Oryza Sativa Subspecies Research Articles

Transcription factor OsDOF1 enhances cold‐stress tolerance potentially through interactions with OsICE1 and its target genes in rice (Oryza sativa L.)

AbstractRice plants are generally sensitive to cold temperatures, which adversely affect their yield. Japonica rice, a major subspecies of Oryza sativa L. (Asian cultivated rice), exhibits cold tolerance. Recently, we identified that the plant‐specific transcription factor DNA binding with one finger 1 (Dof1) was associated with the cold resistance of Japonica rice. The purpose of this study was to delineate the role of the OsDof1 gene in cold‐stress tolerance and its potential molecular pathways. Two japonica rice varieties, including the cold‐resistant line LD5 and the cold‐sensitive line LJ11, were used. The protein–protein interactions were determined by yeast two‐hybrid (Y2H), tag‐based pull‐down, and bimolecular fluorescence complementation (BiFC) assays. Truncated DsDOF1 protein was identified in LJ11 as a consequence of a point mutation, and OsDOF1‐T failed to be transported into the nucleus. Furthermore, OsDREB1B, OsDREB2A, OsNF‐YC, OsLEA3, OsRAB16A, OsLIP9, OsNCED3, OsABI2, OsMYBS3, and OsBS3R‐2 were differentially expressed in OsDof1‐overexpressing transgenic versus wild‐type rice lines. Notably, OsICE1 was found to be the target protein of OsDOF1, and subsequent Y2H, BiFC, and tag‐based pull‐down assays showed a direct interaction between the two proteins. In conclusion, OsDOF1 exerts a critical role in cold tolerance potentially through direct interactions with OsICE1 and its target genes in rice. These findings may help to better understand the molecular basis of rice adaptation to cold stress, and OsDof1 could be a target gene in the molecular breeding of rice for better adaptation to a cold‐climate environment, which might eventually improve the yield of rice.

The role of gene presence-absence variations on genetic incompatibility in Asian rice.

Genetic incompatibilities are widespread between species. However, it remains unclear whether they all originated after population divergence as suggested by the Bateson-Dobzhansky-Muller model, and if not, what is their prevalence and distribution within populations. The gene presence-absence variations (PAVs) provide an opportunity for investigating gene-gene incompatibility. Here, we searched for the repulsion of coexistence between gene PAVs to identify the negative interaction of gene functions separately in two Oryza sativa subspecies. Many PAVs are involved in subspecies-specific negative epistasis and segregate at low-to-intermediate frequencies in focal subspecies but at low or high frequencies in the other subspecies. Incompatible PAVs are enriched in two functional groups, defense response and protein phosphorylation, which are associated with plant immunity and consistent with autoimmunity being a known mechanism of hybrid incompatibility in plants. Genes in the two enriched functional groups are older and seldom directly interact with each other. Instead, they interact with other younger gene PAVs with diverse functions. Our results illustrate the landscape of genetic incompatibility at gene PAVs in rice, where many incompatible pairs have already segregated as polymorphisms within subspecies, and many are novel negative interactions between older defense-related genes and younger genes with diverse functions.

Molecular Breeding of Water-Saving and Drought-Resistant Rice for Blast and Bacterial Blight Resistance.

Rice production is often affected by biotic and abiotic stressors. The breeding of resistant cultivars is a cost-cutting and environmentally friendly strategy to maintain a sustainable high production level. An elite water-saving and drought-resistant rice (WDR), Hanhui3, is susceptible to blast and bacterial blight (BB). This study was conducted to introgress three resistance genes (Pi2, xa5, and Xa23) for blast and BB into Hanhui3, using marker-assisted selection (MAS) for the foreground selection and a whole-genome single-nucleotide polymorphism (SNP) array for the background selection. As revealed by the whole-genome SNP array, the recurrent parent genome (RPG) recovery of the improved NIL was 94.2%. The resistance levels to blast and BB of the improved NIL and its derived hybrids were higher than that of the controls. In addition, the improved NIL and its derived hybrids retained the desired agronomic traits from Hanhui3, such as yield. The improved NIL could be useful to enhance resistance against biotic stressors and produce stable grain yields in Oryza sativa subspecies indica rice breeding programs.

Genetic Effects of Indica Lineage Introgression on Amylopectin Chain Length Distribution in Japonica Milled Rice

The fine structure of amylopectin affects rice quality; in particular, the amylopectin chain length distribution (ACLD) in milled rice differs between subspecies of Oryza sativa L. However, the correlation between ACLD and quality trait factors, and the genetic basis of ACLD phenotypic variation, are still unknown. Here, the correlations of ACLD with cooking and eating quality and with the rapid viscosity analysis (RVA) index were studied using chromosome segment substitution lines (CSSLs). Clear variations in ACLD were observed in introgression lines: introgression of indica segments of chromosome 3 and 7 increased the proportion of amylopectin Fa, and another segment of chromosome 3 reduced the proportion of amylopectin Fb2. A segment of chromosome 11 decreased the proportion of amylopectin Fa but increased that of Fb3. Correlation analysis with the RVA index further showed that the breakdown viscosity (BDV) was negatively correlated with the proportion of amylopectin Fb1, Fb2, and Fb3 chains, and positively correlated with Fa. Consistency viscosity (CSV) values were negatively correlated with the proportion of amylopectin Fb1, Fb2, and Fb3 chains. We thus clarified the quality trait factors determined by variation in ACLD, and provide key information for pyramiding inter-subspecific genetic superiority in molecular design breeding for rice quality.

Identification of mouldy rice using an electronic nose combined with SPME-GC/MS

Early warning of mildew in grain storage is extremely important to ensure the security of reserves. Japonica rice (Oryza sativa subspecies. Japonica) was used as an example in this study to develop a strategy for the establishment of an early warning and analysis system for grain mildew, that is, an electronic nose (E-nose) is used to analyse the volatile substances produced during the mildewing of japonica rice. The main volatile components of mildewed japonica rice were determined to be alcohol, aldehyde and ketone, to which the W2S sensor of the E-nose is sensitive. A gas chromatography-mass spectrometry (GC-MS) analysis showed that the content of mouldy markers (1-octen-3-ol and 3-octanone) and the response of the W2S sensor were highly correlated with the total colony number. Principal component analysis (PCA) and cluster analysis further verified the excellent performance of the E-nose in identifying japonica rice mildewing. Thus, it is feasible to use the E-nose in combination with GC/MS for the early detection of mould contamination of japonica rice.

Could Japonica Rice Be an Alternative Variety for Increased Global Food Security and Climate Change Mitigation?

The growing importance of rice globally over the past three decades is evident in its strategic place in many countries’ food security planning policies. Still, its cultivation emits substantial greenhouse gases (GHGs). The Indica and Japonica sub-species of Oryza sativa L. are mainly grown, with Indica holding the largest market share. The awareness, economics, and acceptability of Japonica rice in a food-insecure Indica rice-consuming population were surveyed. The impact of parboiling on Japonica rice was studied and the factors which most impacted stickiness were investigated through sensory and statistical analyses. A comparison of the growing climate and greenhouse gas emissions of Japonica and Indica rice was carried out by reviewing previous studies. Survey results indicated that non-adhesiveness and pleasant aroma were the most preferred properties. Parboiling treatment altered Japonica rice’s physical and chemical properties, introducing gelatinization of starch and reducing adhesiveness while retaining micronutrient concentrations. Regions with high food insecurity and high consumption of Indica rice were found to have suitable climatic conditions for growing Japonica rice. Adopting the higher-yielding, nutritious Japonica rice whose cultivation emits less GHG in these regions could help strengthen food security while reducing GHGs in global rice cultivation.

Evolutionary Understanding of Metacaspase Genes in Cultivated and Wild Oryza Species and Its Role in Disease Resistance Mechanism in Rice.

Metacaspases (MCs), a class of cysteine-dependent proteases found in plants, fungi, and protozoa, are predominately involved in programmed cell death processes. In this study, we identified metacaspase genes in cultivated and wild rice species. Characterization of metacaspase genes identified both in cultivated subspecies of Oryza sativa, japonica, and indica and in nine wild rice species was performed. Extensive computational analysis was conducted to understand gene structures, phylogenetic relationships, cis-regulatory elements, expression patterns, and haplotypic variations. Further, the haplotyping study of metacaspase genes was conducted using the whole-genome resequencing data publicly available for 4726 diverse genotype and in-house resequencing data generated for north-east Indian rice lines. Sequence variations observed among wild and cultivated rice species for metacaspase genes were used to understand the duplication and neofunctionalization events. The expression profiles of metacaspase genes were analyzed using RNA-seq transcriptome profiling in rice during different developmental stages and stress conditions. Real-time quantitative PCR analysis of candidate metacaspase genes in rice cultivars Pusa Basmati-1 in response to Magnaporthe oryzae infection indicated a significant role in the disease resistance mechanism. The information provided here will help to understand the evolution of metacaspases and their role under stress conditions in rice.

Genome-wide screening of Oryza sativa ssp. japonica and indica reveals a complex family of proteins with ribosome-inactivating protein domains

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes capable of halting protein synthesis by irreversible modification of ribosomes. Although RIPs are widespread they are not ubiquitous in the plant kingdom. The physiological importance of RIPs is not fully elucidated, but evidence suggests a role in the protection of the plant against biotic and abiotic stresses. Searches in the rice genome revealed a large and highly complex family of proteins with a RIP domain. A comparative analysis retrieved 38 RIP sequences from the genome sequence of Oryza sativa subspecies japonica and 34 sequences from the subspecies indica. The RIP sequences are scattered over different chromosomes but are mostly found on the third chromosome. The phylogenetic tree revealed the pairwise clustering of RIPs from japonica and indica. Molecular modeling and sequence analysis yielded information on the catalytic site of the enzyme, and suggested that a large part of RIP domains probably possess N-glycosidase activity. Several RIPs are differentially expressed in plant tissues and in response to specific abiotic stresses. This study provides an overview of RIP motifs in rice and will help to understand their biological role(s) and evolutionary relationships.

Characterization of indica–japonica subspecies-specific InDel loci in wild relatives of rice (Oryza sativa L. subsp. indica Kato and subsp. japonica Kato)

Insertion/deletion (InDel) polymorphisms are generally irreversible and, thus, are useful for evaluating the genetic relationships within the genus Oryza. Moreover, subspecies-specific (SS) InDel markers linked to conserved genomic regions specific to the indica and japonica subspecies of Oryza sativa can provide insight into the genetic relationships between cultivated and wild rice. The evolutionary relationship among Oryza species in respect to their indica and japonica alleles was investigated using 67 selected indica–japonica InDel SS-STS primers across 290 accessions, including 61 Asian cultivated rice (O. sativa) cultivars, 27 African cultivated rice (O. glaberrima) accessions, and 202 accessions of wild Orzya species. The average SS allele frequency of the various Oryza species, from AA-genome to BB ~ EE, and FF ~ HHKK showed an increased proportion of non-O. sativa and null alleles in the more distantly related wild species. Most of the wild species, except the more distant EE, GG, HHJJ, and HHKK genome accessions, consisted of relatively more indica than japonica alleles of SS markers. To validate the SS-STS study, PCR products of nine markers were sequenced across 24–33 accessions. Sequencing results revealed that Oryza species share indica or japonica-like conserved InDel regions even across the different genomes. The presence of some japonica alleles beyond the AA genome at some SS InDel loci also suggests that japonica-specific alleles occurred early in the history of the Oryza genus. The O. sativa sub-species specific markers thus provide further insight into the evolutionary pathway in the genus Oryza and the process of differentiation between indica and japonica.

Genome-wide cataloging and analysis of alternatively spliced genes in cereal crops.

BackgroundProtein functional diversity at the post-transcriptional level is regulated through spliceosome mediated pre-mRNA alternative splicing (AS) events and that has been widely demonstrated to be a key player in regulating the functional diversity in plants. Identification and analysis of AS genes in cereal crop plants are critical for crop improvement and understanding regulatory mechanisms.ResultsWe carried out the comparative analyses of the functional landscapes of the AS using the consensus assembly of expressed sequence tags and available mRNA sequences in four cereal plants. We identified a total of 8,734 in Oryza sativa subspecies (ssp) japonica, 2,657 in O. sativa ssp indica, 3,971 in Sorghum bicolor, and 10,687 in Zea mays AS genes. Among the identified AS events, intron retention remains to be the dominant type accounting for 23.5 % in S. bicolor, and up to 55.8 % in O. sativa ssp indica. We identified a total of 887 AS genes that were conserved among Z. mays, S. bicolor, and O. sativa ssp japonica; and 248 AS genes were found to be conserved among all four studied species or ssp. Furthermore, we identified 53 AS genes conserved with Brachypodium distachyon. Gene Ontology classification of AS genes revealed functional assignment of these genes in many biological processes with diverse molecular functions.ConclusionsAS is common in cereal plants. The AS genes identified in four cereal crops in this work provide the foundation for further studying the roles of AS in regulation of cereal plant growth and development. The data can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/).Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1914-5) contains supplementary material, which is available to authorized users.

Whole genome sequencing of elite rice cultivars as a comprehensive information resource for marker assisted selection.

Current advances in sequencing technologies and bioinformatics revealed the genomic background of rice, a staple food for the poor people, and provided the basis to develop large genomic variation databases for thousands of cultivars. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to aid the development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 104 rice varieties belonging to the major subspecies of Oryza sativa. We identified repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. Genotyping of over 18 million polymorphic locations within O. sativa allowed us to reconstruct the individual haplotype patterns shaping the genomic background of elite varieties used by farmers throughout the Americas. Based on a reconstruction of the alleles for the gene GBSSI, we could identify novel genetic markers for selection of varieties with high amylose content. We expect that both the analysis methods and the genomic information described here would be of great use for the rice research community and for other groups carrying on similar sequencing efforts in other crops.

Genetic Characterization of Indigenous Rice Varieties in Eastern Himalayan Region of Northeast India

The eastern Himalayan region of Northeast (NE) India is home to a large number of indigenous rice varieties, which are traditionally classified as Oryza sativa subspecies indica, japonica or intermediate types. The classification based on traditional Cheng's index is often inconclusive due to phenotypic plasticity of morphological characters, which are influenced by environmental conditions. We used molecular markers specific for indica and japonica subspecies to assess the degree of genetic relatedness of indigenous rice varieties in NE India. The results revealed that majority of upland (jum) and glutinous rice varieties, traditionally considered as japonica, were genetically close to the subspecies indica. All varieties of boro ecotype were found to be indica type, and only a few varieties cultivated in lowland and upland areas were japonica type. Some of the lowland varieties of the sali ecotype were intermediate between indica and japonica, and they showed a closer genetic affinity to O. rufipogon.

Rice varietal improvement for Mediterranean France

In France, rice is grown mainly near the Mediterranean Sea, in the Camargue. An irrigated system based on direct seeding on wet land is used. Occurrence of cold periods throughout the crop cycle (April/May-September/October) and the risk of soil salinization are the most important constraints. Since 1988, CIRAD and CFR have been implementing a rice breeding program for the Camargue. It aims at creating cultivars fitting the needs of rice farmers (yield potential and stability), the rice industry (milling and processing qualities) and consumers (cooking and taste qualities). Varieties grown in the region belong to the japonica subspecies of Oryza sativa. Hybridization is the basis for creation of variability. Progenies are selected using the pedigree method associated or not with haploïdipolidisation applied to the first generation. Marker-assisted selection has recently been introduced to improve selection efficacy for resistance to fungal diseases occurring erratically in relation to climate. So far, 25 varieties have been registered in the European Official Catalogue, of which 5 have proven particularly successful among farmers. Progress achieved for germination in anaerobic conditions, resistance to stem borers, grain shape and other qualities, including aroma, are presented. Prospects in relation to new objectives such as weed control and adaptation to climate change are also presented.

Differentiation in wild-type allele of the sd1 locus concerning culm length between indica and japonica subspecies of Oryza sativa (rice)

A dwarfing gene (allele) sd1-d has been intensively utilized to develop short-culm indica varieties in southeast Asia up to now. Before the first sd1-d-carrying variety IR8 was released, rice researchers had recognized the general tendency that culm length is higher in indica varieties than in temperate-japonica ones. Inter-subspecific difference of the tall (wild-type) allele SD1 at the sd1 locus was examined on the common genetic background, using five isogenic lines developed by substituting sd1-d of the recurrent parent IR36 by SD1s of two indica varieties, two temperate-japonica varieties and one tropical-japonica variety. The two indica -donor isogenic lines had longer culms than the three japonica-donor isogenic lines consistently in two different environmental conditions. Moreover, nonsynonymous single-nucleotide polymorphism between the two subspecies was detected at two sites in Exon 1 and Exon 3 of the sd1 locus. It is demonstrated that the inter-subspecific differentiation of SD1 contributes height difference between indica and japonica. The indica-originating and japonica-originating alleles at the sd1 locus were designated as SD1-in(t) and SD1-ja(t), respectively.

The complete rice genome sequence: a gold mine for future rice research

The map-based complete rice genome sequence is now freely available to researchers worldwide, providing the most fundamental tool that should further accelerate efforts to improve the staple crop that feeds more than half the world’s population. The finished-quality sequence covers 95% of the 389-Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified. The complete genetic information on rice will serve as a gold mine for genomic research in rice and other cereal species. It will facilitate the identification of many important genes by both forward and reverse genetics strategies, and clarify the relationships between sequence variation and phenotypes. The genome sequence derived from Oryza sativa subspecies japonica can be used as a reference sequence for comparative analysis among Oryza species that will help in understanding the major factors involved in speciation and searching for useful genetic resources. Furthermore, the completed sequence will also serve as a standard for cereal genome comparison and identification of rice orthologous genes in other cereal crops, thereby providing a platform for establishing the genomics of each cereal species.

A collection of 10,096 indica rice full-length cDNAs reveals highly expressed sequence divergence between Oryza sativa indica and japonica subspecies

Relatively few indica rice full-length cDNAs were available to aid in the annotation of rice genes. The data presented here described the sequencing and analysis of 10,096 full-length cDNAs from Oryza sativa subspecies indica Guangluai 4. Of them, 9,029 matched rice genomic sequences in publicly-available databases, and 1,200 were identified as new rice genes. Comparison with the knowledge-based Oryza Molecular Biological Encyclopedia japonica cDNA collection indicated that 3,316 (41.6%) of the 7,965 indica-japonica cDNA pairs showed no distinct variations at protein level (2,117 indica-japonica cDNA pairs showed fully identical and 1,199 indica-japonica cDNA pairs showed no frame shift). Moreover, 3,645 (45.8%) of the indica-japonica pairs showed substantial differences at the protein level due to single nucleotide polymorphisms (SNPs), insertions or deletions, and sequence-segment variations between indica and japonica subspecies. Further experimental verifications using PCR screening and quantitative reverse transcriptional PCR revealed unique transcripts for indica subspecies. Comparative analysis also showed that most of rice genes were evolved under purifying selection. These variations might distinguish the phenotypic changes of the two cultivated rice subspecies indica and japonica. Analysis of these cDNAs extends known rice genes and identifies new ones in rice.

Haplotype variation in structure and expression of a gene cluster associated with a quantitative trait locus for improved yield in rice

By constructing nearly isogenic lines (NILs) that differ only at a single quantitative trait locus (QTL), we fine-mapped the yield-improving QTL qGY2-1 to a 102.9-kb region on rice chromosome 2. Comparison analysis of the genomic sequences in the mapped QTL region between the donor (Dongxiang wild rice, Oryza rufipogon Griff.) and recurrent (Guichao2, Oryza sativa ssp. indica) parents used for the development of NILs identified the haplotypes of a leucine-rich repeat receptor kinase gene cluster, which showed extensive allelic variation. The sequences between genes in the cluster had a very high rate of divergence. More importantly, the genes themselves also differed between two haplotypes: Only 92% identity was observed for one allele, and another allele was found to have completely lost its allelic counterpart in Guichao2. The other six shared genes all showed >98% identity, and four of these exhibited obvious regulatory variation. The same haplotype segments also differed in length (43.9-kb in Guichao2 vs. 52.6-kb in Dongxiang wild rice). Such extensive sequence variation was also observed between orthologous regions of indica (cv. 93-11) and japonica (cv. Nipponbare) subspecies of Oryza sativa. Different rates of sequence divergence within the cluster have resulted in haplotype variability in 13 rice accessions. We also detected allelic expression variation in this gene cluster, in which some genes gave unequal expression of alleles in hybrids. These allelic variations in structure and expression suggest that the leucine-rich repeat receptor kinase gene cluster identified in our study should be a particularly good candidate for the source of the yield QTL.

Phylogenetic relationships among A‐genome species of the genus Oryza revealed by intron sequences of four nuclear genes

The A-genome group in Oryza consists of eight diploid species and is distributed world-wide. Here we reconstructed the phylogeny among the A-genome species based on sequences of nuclear genes and MITE (miniature inverted-repeat transposable elements) insertions. Thirty-seven accessions representing two cultivated and six wild species from the A-genome group were sampled. Introns of four nuclear single-copy genes on different chromosomes were sequenced and analysed by both maximum parsimony (MP) and Bayesian inference methods. All the species except for Oryza rufipogon and Oryza nivara formed a monophyletic group and the Australian endemic Oryza meridionalis was the earliest divergent lineage. Two subspecies of Oryza sativa (ssp. indica and ssp. japonica) formed two separate monophyletic groups, suggestive of their polyphyletic origin. Based on molecular clock approach, we estimated that the divergence of the A-genome group occurred c. 2.0 million years ago (mya) while the two subspecies (indica and japonica) separated c. 0.4 mya. Intron sequences of nuclear genes provide sufficient resolution and are informative for phylogenetic inference at lower taxonomic levels.

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.

Rapid recent growth and divergence of rice nuclear genomes.

By employing the nuclear DNA of the African rice Oryza glaberrima as a reference genome, the timing, natures, mechanisms, and specificities of recent sequence evolution in the indica and japonica subspecies of Oryza sativa were identified. The data indicate that the genome sizes of both indica and japonica have increased substantially, >2% and >6%, respectively, since their divergence from a common ancestor, mainly because of the amplification of LTR-retrotransposons. However, losses of all classes of DNA sequence through unequal homologous recombination and illegitimate recombination have attenuated the growth of the rice genome. Small deletions have been particularly frequent throughout the genome. In >1 Mb of orthologous regions that we analyzed, no cases of complete gene acquisition or loss from either indica or japonica were found, nor was any example of precise transposon excision detected. The sequences between genes were observed to have a very high rate of divergence, indicating a molecular clock for transposable elements that is at least 2-fold more rapid than synonymous base substitutions within genes. We found that regions prone to frequent insertions and deletions also exhibit higher levels of point mutation. These results indicate a highly dynamic rice genome with competing processes for the generation and removal of genetic variation.