Natural Diversity in Stomatal Features of Cultivated and Wild Oryza Species.

BACKGROUND: Stomata in rice control a number of physiological processes by regulating gas and water exchange between the atmosphere and plant tissues. The impact of the structural diversity of these micropores on its conductance level is an important area to explore before introducing stomatal traits into any breeding program in order to increase photosynthesis and crop yield. Therefore, an intensive measurement of structural components of stomatal complex (SC) of twenty three Oryza species spanning the primary, secondary and tertiary gene pools of rice has been conducted. RESULTS: Extensive diversity was found in stomatal number and size in different Oryza species and Oryza complexes. Interestingly, the dynamics of stomatal traits in Oryza family varies differently within different Oryza genetic complexes. Example, the Sativa complex exhibits the greatest diversity in stomatal number, while the Officinalis complex is more diverse for its stomatal size. Combining the structural information with the Oryza phylogeny revealed that speciation has tended towards increasing stomatal density rather than stomatal size in rice family. Thus, the most recent species (i.e. the domesticated rice) eventually has developed smaller yet numerous stomata. Along with this, speciation has also resulted in a steady increase in stomatal conductance (anatomical, gmax) in different Oryza species. These two results unambiguously prove that increasing stomatal number (which results in stomatal size reduction) has increased the stomatal conductance in rice. Correlations of structural traits with the anatomical conductance, leaf carbon isotope discrimination (∆13C) and major leaf morphological and anatomical traits provide strong supports to untangle the ever mysterious dependencies of these traits in rice. The result displayed an expected negative correlation in the number and size of stomata; and positive correlations among the stomatal length, width and area with guard cell length, width on both abaxial and adaxial leaf surfaces. In addition, gmax is found to be positively correlated with stomatal number and guard cell length. The ∆13C values of rice species showed a positive correlation with stomatal number, which suggest an increased water loss with increased stomatal number. Interestingly, in contrast, the ∆13C consistently shows a negative relationship with stomatal and guard cell size, which suggests that the water loss is less when the stomata are larger. Therefore, we hypothesize that increasing stomatal size, instead of numbers, is a better approach for breeding programs in order to minimize the water loss through stomata in rice. CONCLUSION: Current paper generates useful data on stomatal profile of wild rice that is hitherto unknown for the rice science community. It has been proved here that the speciation has resulted in an increased stomatal number accompanied by size reduction during Oryza's evolutionary course; this has resulted in an increased gmax but reduced water use efficiency. Although may not be the sole driver of water use efficiency in rice, our data suggests that stomata are a potential target for modifying the currently low water use efficiency in domesticated rice. It is proposed that Oryza barthii can be used in traditional breeding programs in enhancing the stomatal size of elite rice cultivars.

Novel cis-acting regulatory elements in wild Oryza species impart improved rice bran quality by lowering the expression of phospholipase D alpha1 enzyme (OsPLDα1).

Rice bran oil is good quality edible oil, rich in antioxidants and comprised typically of oleic-linoleic type fatty acids. However, presence of a highly lipolytic enzyme Phospholipase D alpha1 (OsPLDα1) increases free fatty acid content in the oil which further leads to stale flavor and rancidity of the oil, making it unfit for human consumption. In this study, we compared the upstream regions of OsPLDα1 orthologs across 34 accessions representing 5 wild Oryza species and 8 cultivars, to uncover sequence variations and identify cis-elements involved in differential transcription of orthologs. Alignment of the upstream sequences to the Nipponbare OsPLDα1 reference sequence revealed the presence of 39 SNPs. Phylogenetic analysis showed that all the selected cultivars and wild species accessions are closely related to the reference except for three accessions of O. rufipogon (IRGC89224, IRGC104425, and IRGC105902). Furthermore, using exon-specific qRT-PCR, OsPLDα1 expression patterns in immature grains indicated significant differences in transcript abundance between the wild species accessions. In comparison to the control, lowest gene expression was observed in IRGC89224 accession (0.20-fold) followed by IRGC105902 (0.26-fold) and IRGC104425 (0.41-fold) accessions. In-silico analysis of the OsPLDα1 promoter revealed that the copy number variations of CGCGBOXAT, GT1CONSENSUS, IBOXCORE, NODCON2GM, OSE2ROOTNODULE, SURECOREATSULTR11, and SORLIP1AT cis-elements play an important role in the transcriptional activities of orthologous genes. Owing to the presence of ARFAT and SEBF elements only in the IRGC89224 accession, which had the lowest gene expression as well, these putative upstream regulatory sequences have been identified as novel cis-elements which may act as repressors in regulating the OsPLDα1 gene expression. The accessions identified with low OsPLDα1 expressions could be further deployed as potential donors of ideal OsPLDα1 allele for transfer of the desired trait into elite rice cultivars.

The Development and Characterization of Near-Isogenic and Pyramided Lines Carrying Resistance Genes to Brown Planthopper with the Genetic Background of Japonica Rice (Oryza sativa L.).

The brown planthopper (BPH: Nilaparvata lugens Stål.) is a major pest of rice, Oryza sativa, in Asia. Host plant resistance has tremendous potential to reduce the damage caused to rice by the planthopper. However, the effectiveness of resistance genes varies spatially and temporally according to BPH virulence. Understanding patterns in BPH virulence against resistance genes is necessary to efficiently and sustainably deploy resistant rice varieties. To survey BPH virulence patterns, seven near-isogenic lines (NILs), each with a single BPH resistance gene (BPH2-NIL, BPH3-NIL, BPH17-NIL, BPH20-NIL, BPH21-NIL, BPH32-NIL and BPH17-ptb-NIL) and fifteen pyramided lines (PYLs) carrying multiple resistance genes were developed with the genetic background of the japonica rice variety, Taichung 65 (T65), and assessed for resistance levels against two BPH populations (Hadano-66 and Koshi-2013 collected in Japan in 1966 and 2013, respectively). Many of the NILs and PYLs were resistant against the Hadano-66 population but were less effective against the Koshi-2013 population. Among PYLs, BPH20+BPH32-PYL and BPH2+BPH3+BPH17-PYL granted relatively high BPH resistance against Koshi-2013. The NILs and PYLs developed in this research will be useful to monitor BPH virulence prior to deploying resistant rice varieties and improve rice's resistance to BPH in the context of regionally increasing levels of virulence.

Production and cytological characterization of a synthetic amphiploid derived from a cross between Oryza sativa and Oryza punctata.

Oryza punctata Kotschy ex Steud. (BB, 2n = 24) is a wild species of rice that has many useful agronomic traits. An interspecific hybrid (AB, 2n = 24) was produced by crossing O. punctata and Oryza sativa variety Punjab Rice 122 (PR122, AA, 2n = 24) to broaden the narrow genetic base of cultivated rice. Cytological analysis of the pollen mother cells (PMCs) of the interspecific hybrids confirmed that they have 24 chromosomes. The F1 hybrids showed the presence of 19-20 univalents and 1-3 bivalents. The interspecific hybrid was treated with colchicine to produce a synthetic amphiploid (AABB, 2n = 48). Pollen fertility of the synthetic amphiploid was found to be greater than 50% and partial seed set was observed. Chromosome numbers in the PMCs of the synthetic amphiploid were 24II, showing normal pairing. Flow cytometric analysis also confirmed doubled genomic content in the synthetic amphiploid. Leaf morphological and anatomical studies of the synthetic amphiploid showed higher chlorophyll content and enlarged bundle sheath cells as compared with both of its parents. The synthetic amphiploid was backcrossed with PR122 to develop a series of addition and substitution lines for the transfer of useful genes from O. punctata with least linkage drag.

Potential of Oryza officinalis to augment the cold tolerance genetic mechanisms of Oryza sativa by network complementation.

Oryza officinalis is an accessible alien donor for genetic improvement of rice. Comparison across a representative panel of Oryza species showed that the wild O. officinalis and cultivated O. sativa ssp. japonica have similar cold tolerance potentials. The possibility that either distinct or similar genetic mechanisms are involved in the low temperature responses of each species was addressed by comparing their transcriptional networks. General similarities were supported by shared transcriptomic signatures indicative of equivalent metabolic, hormonal, and defense status. However, O. officinalis has maintained an elaborate cold-responsive brassinosteroid-regulated BES1-network that appeared to have been fragmented in O. sativa. BES1-network is potentially important for integrating growth-related responses with physiological adjustments and defenses through the protection of photosynthetic machinery and maintenance of stomatal aperture, oxidative defenses, and osmotic adjustment. Equivalent physiological processes are functional in O. sativa but their genetic mechanisms are under the direct control of ABA-dependent, DREB-dependent and/or oxidative-mediated networks uncoupled to BES1. While O. officinalis and O. sativa represent long periods of speciation and domestication, their comparable cold tolerance potentials involve equivalent physiological processes but distinct genetic networks. BES1-network represents a novel attribute of O. officinalis with potential applications in diversifying or complementing other mechanisms in the cultivated germplasm.

High-resolution genetic mapping of a novel brown planthopper resistance locus, Bph34 in Oryza sativa L. X Oryza nivara (Sharma & Shastry) derived interspecific F2 population.

KEY MESSAGE: A BPH-resistant locus designated as Bph34 identified in Oryza nivara acc. IRGC104646 on long arm of chromosome 4 using high-resolution mapping with 50 K SNP chip. BPH resistance contributed by locus showed dominant inheritance in F2 and F3. The Bph34 locus is 91 kb in size and contains 11 candidate genes. In addition to SNP markers, SSR markers, RM16994 and RM17007 co-segregated with the BPH resistance. These two SSR markers can facilitate marker-assisted transfer of the Bph34 locus into elite rice cultivars in all labs. Brown planthopper (BPH, Nilaparvata lugen Stål) is one of the most destructive insects of rice (Oryza sativa L.) causing significant yield losses annually. Exploiting host plant resistance to BPH and incorporating resistant genes in susceptible commercial cultivars is economical and environmentally friendly approach to manage this pest. Here, we report high-resolution mapping of a novel genetic locus for resistance to BPH, designated as Bph34 on long arm of rice chromosome 4. The locus was mapped using an interspecific F2 population derived from a cross between susceptible indica cultivar PR122 and BPH-resistant wild species, O. nivara acc. IRGC104646. Inheritance studies performed using F2 and F2:3 populations revealed the presence of single dominant gene. Construction of high-density linkage map using 50 K SNP chip (OsSNPnks) followed by QTL mapping identified single major locus at 28.8 LOD score between SNP markers, AX-95952039 and AX-95921548. The major locus contributing resistance to BPH designated as Bph34 and explained 68.3% of total phenotypic variance. The Bph34 locus is 91 Kb in size on Nipponbare reference genome-IRGSP-1.0 and contains 11 candidate genes. In addition to associated SNP markers, two SSR markers, RM16994 and RM17007, also co-segregated with the Bph34 which can be used efficiently for markers assisted transfer into elite rice cultivars across the labs.

The Evolutionary Basis of Naturally Diverse Rice Leaves Anatomy.

Rice contains genetically and ecologically diverse wild and cultivated species that show a wide variation in plant and leaf architecture. A systematic characterization of leaf anatomy is essential in understanding the dynamics behind such diversity. Therefore, leaf anatomies of 24 Oryza species spanning 11 genetically diverse rice genomes were studied in both lateral and longitudinal directions and possible evolutionary trends were examined. A significant inter-species variation in mesophyll cells, bundle sheath cells, and vein structure was observed, suggesting precise genetic control over these major rice leaf anatomical traits. Cellular dimensions, measured along three growth axes, were further combined proportionately to construct three-dimensional (3D) leaf anatomy models to compare the relative size and orientation of the major cell types present in a fully expanded leaf. A reconstruction of the ancestral leaf state revealed that the following are the major characteristics of recently evolved rice species: fewer veins, larger and laterally elongated mesophyll cells, with an increase in total mesophyll area and in bundle sheath cell number. A huge diversity in leaf anatomy within wild and domesticated rice species has been portrayed in this study, on an evolutionary context, predicting a two-pronged evolutionary pathway leading to the 'sativa leaf type' that we see today in domesticated species.

Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza.

Long terminal repeat (LTR) retrotransposons constitute a significant portion of most eukaryote genomes and can dramatically change genome size and organization. Although LTR retrotransposon content variation is well documented, the dynamics of genomic flux caused by their activity are poorly understood on an evolutionary time scale. This is primarily because of the lack of an experimental system composed of closely related species whose divergence times are within the limits of the ability to detect ancestrally related retrotransposons. The genus Oryza, with 24 species, ten genome types, different ploidy levels and over threefold genome size variation, constitutes an ideal experimental system to explore genus-level transposon dynamics. Here we present data on the discovery and characterization of an LTR retrotransposon family named RWG in the genus Oryza. Comparative analysis of transposon content (approximately 20 to 27,000 copies) and transpositional history of this family across the genus revealed a broad spectrum of independent and lineage-specific changes that have implications for the evolution of genome size and organization. In particular, we provide evidence that the basal GG genome of Oryza (O. granulata) has expanded by nearly 25% by a burst of the RWG lineage Gran3 subsequent to speciation. Finally we describe the recent evolutionary origin of Dasheng, a large retrotransposon derivative of the RWG family, specifically found in the A, B and C genome lineages of Oryza.

New insights into Oryza genome evolution: high gene colinearity and differential retrotransposon amplification.

An approximately 247-kb genomic region from FF genome of wild rice Oryza brachyantha, possessing the smallest Oryza genome, was compared to the orthologous approximately 450-kb region from AA genome, O. sativa L. ssp. japonica. 37 of 38 genes in the orthologous regions are shared between japonica and O. brachyantha. Analyses of nucleotide substitution in coding regions suggest the two genomes diverged approximately 10 million years ago. Comparisons of transposable elements (TEs) reveal that the density of DNA TEs in O. brachyantha is comparable to O. sativa; however, the density of RNA TEs is dramatically lower. The genomic fraction of RNA TEs in japonica is two times greater than in O. brachyantha. Differences, particularly in RNA TEs, in this region and in BAC end sequences from five wild and two cultivated Oryza species explain major genome size differences between sativa and brachyantha. Gene expression analyses of three ObDREB1 genes in the sequenced region indicate orthologous genes retain similar expression patterns following cold stress. Our results demonstrate that size and number of RNA TEs play a major role in genomic differentiation and evolution in Oryza. Additionally, distantly related O. brachyantha shares colinearity with O. sativa, offering opportunities to use comparative genomics to explore the genetic diversity of wild species to improve cultivated rice.

Doubling genome size without polyploidization: dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice.

Retrotransposons are the main components of eukaryotic genomes, representing up to 80% of some large plant genomes. These mobile elements transpose via a "copy and paste" mechanism, thus increasing their copy number while active. Their accumulation is now accepted as the main factor of genome size increase in higher eukaryotes, besides polyploidy. However, the dynamics of this process are poorly understood. In this study, we show that Oryza australiensis, a wild relative of the Asian cultivated rice O. sativa, has undergone recent bursts of three LTR-retrotransposon families. This genome has accumulated more than 90,000 retrotransposon copies during the last three million years, leading to a rapid twofold increase of its size. In addition, phenetic analyses of these retrotransposons clearly confirm that the genomic bursts occurred posterior to the radiation of the species. This provides direct evidence of retrotransposon-mediated variation of genome size within a plant genus.

The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza.

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

The oryza map alignment project: the golden path to unlocking the genetic potential of wild rice species.

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the 'Oryza Map Alignment Project' (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project's finished reference genome--O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara--thought to be the progenitor of modern cultivated rice.

Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice.

Cultivation-independent studies suggest that roots of rice (Oryza sativa) are colonized by a diverse community of nitrogen-fixing bacteria. Here we report for the first time mRNA-based profiling of nitrogenase (nifH) genes, to study the impact of lowland-rice genotypes at the cultivar level on the functional diversity of root-associated diazotrophs. Root RNA extracts from all plants contained nifH mRNA at levels detectable by reverse transcription polymerase chain reaction (RT-PCR). Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed only small interplant variations. However, RNA- and DNA-based profiles obtained from the same root extractions differed from each other, suggesting that presence of diazotrophs did not necessarily coincide with active transcription of nif genes. Application of N-fertilizer at planting had a long-term effect on the profile of expressed nitrogenase genes. Phylogenetic analysis of a clone library constructed for nifH fragments expressed in wild species of rice roots indicated that active diazotrophs were not related to cultured strains. The composition of active diazotrophic communities was compared for six related cultivars of O. sativa, wild species Oryza brachyantha, and a genetic cross between it and cv. IR56, grown under identical conditions in rice field soil in the Philippines without N-fertilizer application. Remarkable varietal differences in root associated nifH-gene expressing communities were detected. This underlines the importance of mRNA-based approaches to study functional diversity and eventually identify key diazotrophs in a particular environment.

Identification and mapping of the QTL for aluminum tolerance introgressed from the new source, Oryza Rufipogon Griff., into indica rice (Oryza sativa L.).

This study was conducted to identify and map the quantitative trait locus (QTL) controlling Al tolerance in rice using molecular markers. A population of 171 F(6) recombinant inbred lines (RILs) derived from the cross of Oryza sativa (IR64), the Al susceptible parent, and Oryza rufipogon, the Al tolerant parent, was evaluated for Al tolerance using a nutrient solution with and without 40 ppm of active Al(+3). A genetic map, consisting of 151 molecular markers covering 1,755 cM with an average distance of 11.6 cM between loci, was constructed. Nine QTLs were dentified including one for root length under non-stress conditions (CRL), three for root length under Al stress (SRL) and five for relative root length (RRL). O. rufipogon contributed favorable alleles for each of the five QTLs for RRL, which is a primary parameter for Al tolerance, and individually they explained 9.0-24.9% of the phenotypic variation. Epistatic analysis revealed that CRL was conditioned by an epistatic effect, whereas SRL and RRL were controlled by additive effects. Comparative genetic analysis showed that QTLs for RRL, which mapped on chromosomes 1 and 9, appear to be consistent among different rice populations. Interestingly, a major QTL for RRL, which explained 24.9% of the phenotypic variation, was found on chromosome 3 of rice, which is conserved across cereal species. These results indicate the possibilities to use marker-assisted selection and pyramiding QTLs for enhancing Al tolerance in rice. Positional cloning of such QTLs introgressed from O. rufipogon will provide a better understanding of the Al tolerance mechanism in rice and the evolutionary genetics of plant adaptation to acid-soil conditions across cereal species.