The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C-4 traits into C-3 plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C-4 plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C-4 species from C-3 species. To identify genetic factors that specify C-4 leaf anatomy, we generated ethyl methanesulfonate- and -ray-mutagenized populations of the C-4 species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F-2 populations as homozygous recessive alleles. Bulk segregant analysis using next-generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C-4 and C-3 leaf anatomy may arise in part through differential activity of this pathway in the two leaf types. Significance Statement The high vein density associated with Kranz anatomy is a defining characteristic of all plants with two-cell type C-4 photosynthesis. Here we show that the brassinosteroid pathway is important for high vein density in C-4 leaves.

To understand the basis of broad-spectrum disease resistance in rice, we isolated a gamma-ray-induced IR64 mutant G978 that showed enhanced resistance to blast and bacterial blight. The resistance is quantitative and non-race specific against the bacterial and fungal pathogens. The mutation is inherited as a single recessive gene, designated as Bsdr1 and causes shorter stature relative to the wild type; however, it does not show lesion mimics phenotype under the conditions tested. The mutation was mapped as a quantitative trait locus to a 3.8-Mb region on chromosome 12. By comparing the gene expression profiles of the mutant and wild type, we identified a candidate gene encoding a U-box domain-containing protein. The disrupted gene showed a loss of expression in the mutant and co-segregated with mutant phenotype. The mutant provides a useful tool for investigating the important genes responsible for non-race specific resistance to two distinct diseases.

Panicle exsertion, an essential physiological process for obtaining high grain yield in rice is mainly driven by peduncle (uppermost internode) elongation. Drought at heading/panicle emergence prevented peduncle elongation from reaching its maximum length even after re-watering. This inhibitory effect of drought resulted in delayed heading and trapping spikelets lower down the panicle inside the flag-leaf sheath, thus increasing sterility in the lower un-exserted spikelets and also among the upper superior spikelets whose exsertion was delayed. Intermittent drought stress caused a significant reduction in relative water content (RWC) and an increase in the abscisic acid (ABA) level of the peduncles, while both returned to normal levels upon re-watering. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis revealed the down-regulation of GA biosynthetic genes during drought. 2D-PAGE analysis of proteins from peduncles collected under well-watered, drought-stressed, and re-watered plants revealed at least twofold differential changes in expression of 31 proteins in response to drought and most of these changes were largely reversed by re-watering. The results indicate that ABA-GA antagonism is a key focal point for understanding the failure of panicle exsertion under drought stress and the consequent increase in spikelet sterility.

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study. =20

Rice is atypical in that it is an agricultural cereal that is immune to fungal rust diseases. This report demonstrates that several cereal rust species (Puccinia graminis f. sp tritici, P triticina, P. striiformis, and P hordei) can infect rice and produce all the infection structures necessary for plant colonization, including specialized feeding cells (haustoria). Some rust infection sites are remarkably large and many plant cells are colonized, suggesting that nutrient uptake occurs to support this growth. Rice responds with an active, nonhost resistance (NHR) response that prevents fungal sporulation and that involves callose deposition, production of reactive oxygen species, and, occasionally, cell death. Genetic variation for the efficacy of NHR to wheat stem rust and wheat leaf rust was observed. Unlike cereal rusts, the rust pathogen (Melampsora lini) of the dicotyledenous plant flax (Linum usitatissimum) rarely successfully infects rice due to an apparent inability to recognize host-derived signals. Morphologically abnormal infection structures are produced and appressorial-like structures often don't coincide with stomata. These data suggest that basic compatibility is an important determinate of nonhost infection outcomes of rust diseases on cereals, with cereal rusts being more capable of infecting a cereal nonhost species compared with rust species that are adapted for dicot hosts.

Most agronomically important traits, including resistance against pathogens, are governed by quantitative trait loci (QTL). QTL-mediated resistance shows promise of being effective and long-lasting against diverse pathogens. Identification of genes controlling QTL-based disease resistance contributes to breeding for cultivars that exhibit high and stable resistance. Several defense response genes have been successfully used as good predictors and contributors to QTL-based resistance against several devastating rice diseases. In this study, we identified and characterized a rice (Oryza sativa) mutant line containing a 750 bp deletion in the second exon of OsPAL4, a member of the phenylalanine ammonia-lyase gene family. OsPAL4 clusters with three additional OsPAL genes that co-localize with QTL for bacterial blight and sheath blight disease resistance on rice chromosome 2. Self-pollination of heterozygous ospal4 mutant lines produced no homozygous progeny, suggesting that homozygosity for the mutation is lethal. The heterozygous ospal4 mutant line exhibited increased susceptibility to three distinct rice diseases, bacterial blight, sheath blight, and rice blast. Mutation of OsPAL4 increased expression of the OsPAL2 gene and decreased the expression of the unlinked OsPAL6 gene. OsPAL2 function is not redundant because the changes in expression did not compensate for loss of disease resistance. OsPAL6 co-localizes with a QTL for rice blast resistance, and is down-regulated in the ospal4 mutant line; this may explain enhanced susceptibility to Magnoporthe oryzae. Overall, these results suggest that OsPAL4 and possibly OsPAL6 are key contributors to resistance governed by QTL and are potential breeding targets for improved broad-spectrum disease resistance in rice.

Although rice resistance plays an important role in controlling the brown planthopper (BPH), Nilaparvata lugens, not all varieties have the same level of protection against BPH infestation. Understanding the molecular interactions in rice defense response is an important tool to help to reveal unexplained processes that underlie rice resistance to BPH. A proteomics approach was used to explore how wild type IR64 and near-isogenic rice mutants with gain and loss of resistance to BPH respond during infestation. A total of 65 proteins were found markedly altered in wild type IR64 during BPH infestation. Fifty-two proteins associated with 11 functional categories were identified using mass spectrometry. Protein abundance was less altered at 2 and 14 days after infestation (DAI) (T1, T2, respectively), whereas higher protein levels were observed at 28 DAI (T3). This trend diminished at 34 DAI (T4). Comparative analysis of IR64 with mutants showed 22 proteins that may be potentially associated with rice resistance to the brown planthopper (BPH). Ten proteins were altered in susceptible mutant (D1131) whereas abundance of 12 proteins including S-like RNase, Glyoxalase I, EFTu1 and Salt stress root protein "RS1" was differentially changed in resistant mutant (D518). S-like RNase was found in greater quantities in D518 after BPH infestation but remained unchanged in IR64 and decreased in D1131. Taken together, this study shows a noticeable level of protein abundance in the resistant mutant D518 compared to the susceptible mutant D1131 that may be involved in rendering enhanced level of resistance against BPH.

Resistance in rice cultivars to the rice blast fungus Magnaporthe oryzae is complex and is controlled by both major genes and quantitative trait loci (QTLs). We undertook a genome-wide association study (GWAS) using the rice diversity panel 1 (RDP1) that was genotyped using a high-density (700 000 single nucleotide polymorphisms) array and inoculated with five diverse M. oryzae isolates. We identified 97 loci associated with blast resistance (LABRs). Among them, 82 were new regions and 15 co-localized with known blast resistance loci. The top 72 LABRs explained up to 98% of the phenotypic variation. The candidate genes in the LABRs encode nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance proteins, receptor-like protein kinases, transcription factors and defence-related proteins. Among them, LABR_64 was strongly associated with resistance to all five isolates. We analysed the function of candidate genes underlying LABR_64 using RNA interference (RNAi) technology and identified two new resistance alleles at the Pi5 locus. We demonstrate an efficient strategy for rapid allele discovery using the power of GWAS, coupled with RNAi technology, for the dissection of complex blast resistance in rice.

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 (H(2)O(2)) 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.

This paper is a section of the book "Drought phenotyping in crops: from theory to practice" (Monneveux Philippe and Ribaut Jean-Marcel eds, published by CGIAR Generation Challenge Programme. Texcoco, Mexico). The section describes recent experience in drought phenotyping in rice which is one of the most drought-susceptible crops. The section contains genetic and genomic resources for drought adaptation and methods for selection of drought-resistant varieties in rice. In appendix, there is experience from Thailand on integration of direct selection for grain yield and physiological traits to confer drought resistance.

Cereal rusts are a constant disease threat that limits the production of almost all agricultural cereals. Rice is atypical in that it is an intensively grown agricultural cereal that is immune to rust pathogens. This immunity is manifested by nonhost resistance (NHR), the mechanisms of which are poorly understood. As part of the Borlaug Global Rust Initiative (BGRI), studies are being undertaken to dissect the molecular mechanisms that provide rust immunity in rice and determine if they can be transferred to wheat via transgenesis. Microscopic analyses showed that cereal rusts are capable of entering the rice leaf via formation of an appressorium over a stomate and subsequent infection of underlying mesophyll cells. However, there is considerable variation in the extent of colonization at each infection site. Our research effort has focused on screening for increased growth of cereal rust using natural and induced variants of rice. Two collections of rice mutants, T-DNA insertional mutants and chemical/irradiation-induced mutants, and diverse germplasm accessions are being screened for compromised NHR to cereal rusts. Preliminary screening with stripe rust identified several potential mutants that allow increased fungal growth. The confirmation of these lines will serve as the foundation for the isolation of gene(s) responsible for this compromised resistance. Details of the strategies being undertaken and progress to date are provided.

Background: We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results: We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion: We anticipate this microarray-based genotyping platform, based on its low cost-persample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

BACKGROUND: Information on the effect of stress on the allele-specific expression (ASE) profile of rice hybrids is limited. More so, the association of allelically imbalanced genes to important traits is yet to be understood. Here we assessed allelic imbalance (AI) in the heterozygote state of rice under non- and water-stress treatments and determined association of asymmetrically expressed genes with grain yield (GY) under drought stress by in-silico co-localization analysis and selective genotyping. The genotypes IR64, Apo and their F1 hybrid (IR64*Apo) were grown under normal and water-limiting conditions. We sequenced the total RNA transcripts for all genotypes then reconstructed the two chromosomes in the heterozygote.; RESULTS: We are able to estimate the transcript abundance of and the differential expression (DE) between the two parent-specific alleles in the rice hybrids. The magnitude and direction of AI are classified into two categories: (1) symmetrical or biallelic and (2) asymmetrical. The latter can be further classified as either IR64- or Apo-favoring gene. Analysis showed that in the hybrids grown under non-stress conditions, 179 and 183 favor Apo- and IR64-specific alleles, respectively. Hence, the number of IR64- and Apo-favoring genes is relatively equal. Under water-stress conditions, 179 and 255 favor Apo- and IR64-specific alleles, respectively, indicating that the number of allelically imbalanced genes is skewed towards IR64. This is nearly 40-60% preference for Apo and IR64 alleles, respectively, to the hybrid transcriptome. We also observed genes which exhibit allele preference switching when exposed to water-stress conditions. Results of in-silico co-localization procedure and selective genotyping of Apo/IR64 F3:5 progenies revealed significant association of several asymmetrically expressed genes with GY under drought stress conditions.; CONCLUSION: Our data suggest that water stress skews AI on a genome-wide scale towards the IR64 allele, the cross-specific maternal allele. Several asymmetrically expressed genes are strongly associated with GY under drought stress which may shed hints that genes associated with important traits are allelically imbalanced. Our approach of integrating hybrid expression analysis and QTL mapping analysis may be an efficient strategy for shortlisting candidate genes for gene discovery.=20

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.=20

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens. =20