Yidan Ouyang

A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice

Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day–specific male-fertility–associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding.

A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice

Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica–japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene.

A Killer-Protector System Regulates Both Hybrid Sterility and Segregation Distortion in Rice

Conquering Rice Sterility The hybrid sterility occurring among rice species has long been a puzzle and hampers progress in breeding crops with improved performance and yield characteristics. Yang et al. (p. 1336) have identified three linked genes encoding a killer, a partner, and a protector protein. The killer and partner work together to kill female gametes not carrying the functional protector, resulting in preferential transmission of gametes carrying the functional protector, which also causes segregation distortion in the progeny. This explanation for how reproductive isolation is maintained among species of rice, and perhaps other organisms, also offers approaches for boosting yields by intersubspecific heterosis. A genetic locus containing three protein-coding genes underlies the system causing hybrid sterility among rice species. Hybrid sterility is a major form of postzygotic reproductive isolation that restricts gene flow between populations. Cultivated rice (Oryza sativa L.) consists of two subspecies, indica and japonica; inter-subspecific hybrids are usually sterile. We show that a killer-protector system at the S5 locus encoded by three tightly linked genes [Open Reading Frame 3 (ORF3) to ORF5] regulates fertility in indica-japonica hybrids. During female sporogenesis, the action of ORF5+ (killer) and ORF4+ (partner) causes endoplasmic reticulum (ER) stress. ORF3+ (protector) prevents ER stress and produces normal gametes, but ORF3– cannot prevent ER stress, resulting in premature programmed cell death and leads to embryo-sac abortion. Preferential transmission of ORF3+ gametes results in segregation distortion in the progeny. These results add to our understanding of differences between indica and japonica rice and may aid in rice genetic improvement.

Rice APOPTOSIS INHIBITOR5 Coupled with Two DEAD-Box Adenosine 5′-Triphosphate-Dependent RNA Helicases Regulates Tapetum Degeneration

This study examines the role of API5, a homolog of animal antiapoptosis proteins, in the degeneration of the tapetum during the formation of male gametophytes in rice. It describes a previously unknown pathway for regulating programmed cell death, one that may be conserved among eukaryotic organisms. Programmed cell death (PCD) during tapetum degeneration in postmeiotic anthers is critical for the proper development of male gametophytes in flowering plants. Although several genes involved in this process have been identified recently, the molecular mechanism is still poorly understood. Here, we show that knockout of rice (Oryza sativa) APOPTOSIS INHIBITOR5 (API5), which encodes a putative homolog of antiapoptosis protein Api5 in animals, results in delayed degeneration of the tapetum due to inhibition of the tapetal PCD process leading to defects in formation of male gametophytes. Os API5 is a nuclear protein that interacts with two DEAD-box ATP-dependent RNA helicases, API5-INTERACTING PROTEIN1 (AIP1) and AIP2. AIP1 and AIP2 are homologs of yeast (Saccharomyces cerevisiae) Suppressor of Bad Response to Refrigeration1 protein 2 (SUB2p) that have critical roles in transcription elongation and pre-mRNA splicing. Os AIP1 and AIP2 can form dimers and interact directly with the promoter region of CP1, a rice cysteine protease gene. Suppression of Os AIP1/2 leads to down-regulation of CP1, resulting in sterility, which is highly similar to the effects of suppressed expression of Os CP1. Our results uncover a previously unknown pathway for regulating PCD during tapetum degeneration in rice, one that may be conserved among eukaryotic organisms.

Comprehensive sequence and expression profile analysis of Hsp20 gene family in rice

The Hsp20 genes represent the most abundant small heat shock proteins (sHSPs) in plants. Hsp20 gene family has been shown to be involved in preventing heat shock and promoting resistance to environmental stress factors, but very little is known about this gene family in rice. Here, we report the identification and characterization of 39 OsHsp20 genes in rice, describing the gene structure, gene expression, genome localization, and phylogenetic relationship of each member. We have used RT-PCR to perform a characterization of the normal and heat shock-induced expression of selective OsHsp20 genes. A genome-wide microarray based gene expression analysis involving 25 stages of vegetative and reproductive development in three rice cultivars has revealed that 36 OsHsp20 genes were expressed in at least one of the experimental stages studied. Among these, transcripts of OsHsp20 were accumulated differentially during vegetative and reproductive developmental stages and preferentially down-regulated in Shanyou 63. In addition, OsHsp20 genes were identified as showing prominent heterosis in family-level expression. Our results suggest that the expression patterns of the OsHsp20 genes are diversified not only in developmental stages but also in variety level.

RiceVarMap: a comprehensive database of rice genomic variations

Rice Variation Map (RiceVarMap, http:/ricevarmap.ncpgr.cn) is a database of rice genomic variations. The database provides comprehensive information of 6 551 358 single nucleotide polymorphisms (SNPs) and 1 214 627 insertions/deletions (INDELs) identified from sequencing data of 1479 rice accessions. The SNP genotypes of all accessions were imputed and evaluated, resulting in an overall missing data rate of 0.42% and an estimated accuracy greater than 99%. The SNP/INDEL genotypes of all accessions are available for online query and download. Users can search SNPs/INDELs by identifiers of the SNPs/INDELs, genomic regions, gene identifiers and keywords of gene annotation. Allele frequencies within various subpopulations and the effects of the variation that may alter the protein sequence of a gene are also listed for each SNP/INDEL. The database also provides geographical details and phenotype images for various rice accessions. In particular, the database provides tools to construct haplotype networks and design PCR-primers by taking into account surrounding known genomic variations. These data and tools are highly useful for exploring genetic variations and evolution studies of rice and other species.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63

Significance Indica rice accounts for >70% of total rice production worldwide, is genetically highly diverse, and can be divided into two major varietal groups independently bred and widely cultivated in China and Southeast Asia. Here, we generated high-quality genome sequences for two elite rice varieties, Zhenshan 97 and Minghui 63, representing the two groups of indica rice and the parents of a leading rice hybrid. Comparative analyses uncovered extensive structural differences between the two genomes and complementarity in their hybrid transcriptome. These findings have general implications for understanding intraspecific variations of organisms with complex genomes. The availability of the two genomes will serve as a foundation for future genome-based explorations in rice toward both basic and applied goals. Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica. Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059–2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation

Significance Insect pests represent a major constraint that reduces crop yield and quality globally. Host plant resistance is often used as a key tactic to control insect pests, but is frequently overcome by newly emerged insect populations. In nature, plants have developed various strategies for sustainable defense. In this work, we isolated a brown planthopper-resistance gene, BPH9, and show that alleles of this gene locus have been widely used in rice breeding and saved rice production from massive brown planthopper (BPH) damage. Allelic diversity in this gene locus has provided resistance to rice against different BPH populations. Manipulating allelic diversity of the gene may provide a strategy for developing resistant varieties to cope with evolving insect populations with new virulence variation. Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most devastating insect pests of rice (Oryza sativa L.). Currently, 30 BPH-resistance genes have been genetically defined, most of which are clustered on specific chromosome regions. Here, we describe molecular cloning and characterization of a BPH-resistance gene, BPH9, mapped on the long arm of rice chromosome 12 (12L). BPH9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR)-containing protein that localizes to the endomembrane system and causes a cell death phenotype. BPH9 activates salicylic acid- and jasmonic acid-signaling pathways in rice plants and confers both antixenosis and antibiosis to BPH. We further demonstrated that the eight BPH-resistance genes that are clustered on chromosome 12L, including the widely used BPH1, are allelic with each other. To honor the priority in the literature, we thus designated this locus as BPH1/9. These eight genes can be classified into four allelotypes, BPH1/9-1, -2, -7, and -9. These allelotypes confer varying levels of resistance to different biotypes of BPH. The coding region of BPH1/9 shows a high level of diversity in rice germplasm. Homologous fragments of the nucleotide-binding (NB) and leucine-rich repeat (LRR) domains exist, which might have served as a repository for generating allele diversity. Our findings reveal a rice plant strategy for modifying the genetic information to gain the upper hand in the struggle against insect herbivores. Further exploration of natural allelic variation and artificial shuffling within this gene may allow breeding to be tailored to control emerging biotypes of BPH.

Aspartic proteases gene family in rice: Gene structure and expression, predicted protein features and phylogenetic relation.

Aspartic proteases constitute a large family in eukaryotes, which play fundamental roles in protein processing, maturation and degradation. In this study, we identified 96 OsAP genes in rice (Oryza sativa L.), the model plant for monocots, by a reiterative database search. The analysis of the complete set of OsAP genes is presented, including chromosomal location, phylogenetic relationships, classification and gene structure. Moreover, a comprehensive expression analysis of OsAP family genes was performed using 24 tissues during the plant life cycle of two rice cultivars. Sixty-six OsAP genes were found to be expressed in at least one of the examined developmental stages, which were divided into 3 classes based on their transcript levels. OsAP genes were also found to be differentially up- or down-regulated in rice seedlings in response to treatments with phytohormones, as well as in plumules/radicles under light/dark conditions. The comprehensive annotation and expression profiling undertaken in this research add to our understanding of OsAP family genes in rice growth and development. Our results also provide a basis for selection of candidate genes for functional validation in future studies.

Genome-wide analysis of endosperm-specific genes in rice.

The endosperm of the cereal crop is an important nutrient source for humans. It also acts as a critical integrator of plant seed growth and development. Despite its importance, the comprehensive understanding in regulating of endosperm development in rice remains elusive. Here, we performed a genomic survey comprising the identification and functional characterization of the endosperm-specific genes (OsEnS) in rice using Affymetrix microarray data and Gene Ontology (GO) analysis. A total of 151 endosperm-specific genes were identified, and the expression patterns of 13 selected genes were confirmed by qRT-PCR analysis. Promoter regions of the endosperm-specific expression genes were analyzed by PLACE Signal Scan Search. The results indicated that some motifs were involved in endosperm-specific expression regulation, and some cis-elements were responsible for hormone regulation. The bootstrap analysis indicated that the RY repeat (CATGCA box) was over-represented in promoter regions of endosperm-specific expression genes. GO analysis indicated that these genes could be classified into 12 groups, namely, transcription factor, stress/defense, seed storage protein (SSP), carbohydrate and energy metabolism, seed maturation, protein metabolism, lipid metabolism, transport, cell wall related, hormone related, signal transduction, and one unclassified group. Taken together, our results provide informative clues for further functional characterization of the endosperm-specific genes, which facilitate the understanding of the molecular mechanism in rice endosperm development.