distances of 11 to 133 Mb. At least two flanking single-nucleotide polymorphism markers were identified within 10 cM of each QTL that could be used in marker-assisted introgression, pyramiding, and selection. The additive effects of the QTLs in each population were determined using single-nucleotide polymorphism haplotype data, which showed a significantly lower coefficient of infection in lines homozygous for the resistant alleles. Analysis of pairwise linkage disequilibrium also revealed high correlation of markers and presence of linkage blocks in the significant regions. To further facilitate marker-assisted breeding, polymerase chain reaction allelic competitive extension (PACE) markers for the adult plant resistance loci were developed. Putative candidate genes were also identified in each of the significant regions, which include resistance gene analogs that encode for kinases, ligases, and predicted receptors of avirulence proteins from pathogens.

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.

Three decades of rust surveys in the United States reveal drastic virulence changes in oat crown rust.

To better understand how the pathogenicity of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca), has changed in the United States, 30 years of USDA survey isolates (n=5,456) tested on 30-40 differential lines were analyzed for overall and Pc resistance gene specific virulence trends and correlations. Pca is incredibly pathologically diverse with 88% of races represented by a single isolate. There are a slightly higher proportion of unique races from the Northern region of the United States and for one fourth of the years, Northern region isolates were significantly more virulent than Southern isolates which supports the idea that sexual recombination in this region is mediated by the alternate host as a major factor in creating new races. However, there is also support for regular isolate movement between North and South regions as isolates in the United States are steadily accumulating virulences at a rate of 0.35 virulences per year. Virulence significantly increased for 23 and decreased for 4 of the 40 differential lines. In the past few years, virulence has reached 90% or greater for 16 differential lines. There were also strong correlations in virulence for certain Pc genes that are likely identical, allelic, or target the same or closely linked pathogen effectors (e.g. Pc39, Pc55, and Pc71), and the results were largely in concordance with recent GWAS effector studies using USDA isolate subsets. Understanding changes in Pca pathogenicity is essential for the responsible deployment and management of Pc resistance genes for sustainable and profitable oat production.

A reference-anchored oat linkage map reveals quantitative trait loci conferring adult plant resistance to crown rust (Puccinia coronata f. sp. avenae).

KEY MESSAGE: We mapped three adult plant resistance (APR) loci on oat chromosomes 4D and 6C and developed flanking KASP/PACE markers for marker-assisted selection and gene pyramiding. Using sequence orthology search and the available oat genomic and transcriptomic data, we surveyed these genomic regions for genes that may control disease resistance. Sources of durable disease resistance are needed to minimize yield losses in cultivated oat caused by crown rust (Puccinia coronata f. sp. avenae). In this study, we developed five oat recombinant inbred line mapping populations to identify sources of adult plant resistance from crosses between five APR donors and Otana, a susceptible variety. The preliminary bulk segregant mapping based on allele frequencies showed two regions in linkage group Mrg21 (Chr4D) that are associated with the APR phenotype in all five populations. Six markers from these regions in Chr4D were converted to high-throughput allele specific PCR assays and were used to genotype all individuals in each population. Simple interval mapping showed two peaks in Chr4D, named QPc.APR-4D.1 and QPc.APR-4D.2, which were detected in the OtanaA/CI4706-2 and OtanaA/CI9416-2 and in the Otana/PI189733, OtanaD/PI260616, and OtanaA/CI8000-4 populations, respectively. These results were validated by mapping two entire populations, Otana/PI189733 and OtanaA/CI9416, genotyped using Illumina HiSeq, in which polymorphisms were called against the OT3098 oat reference genome. Composite interval mapping results confirmed the presence of the two quantitative trait loci (QTL) located on oat chromosome 4D and an additional QTL with a smaller effect located on chromosome 6C. This mapping approach also narrowed down the physical intervals to between 5 and 19 Mb, and indicated that QPc.APR-4D.1, QPc.APR-4D.2, and QPc.APR-6C explained 43.4%, 38.5%, and 21.5% of the phenotypic variation, respectively. In a survey of the gene content of each QTL, several clusters of disease resistance genes that may contribute to APR were found. The allele specific PCR markers developed for these QTL regions would be beneficial for marker-assisted breeding, gene pyramiding, and future cloning of resistance genes from oat.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Identification, introgression, and molecular marker genetic analysis and selection of a highly effective novel oat crown rust resistance from diploid oat, Avena strigosa.

KEY MESSAGE: Oat crown rust is one of the most damaging diseases of oat. We identified a new source of resistance and developed KASP and TaqMan markers for selection in breeding programs. A new highly effective resistance to oat crown rust (Puccinia coronata f. sp. avenae) was identified in the diploid oat Avena strigosa PI 258731 and introgressed into hexaploid cultivated oat. Young plants with this resistance show moderate susceptibility, whereas older plant tissues and adult plants are resistant with no virulent isolates encountered in over 8 years of testing. Resistance was incorporated into hexaploid oat by embryo rescue, colchicine chromosome doubling followed by backcrosses with a hexaploid parent, and selection for stable transmission of resistance. To mitigate flag leaf and panicle chlorosis/necrosis associated with the resistance, crosses were made with derived resistant lines to breeding lines of divergent parentage followed by selection. Subsequently, two F2 sister lines, termed MNBT1020-1 and MNBT1021-1, were identified in which the chlorosis/necrosis was reduced. These two lines performed well in replicated multi-location state trials in 2015 and 2016 out-yielding all cultivar entries. Segregating F2:3 plants resulting from crosses of MNBT lines to susceptible parents were genotyped with the oat 6K SNP array, and SNP loci with close linkage to the resistance were identified. KASP assays generated from linked SNPs showed accurate discrimination of the resistance in derivatives of the resistant MNBT lines crossed to susceptible breeding lines. A TaqMan marker was developed and correctly identified homozygous resistance in over 95% of 379 F4 plants when rust was scored in F4:5 plants in the field. Thus, a novel highly effective resistance and associated molecular markers are available for use in breeding, genetic analysis, and functional studies.

Detection of Race-Specific Resistance Against Puccinia coronata f. sp. avenae in Brachypodium Species.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in nonhost species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine whether effector-triggered immunity contributes to nonhost resistance to P. coronata f. sp. avenae in Brachypodium spp.

Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants.

Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversity. The magnitude and essence of intergenomic interactions between nuclear and cytoplasmic genomes remain unknown due to the direction of many crosses. This study was conducted to address the role of nuclear-cytoplasmic interactions as a source of variation upon hybridization. Wheat (Triticum aestivum) alloplasmic lines carrying the cytoplasm of Aegilops mutica along with an integrated approach utilizing comparative quantitative trait locus (QTL) and epigenome analysis were used to dissect this interaction. The results indicate that cytoplasmic genomes can modify the magnitude of QTL controlling certain physiological traits such as dry matter weight. Furthermore, methylation profiling analysis detected eight polymorphic regions affected by the cytoplasm type. In general, these results indicate that novel nuclear-cytoplasmic interactions can potentially trigger an epigenetic modification cascade in nuclear genes which eventually change the genetic network controlling physiological traits. These modified genetic networks can serve as new sources of variation to accelerate the evolutionary process. Furthermore, this variation can synthetically be produced by breeders in their programs to develop epigenomic-segregating lines.

Fast-forward genetics by radiation hybrids to saturate the locus regulating nuclear-cytoplasmic compatibility in Triticum.

The nuclear-encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano-mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear-cytoplasmic compatibility in wheat.

Toward a better understanding of the genomic region harboring Fusarium head blight resistance QTL Qfhs.ndsu-3AS in durum wheat.

KEY MESSAGE: New molecular markers were developed and mapped to the FHB resistance QTL region in high resolution. Micro-collinearity of the QTL region with rice and Brachypodium was revealed for a better understanding of the genomic region. The wild emmer wheat (Triticum dicoccoides)-derived Fusarium head blight (FHB) resistance quantitative trait locus (QTL) Qfhs.ndsu-3AS previously mapped to the short arm of chromosome 3A (3AS) in a population of recombinant inbred chromosome lines (RICLs). This study aimed to attain a better understanding of the genomic region harboring Qfhs.ndsu-3AS and to improve the utility of the QTL in wheat breeding. Micro-collinearity of the QTL region with rice chromosome 1 and Brachypodium chromosome 2 was identified and used for marker development in saturation mapping. A total of 42 new EST-derived sequence tagged site (STS) and simple sequence repeat (SSR) markers were developed and mapped to the QTL and nearby regions on 3AS. Further comparative analysis revealed a complex collinearity of the 3AS genomic region with their collinear counterparts of rice and Brachypodium. Fine mapping of the QTL region resolved five co-segregating markers (Xwgc1186/Xwgc716/Xwgc1143/Xwgc501/Xwgc1204) into three distinct loci proximal to Xgwm2, a marker previously reported to be closely linked to the QTL. Four other markers (Xwgc1226, Xwgc510, Xwgc1296, and Xwgc1301) mapped farther proximal to the above markers in the QTL region with a higher resolution. Five homozygous recombinants with shortened T. dicoccoides chromosomal segments in the QTL region were recovered by molecular marker analysis and evaluated for FHB resistance. Qfhs.ndsu-3AS was positioned to a 5.2 cM interval flanked by the marker Xwgc501 and Xwgc510. The recombinants containing Qfhs.ndsu-3AS and new markers defining the QTL will facilitate utilization of this resistance source in wheat breeding.

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

Radiation hybrid maps of the D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes.

BACKGROUND: The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay. METHODS: In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii. RESULTS: A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome. CONCLUSION: This study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.

Development of a D genome specific marker resource for diploid and hexaploid wheat.

BACKGROUND: Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome. RESULTS: In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci. CONCLUSIONS: In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website ( http://probes.pw.usda.gov/ATRJM/ ) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.

Deletion mutagenesis identifies a haploinsufficient role for γ-zein in opaque2 endosperm modification.

Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant opaque2. Using γ-irradiation, we created opaque QPM variants to identify opaque2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize (Zea mays) functional genomics tool. A K0326Y QPM deletion mutant was null for the 27- and 50-kD γ-zeins and abolished vitreous endosperm formation. Illumina exon and RNA sequencing revealed a 1.2-megabase pair deletion encompassing the 27- and 50-kD γ-zein genes on chromosome 7 and a deletion of at least 232 kb on chromosome 9. Protein body number was reduced by over 90%, while protein body size is similar to the wild type. Kernels hemizygous for the γ-zein deletion had intermediate 27- and 50-kD γ-zein levels and were semivitreous, indicating haploinsufficiency of these gene products in opaque2 endosperm modification. The γ-zein deletion further increased lysine in QPM in its homozygous and hemizygous states. This work identifies 27-kD γ-zein as an opaque2 modifier gene within the largest QPM quantitative trait locus and may suggest the 50-kD γ-zein also contributes to this quantitative trait locus. It further demonstrates that genome-wide deletions in nonreference maize lines can be identified through a combination of assembly of Illumina reads against the B73 genome and integration of RNA sequencing data.

PIECE: a database for plant gene structure comparison and evolution.

Gene families often show degrees of differences in terms of exon-intron structures depending on their distinct evolutionary histories. Comparative analysis of gene structures is important for understanding their evolutionary and functional relationships within plant species. Here, we present a comparative genomics database named PIECE (http://wheat.pw.usda.gov/piece) for Plant Intron and Exon Comparison and Evolution studies. The database contains all the annotated genes extracted from 25 sequenced plant genomes. These genes were classified based on Pfam motifs. Phylogenetic trees were pre-constructed for each gene category. PIECE provides a user-friendly interface for different types of searches and a graphical viewer for displaying a gene structure pattern diagram linked to the resulting bootstrapped dendrogram for each gene family. The gene structure evolution of orthologous gene groups was determined using the GLOOME, Exalign and GECA software programs that can be accessed within the database. PIECE also provides a web server version of the software, GSDraw, for drawing schematic diagrams of gene structures. PIECE is a powerful tool for comparing gene sequences and provides valuable insights into the evolution of gene structure in plant genomes.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.

BACKGROUND: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. RESULTS: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. CONCLUSIONS: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.