Michael O. Pumphrey

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

Megabase Level Sequencing Reveals Contrasted Organization and Evolution Patterns of the Wheat Gene and Transposable Element Spaces

This article describes the molecular analysis of large contiguous sequences produced from the bread wheat genome. It provides novel insights into the number, distribution, and density of genes along chromosome 3B and reveals an unexpectedly high amount of noncollinear genes compared to model grass genomes. To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.

Nonadditive Expression of Homoeologous Genes Is Established Upon Polyploidization in Hexaploid Wheat

Effects of polyploidy in allohexaploid wheat (Triticum aestivum L.) have primarily been ascribed to increases in coding sequence variation and potential to acquire new gene functions through mutation of redundant loci. However, regulatory variation that arises through new promoter and transcription factor combinations or epigenetic events may also contribute to the effects of polyploidization. In this study, gene expression was characterized in a synthetic T. aestivum line and the T. turgidum and Aegilops tauschii parents to establish a timeline for such regulatory changes and estimate the frequency of nonadditive expression of homoeologous transcripts in newly formed T. aestivum. Large-scale analysis of nonadditive gene expression was assayed by microarray expression experiments, where synthetic T. aestivum gene expression was compared to additive model values (mid-parent) calculated from parental T. turgidum and Ae. tauschii expression levels. Approximately 16% of genes were estimated to display nonadditive expression in synthetic T. aestivum. A certain fraction of the genes (2.9%) showed overdominance or underdominance. cDNA–single strand conformation polymorphism analysis was applied to measure expression of homoeologous transcripts and further verify microarray data. The results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and nonadditive gene expression in T. aestivum.

A Genome-Wide Association Study of Resistance to Stripe Rust (Puccinia striiformis f. sp. tritici) in a Worldwide Collection of Hexaploid Spring Wheat (Triticum aestivum L.)

New races of Puccinia striiformis f. sp. tritici (Pst), the causal pathogen of wheat stripe rust, show high virulence to previously deployed resistance genes and are responsible for large yield losses worldwide. To identify new sources of resistance we performed a genome-wide association study (GWAS) using a worldwide collection of 1000 spring wheat accessions. Adult plants were evaluated under field conditions in six environments in the western United States, and seedlings were tested with four Pst races. A single-nucleotide polymorphism (SNP) Infinium 9K-assay provided 4585 SNPs suitable for GWAS. High correlations among environments and high heritabilities were observed for stripe rust infection type and severity. Greater levels of Pst resistance were observed in a subpopulation from Southern Asia than in other groups. GWAS identified 97 loci that were significant for at least three environments, including 10 with an experiment-wise adjusted Bonferroni probability < 0.10. These 10 quantitative trait loci (QTL) explained 15% of the phenotypic variation in infection type, a percentage that increased to 45% when all QTL were considered. Three of these 10 QTL were mapped far from previously identified Pst resistance genes and QTL, and likely represent new resistance loci. The other seven QTL mapped close to known resistance genes and allelism tests will be required to test their relationships. In summary, this study provides an integrated view of stripe rust resistance resources in spring wheat and identifies new resistance loci that will be useful to diversify the current set of resistance genes deployed to control this devastating disease.

A novel Robertsonian translocation event leads to transfer of a stem rust resistance gene (Sr52) effective against race Ug99 from Dasypyrum villosum into bread wheat

Stem rust (Puccinia graminis f. sp. tritici Eriks. & E. Henn.) (the causal agent of wheat stem rust) race Ug99 (also designated TTKSK) and its derivatives have defeated several important stem rust resistance genes widely used in wheat (Triticum aestivum L.) production, rendering much of the worldwide wheat acreage susceptible. In order to identify new resistance sources, a large collection of wheat relatives and genetic stocks maintained at the Wheat Genetic and Genomic Resources Center was screened. The results revealed that most accessions of the diploid relative Dasypyrum villosum (L.) Candargy were highly resistant. The screening of a set of wheat–D. villosum chromosome addition lines revealed that the wheat–D. villosum disomic addition line DA6V#3 was moderately resistant to race Ug99. The objective of the present study was to produce and characterize compensating wheat–D. villosum whole arm Robertsonian translocations (RobTs) involving chromosomes 6D of wheat and 6V#3 of D. villosum through the mechanism of centric breakage-fusion. Seven 6V#3-specific EST–STS markers were developed for screening F2 progeny derived from plants double-monosomic for chromosomes 6D and 6V#3. Surprisingly, although 6D was the target chromosome, all recovered RobTs involved chromosome 6A implying a novel mechanism for the origin of RobTs. Homozygous translocations (T6AS·6V#3L and T6AL·6V#3S) with good plant vigor and full fertility were selected from F3 families. A stem rust resistance gene was mapped to the long arm 6V#3L in T6AS·6V#3L and was designated as Sr52. Sr52 is temperature-sensitive and is most effective at 16°C, partially effective at 24°C, and ineffective at 28°C. The T6AS·6V#3L stock is a new source of resistance to Ug99, is cytogenetically stable, and may be useful in wheat improvement.

Discovery and molecular mapping of a new gene conferring resistance to stem rust, Sr53, derived from Aegilops geniculata and characterization of spontaneous translocation stocks with reduced alien chromatin.

This study reports the discovery and molecular mapping of a resistance gene effective against stem rust races RKQQC and TTKSK (Ug99) derived from Aegilops geniculata (2n = 4x = 28, UgUgMgMg). Two populations from the crosses TA5599 (T5DL-5MgL·5MgS)/TA3809 (ph1b mutant in Chinese Spring background) and TA5599/Lakin were developed and used for genetic mapping to identify markers linked to the resistance gene. Further molecular and cytogenetic characterization resulted in the identification of nine spontaneous recombinants with shortened Ae. geniculata segments. Three of the wheat–Ae. geniculata recombinants (U6154-124, U6154-128, and U6200-113) are interstitial translocations (T5DS·5DL-5MgL-5DL), with 20–30% proximal segments of 5MgL translocated to 5DL; the other six are recombinants (T5DL-5MgL·5MgS) have shortened segments of 5MgL with fraction lengths (FL) of 0.32–0.45 compared with FL 0.55 for the 5MgL segment in the original translocation donor, TA5599. Recombinants U6200-64, U6200-117, and U6154-124 carry the stem rust resistance gene Sr53 with the same infection type as TA5599, the resistance gene donor. All recombinants were confirmed to be genetically compensating on the basis of genomic in situ hybridization and molecular marker analysis with chromosome 5D- and 5Mg-specific SSR/STS-PCR markers. These recombinants between wheat and Ae. geniculata will provide another source for wheat stem rust resistance breeding and for physical mapping of the resistance locus and crossover hot spots between wheat chromosome 5D and chromosome 5MgL of Ae. geniculata.

Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat and barley that leads to reduced yield and mycotoxin contamination of grain, making it unfit for human consumption. FHB is a global problem, with outbreaks in the United States, Canada, Europe, Asia and South America. In the United States alone, total direct and secondary economic losses from 1993 to 2001 owing to FHB were estimated at

Development and characterization of wheat-Ae. searsii Robertsonian translocations and a recombinant chromosome conferring resistance to stem rust

7.67 billion. Fhb1 is the most consistently reported quantitative trait locus (QTL) for FHB resistance breeding. Here we report the map-based cloning of Fhb1 from a Chinese wheat cultivar Sumai 3. By mutation analysis, gene silencing and transgenic overexpression, we show that a pore-forming toxin-like (PFT) gene at Fhb1 confers FHB resistance. PFT is predicted to encode a chimeric lectin with two agglutinin domains and an ETX/MTX2 toxin domain. Our discovery identifies a new type of durable plant resistance gene conferring quantitative disease resistance to plants against Fusarium species.

Near-Infrared Spectroscopic Method for Identification of Fusarium Head Blight Damage and Prediction of Deoxynivalenol in Single Wheat Kernels

The emergence of a new highly virulent race of stem rust (Puccinia graminis tritici), Ug99, rapid evolution of new Ug99 derivative races overcoming resistance of widely deployed genes, and spread towards important wheat growing areas now potentially threaten world food security. Exploiting novel genes effective against Ug99 from wild relatives of wheat is one of the most promising strategies for the protection of the wheat crop. A new source of resistance to Ug99 was identified in the short arm of the Aegilopssearsii chromosome 3Ss by screening wheat- Ae.searsii introgression libraries available as individual chromosome and chromosome arm additions to the wheat genome. For transferring this resistance gene into common wheat, we produced three double-monosomic chromosome populations (3A/3Ss, 3B/3Ss and 3D/3Ss) and then applied integrated stem rust screening, molecular maker analysis, and cytogenetic analysis to identify resistant wheat-Ae. searsii Robertsonian translocation. Three Robertsonian translocations (T3AL·3SsS, T3BL·3SsS and T3DL·3SsS) and one recombinant (T3DS-3SsS·3SsL) with stem rust resistance were identified and confirmed to be genetically compensating on the basis of genomic in situ hybridization, analysis of 3A, 3B, 3D and 3SsS-specific SSR/STS-PCR markers, and C-banding. In addition, nine SSR/STS-PCR markers of 3SsS-specific were developed for marker-assisted selection of the resistant gene. Efforts to reduce potential linkage drag associated with 3SsS of Ae. searsii are currently under way.

Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99)

Cereal Chem. 87(6):511–517 Fusarium Head Blight (FHB), or scab, can result in significant crop yield losses and contaminated grain in wheat (Triticum aestivum L.). Growing less susceptible cultivars is one of the most effective methods for managing FHB and for reducing deoxynivalenol (DON) levels in grain, but breeding programs lack a rapid and objective method for identifying the fungi and toxins. It is important to estimate proportions of sound kernels and Fusarium-damaged kernels (FDK) in grain and to estimate DON levels of FDK to objectively assess the resistance of a cultivar. An automated single kernel near-infrared (SKNIR) spectroscopic method for identification of FDK and for estimating DON levels was evaluated. The SKNIR system classified visually sound and FDK with an accuracy of 98.8 and 99.9%, respectively. The sound fraction had no or very little accumulation of DON. The FDK fraction was sorted into fractions with high or low DON content. The kernels identified as FDK by the SKNIR system had better correlation with other FHB assessment indices such as FHB severity, FHB incidence and kernels/g than visual FDK%. This technique can be successfully employed to nondestructively sort kernels with Fusarium damage and to estimate DON levels of those kernels. Single kernels could be predicted as having low ( 60 ppm) DON with ≈96% accuracy. Single kernel DON levels of the high DON kernels could be estimated with R 2 = 0.87 and standard error of prediction (SEP) of 60.8 ppm. Because the method is nondestructive, seeds may be saved for generation advancement. The automated method is rapid (1 kernel/sec) and sorting grains into several fractions depending on DON levels will provide breeders with more information than techniques that deliver average DON levels from bulk seed samples.