Rebecca M. Davidson

Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data

BackgroundRice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa (japonica group).ResultsThe Nipponbare genome assembly was updated by revising and validating the minimal tiling path of clones with the optical map for rice. Sequencing errors in the revised genome assembly were identified by re-sequencing the genome of two different Nipponbare individuals using the Illumina Genome Analyzer II/IIx platform. A total of 4,886 sequencing errors were identified in 321 Mb of the assembled genome indicating an error rate in the original IRGSP assembly of only 0.15 per 10,000 nucleotides. A small number (five) of insertions/deletions were identified using longer reads generated using the Roche 454 pyrosequencing platform. As the re-sequencing data were generated from two different individuals, we were able to identify a number of allelic differences between the original individual used in the IRGSP effort and the two individuals used in the re-sequencing effort. The revised assembly, termed Os-Nipponbare-Reference-IRGSP-1.0, is now being used in updated releases of the Rice Annotation Project and the Michigan State University Rice Genome Annotation Project, thereby providing a unified set of pseudomolecules for the rice community.ConclusionsA revised, error-corrected, and validated assembly of the Nipponbare cultivar of rice was generated using optical map data, re-sequencing data, and manual curation that will facilitate on-going and future research in rice. Detection of polymorphisms between three different Nipponbare individuals highlights that allelic differences between individuals should be considered in diversity studies.

Genomewide SNP variation reveals relationships among landraces and modern varieties of rice.

Rice, the primary source of dietary calories for half of humanity, is the first crop plant for which a high-quality reference genome sequence from a single variety was produced. We used resequencing microarrays to interrogate 100 Mb of the unique fraction of the reference genome for 20 diverse varieties and landraces that capture the impressive genotypic and phenotypic diversity of domesticated rice. Here, we report the distribution of 160,000 nonredundant SNPs. Introgression patterns of shared SNPs revealed the breeding history and relationships among the 20 varieties; some introgressed regions are associated with agronomic traits that mark major milestones in rice improvement. These comprehensive SNP data provide a foundation for deep exploration of rice diversity and gene–trait relationships and their use for future rice improvement.

A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice.

Plant disease resistance governed by quantitative trait loci (QTL) is predicted to be effective against a broad spectrum of pathogens and long lasting. Use of these QTL to improve crop species, however, is hindered because the genes contributing to the trait are not known. Five disease resistance QTL that colocalized with defense response genes were accumulated by marker-aided selection to develop blast-resistant varieties. One advanced backcross line carrying the major-effect QTL on chromosome (chr) 8, which included a cluster of 12 germin-like protein (OsGLP) gene members, exhibited resistance to rice (Oryza sativa) blast disease over 14 cropping seasons. To determine if OsGLP members contribute to resistance and if the resistance was broad spectrum, a highly conserved portion of the OsGLP coding region was used as an RNA interference trigger to silence a few to all expressed chr 8 OsGLP family members. Challenge with two different fungal pathogens (causal agents of rice blast and sheath blight diseases) revealed that as more chr 8 OsGLP genes were suppressed, disease susceptibility of the plants increased. Of the 12 chr 8 OsGLPs, one clustered subfamily (OsGER4) contributed most to resistance. The similarities of sequence, gene organization, and roles in disease resistance of GLP family members in rice and other cereals, including barley (Hordeum vulgare) and wheat (Triticum aestivum), suggest that resistance contributed by the chr 8 OsGLP is a broad-spectrum, basal mechanism conserved among the Gramineae. Natural selection may have preserved a whole gene family to provide a stepwise, flexible defense response to pathogen invasion.

Comparative transcriptomics of three Poaceae species reveals patterns of gene expression evolution

The Poaceae family, also known as the grasses, includes agronomically important cereal crops such as rice, maize, sorghum, and wheat. Previous comparative studies have shown that much of the gene content is shared among the grasses; however, functional conservation of orthologous genes has yet to be explored. To gain an understanding of the genome-wide patterns of evolution of gene expression across reproductive tissues, we employed a sequence-based approach to compare analogous transcriptomes in species representing three Poaceae subgroups including the Pooideae (Brachypodium distachyon), the Panicoideae (sorghum), and the Ehrhartoideae (rice). Our transcriptome analyses reveal that only a fraction of orthologous genes exhibit conserved expression patterns. A high proportion of conserved orthologs include genes that are upregulated in physiologically similar tissues such as leaves, anther, pistil, and embryo, while orthologs that are highly expressed in seeds show the most diverged expression patterns. More generally, we show that evolution of gene expression profiles and coding sequences in the grasses may be linked. Genes that are highly and broadly expressed tend to be conserved at the coding sequence level while genes with narrow expression patterns show accelerated rates of sequence evolution. We further show that orthologs in syntenic genomic blocks are more likely to share correlated expression patterns compared with non-syntenic orthologs. These findings are important for agricultural improvement because sequence information is transferred from model species, such as Brachypodium, rice, and sorghum to crop plants without sequenced genomes.

Utility of RNA Sequencing for Analysis of Maize Reproductive Transcriptomes

Transcriptome sequencing is a powerful method for studying global expression patterns in large, complex genomes. Evaluation of sequence‐based expression profiles during reproductive development would provide functional annotation to genes underlying agronomic traits. We generated transcriptome profiles for 12 diverse maize (Zea mays L.) reproductive tissues representing male, female, developing seed, and leaf tissues using high throughput transcriptome sequencing. Overall, ∼80% of annotated genes were expressed. Comparative analysis between sequence and hybridization‐based methods demonstrated the utility of ribonucleic acid sequencing (RNA‐seq) for expression determination and differentiation of paralagous genes (∼85% of maize genes). Analysis of 4975 gene families across reproductive tissues revealed expression divergence is proportional to family size. In all pairwise comparisons between tissues, 7 (pre‐ vs. postemergence cobs) to 48% (pollen vs. ovule) of genes were differentially expressed. Genes with expression restricted to a single tissue within this study were identified with the highest numbers observed in leaves, endosperm, and pollen. Coexpression network analysis identified 17 gene modules with complex and shared expression patterns containing many previously described maize genes. The data and analyses in this study provide valuable tools through improved gene annotation, gene family characterization, and a core set of candidate genes to further characterize maize reproductive development and improve grain yield potential.

Gene Coexpression Network Analysis as a Source of Functional Annotation for Rice Genes

With the existence of large publicly available plant gene expression data sets, many groups have undertaken data analyses to construct gene coexpression networks and functionally annotate genes. Often, a large compendium of unrelated or condition-independent expression data is used to construct gene networks. Condition-dependent expression experiments consisting of well-defined conditions/treatments have also been used to create coexpression networks to help examine particular biological processes. Gene networks derived from either condition-dependent or condition-independent data can be difficult to interpret if a large number of genes and connections are present. However, algorithms exist to identify modules of highly connected and biologically relevant genes within coexpression networks. In this study, we have used publicly available rice (Oryza sativa) gene expression data to create gene coexpression networks using both condition-dependent and condition-independent data and have identified gene modules within these networks using the Weighted Gene Coexpression Network Analysis method. We compared the number of genes assigned to modules and the biological interpretability of gene coexpression modules to assess the utility of condition-dependent and condition-independent gene coexpression networks. For the purpose of providing functional annotation to rice genes, we found that gene modules identified by coexpression analysis of condition-dependent gene expression experiments to be more useful than gene modules identified by analysis of a condition-independent data set. We have incorporated our results into the MSU Rice Genome Annotation Project database as additional expression-based annotation for 13,537 genes, 2,980 of which lack a functional annotation description. These results provide two new types of functional annotation for our database. Genes in modules are now associated with groups of genes that constitute a collective functional annotation of those modules. Additionally, the expression patterns of genes across the treatments/conditions of an expression experiment comprise a second form of useful annotation.

The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputs

The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein–protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses.

Rice Germin-Like Proteins: Allelic Diversity and Relationships to Early Stress Responses

Germin-like protein (GLP) markers were associated with quantitative trait loci (QTL) for resistance to the rice blast pathogen, Magnaporthe oryzae in multiple rice (Oryza sativa) mapping populations. Twelve paralogous OsGLP gene family members are located within the physical QTL region on chromosome 8, and gene silencing studies suggest that they contribute collectively to the resistance phenotype. We compared sequence and expression profiles of OsGLP alleles in two resistant and two susceptible parental rice lines to find functional polymorphisms that correlated with the resistant phenotype. Based on coding and promoter sequences, the genes belong to two germin subfamily groups (GER3 and GER4). OsGLP members from both subfamilies were constitutively expressed and developmentally regulated in all cultivars. Transient induction above constitutive levels was observed for some OsGLPs, especially GER4 subfamily members, at early time points after M. oryzae infection and mechanical wounding. Varying 5′ regulatory regions and differential expression of some family members between resistant and susceptible cultivars corresponded with differential hydrogen peroxide (H2O2) accumulation after the same stimuli. OsGLP of both GER subfamilies localized to the plant cell wall. The protein location and early gene induction suggest that OsGLPs protect rice leaves at early stages of infection before fungal penetration and subsequent ingress. Our data suggest that regulation of OsGLP genes defines resistant versus susceptible phenotypes.

Understanding broad-spectrum durable resistance in rice

A long-standing goal in rice disease control is to identify and incorporate broadspectrum durable resistance (BSDR). Although quantitative resistance can potentially contribute to BSDR, neither the genes responsible for quantitative resistance nor the pathways or mechanisms by which they may function to contribute to BSDR are understood. Using varieties that show durable resistance historically, we have identified rice genes that are candidates for contributing to BSDR through co-localization with disease resistance QTLs in mapping studies. Several of these genes are known as disease defense response genes (e.g., oxalate oxidase, chitinase, PR1, etc.), whereas others are of unknown function. Genome-wide expression analyses at critical stages of host-pathogen interactions are also being used to reveal additional genes that may play a role in quantitative resistance. By combining chromosomal segments associated with five different candidate genes by marker-assisted selection, rice lines were produced that exhibited a high level of resistance to rice blast in multilocation trials. The current challenge is to understand if and how these candidate genes contribute to BSDR as well as the allelic variation that accounts for function in some lines but not in others. Targeted gene expression and functional analyses of candidate gene family members, for example, the oxalate oxidase gene families, are being used to focus on gene members involved in BSDR, and to determine what gene structural features are key to involvement. Sequence comparisons are providing clues as to critical allelic variation in rice germplasm. Finally, analysis of mutants exhibiting inappropriate activation of defense pathways is guiding the selection of candidate genes or genic regions. The integration of expression, mapping, and allelic diversity data is expected to unveil genes or gene interactions with significant phenotypic effects that can be used in breeding programs.