Development and characterization of near-isogenic lines for brown planthopper resistance genes in the genetic background of japonica rice 'Sagabiyori'.

The brown planthopper (BPH: Nilaparvata lugens Stål) is one of the most destructive insects in rice production. The use of host plant resistance has potential to reduce damage caused by BPH. The heat tolerance japonica rice 'Sagabiyori', with superior grain quality and high soluble starch in the stem, is highly susceptible to damage by BPH. Here, to enhance its BPH resistance, we developed seven near-isogenic lines (NILs) carrying BPH2, BPH17-ptb, BPH32, BPH3, BPH17, BPH20, and BPH21 through marker-assisted selection and evaluated resistance to two BPH populations. Most lines were more resistant to the Hadano-1966 BPH population than Sagabiyori but were less effective against the highly virulent Koshi-2013 population. Nevertheless, in antixenosis tests, Koshi-2013 settled less on all NILs than on Sagabiyori. In addition, adult mortality and the percentage of fresh weight loss of lines carrying BPH17 and BPH3 indicated that these lines have higher resistance to Koshi-2013 than Sagabiyori. Current study revealed that BPH resistance of Sagabiyori became stronger by transferring BPH3 and BPH17 genes. Thus, BPH3 and BPH17 might be valuable for breeding programs to enhance BPH resistance of high grain quality rice varieties with heat tolerance.

Regulator of Awn Elongation 3, an E3 ubiquitin ligase, is responsible for loss of awns during African rice domestication.

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima, have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima. We identify Regulator of Awn Elongation 3 (RAE3) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima. A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

Collection, preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE).

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

Development of an Aus-Derived Nested Association Mapping (Aus-NAM) Population in Rice.

A genetic resource for studying genetic architecture of agronomic traits and environmental adaptation is essential for crop improvements. Here, we report the development of a rice nested association mapping population (aus-NAM) using 7 aus varieties as diversity donors and T65 as the common parent. Aus-NAM showed broad phenotypic variations. To test whether aus-NAM was useful for quantitative trait loci (QTL) mapping, known flowering genes (Ehd1, Hd1, and Ghd7) in rice were characterized using single-family QTL mapping, joint QTL mapping, and the methods based on genome-wide association study (GWAS). Ehd1 was detected in all the seven families and all the methods. On the other hand, Hd1 and Ghd7 were detected in some families, and joint QTL mapping and GWAS-based methods resulted in weaker and uncertain peaks. Overall, the high allelic variations in aus-NAM provide a valuable genetic resource for the rice community.

Substitution Mapping of a Locus Responsible for Hybrid Breakdown in Populations Derived From Interspecific Introgression Line.

Hybrid breakdown, a form of postzygotic reproductive barrier, has been reported to hinder gene flow in many crosses between wild and cultivated rice. Here, the phenomenon of hybrid breakdown was observed as low-tillering (i.e., low tiller number) in some progeny of an interspecific cross produced in an attempt to introduce Oryza meridionalis Ng (W1625) chromosomal segments into Oryza sativa L. ssp. japonica "Taichung 65" (T65). Low-tillering lines were obtained in BC4-derived progeny from a cross between W1625 and "Taichung 65," but the locus for low-tillering could not be mapped in segregating populations. As a second approach to map the locus for low-tillering, we analyzed an F2 population derived from a cross between the low-tillering lines and a high-yielding indica cultivar, "Takanari." A major QTL for low-tillering, qLTN4, was detected between PCR-based markers MS10 and RM307 on the long arm of chromosome 4, with a LOD score of 15.6. The low-tillering phenotype was associated with weak growth and pale yellow phenotype; however, low-tillering plant had less reduction of grain fertility. In an F4 population (4896 plants), 563 recombinant plants were identified and the low-tillering locus was delimited to a 4.6-Mbp region between markers W1 and C5-indel3729. This region could not be further delimited because recombination is restricted in this region of qLTN4, which is near the centromere. Understanding the genetic basis of hybrid breakdown, including the low-tillering habit, will be important for improving varieties in rice breeding.

Exploring the Loci Responsible for Awn Development in Rice through Comparative Analysis of All AA Genome Species.

Wild rice species have long awns at their seed tips, but this trait has been lost through rice domestication. Awn loss mitigates harvest and seed storage; further, awnlessness increases the grain number and, subsequently, improves grain yield in Asian cultivated rice, highlighting the contribution of the loss of awn to modern rice agriculture. Therefore, identifying the genes regulating awn development would facilitate the elucidation of a part of the domestication process in rice and increase our understanding of the complex mechanism in awn morphogenesis. To identify the novel loci regulating awn development and understand the conservation of genes in other wild rice relatives belonging to the AA genome group, we analyzed the chromosome segment substitution lines (CSSL). In this study, we compared a number of CSSL sets derived by crossing wild rice species in the AA genome group with the cultivated species Oryza sativa ssp. japonica. Two loci on chromosomes 7 and 11 were newly discovered to be responsible for awn development. We also found wild relatives that were used as donor parents of the CSSLs carrying the functional alleles responsible for awn elongation, REGULATOR OF AWN ELONGATION 1 (RAE1) and RAE2. To understand the conserveness of RAE1 and RAE2 in wild rice relatives, we analyzed RAE1 and RAE2 sequences of 175 accessions among diverse AA genome species retrieved from the sequence read archive (SRA) database. Comparative sequence analysis demonstrated that most wild rice AA genome species maintained functional RAE1 and RAE2, whereas most Asian rice cultivars have lost either or both functions. In addition, some different loss-of-function alleles of RAE1 and RAE2 were found in Asian cultivated species. These findings suggest that different combinations of dysfunctional alleles of RAE1 and RAE2 were selected after the speciation of O. sativa, and that two-step loss of function in RAE1 and RAE2 contributed to awnlessness in Asian cultivated rice.

Substitution mapping and characterization of brown planthopper resistance genes from indica rice variety, 'PTB33' (Oryza sativa L.).

Rice (Oryza sativa L.) yield is severely reduced by the brown planthopper (BPH), Nilaparvata lugens Stål, in Asian countries. Increasing resistance in rice against BPH can mitigate yield loss. Previous reports indicated the presence of three BPH resistance genes, BPH2, BPH17-ptb, and BPH32, in durable resistant indica rice cultivar 'PTB33'. However, several important questions remain unclear; the genetic locations of BPH resistance genes on rice chromosomes and how these genes confer resistance, especially with relationship to three major categories of resistance mechanisms; antibiosis, antixenosis or tolerance. In this study, locations of BPH2, BPH17-ptb, and BPH32 were delimited using chromosome segment substitution lines derived from crosses between 'Taichung 65' and near-isogenic lines for BPH2 (BPH2-NIL), BPH17-ptb (BPH17-ptb-NIL), and BPH32 (BPH32-NIL). BPH2 was delimited as approximately 247.5 kbp between RM28449 and ID-161-2 on chromosome 12. BPH17-ptb and BPH32 were located between RM1305 and RM6156 on chromosome 4 and RM508 and RM19341 on chromosome 6, respectively. The antibiosis, antixenosis, and tolerance were estimated by several tests using BPH2-NIL, BPH17-ptb-NIL, and BPH32-NIL. BPH2 and BPH17-ptb showed resistance to antibiosis and antixenosis, while BPH17-ptb and BPH32 showed tolerance. These results contribute to the development of durable BPH resistance lines using three resistance genes from 'PTB33'.

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species.

In F1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer-protector model.

Domain Unknown Function DUF1668-Containing Genes in Multiple Lineages Are Responsible for F1 Pollen Sterility in Rice.

Postzygotic reproductive isolation maintains species integrity and uniformity and contributes to speciation by restricting the free gene flow between divergent species. In this study we identify causal genes of two Mendelian factors S22A and S22B on rice chromosome 2 inducing F1 pollen sterility in hybrids between Oryza sativa japonica-type cultivar Taichung 65 (T65) and a wild relative of rice species Oryza glumaepatula. The causal gene of S22B in T65 encodes a protein containing DUF1668 and gametophytically expressed in the anthers, designated S22B_j. The O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region. Transcription level of S22B-j and/or S22B_g did not solely determine the fertility of pollen grains by genotypes at S22B. Western blotting of S22B found that one major band with approximately 46 kDa appeared only at the mature stage and was reduced on semi-sterile heterozygotes at S22B, implying that the 46 kDa band may associated in hybrid sterility. In addition, causal genes of S22A in T65 were found to be S22A_j1 and S22A_j3 encoding DUF1668-containing protein. The allele of a wild rice species Oryza meridionalis Ng at S22B, designated S22B_m, is a loss-of-function allele probably due to large deletion of the gene lacking DUF1668 domain and evolved from the different lineage of O. glumaepatula. Phylogenetic analysis of DUF1668 suggested that many gene duplications occurred before the divergence of current crops in Poaceae, and loss-of-function mutations of DUF1668-containing genes represent the candidate causal genetic events contributing to hybrid incompatibilities. The duplicated DUF1668-domain gene may provide genetic potential to induce hybrid incompatibility by consequent mutations after divergence.

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.

Identification of Anther Length QTL and Construction of Chromosome Segment Substitution Lines of Oryza longistaminata.

Life histories and breeding systems strongly affect the genetic diversity of seed plants, but the genetic architectures that promote outcrossing in Oryza longistaminata, a perennial wild species in Africa, are not understood. We conducted a genetic analysis of the anther length of O. longistaminata accession W1508 using advanced backcross quantitative trait locus (QTL) analysis and chromosomal segment substitution lines (CSSLs) in the genetic background of O. sativa Taichung 65 (T65), with simple sequence repeat markers. QTL analysis of the BC3F1 population (n = 100) revealed that four main QTL regions on chromosomes 3, 5, and 6 were associated to anther length. We selected a minimum set of BC3F2 plants for the development of CSSLs to cover as much of the W1508 genome as possible. The additional minor QTLs were suggested in the regional QTL analysis, using 21 to 24 plants in each of the selected BC3F2 population. The main QTLs found on chromosomes 3, 5, and 6 were validated and designated qATL3, qATL5, qATL6.1, and qATL6.2, as novel QTLs identified in O. longistaminata in the mapping populations of 94, 88, 70, and 95 BC3F4 plants. qATL3, qATL5, and qATL6.1 likely contributed to anther length by cell elongation, whereas qATL6.2 likely contributed by cell multiplication. The QTLs were confirmed again in an evaluation of the W1508ILs. In several chromosome segment substitution lines without the four validated QTLs, the anthers were also longer than those of T65, suggesting that other QTLs also increase anther length in W1508. The cloning and diversity analyses of genes conferring anther length QTLs promotes utilization of the genetic resources of wild species, and the understanding of haplotype evolution on the differentiation of annuality and perenniality in the genus Oryza.

High-resolution mapping of GRH6, a gene from Oryza nivara (Sharma et Shastry) conferring resistance to green rice leafhopper (Nephotettix cincticeps Uhler).

The green rice leafhopper (GRH), Nephotettix cincticeps Uhler, is a major insect pest of cultivated rice, Oryza sativa L., throughout the temperate regions of East Asia. GRH resistance had been reported in the wild species Oryza nivara but genetic basis of GRH resistance in wild rice accession has not been clarified. Here, we found a major QTL, qGRH4.2, on chromosome 4 conferred GRH resistance with 14.1 of the logarithm of odds (LOD) score explaining 67.6% of phenotypic variance in the BC1F1 population derived from a cross between the susceptible japonica cultivar 'Taichung 65' (T65) and O. nivara accession IRGC105715. qGRH4.2 has been identified as GRH6 between the markers RM5414 and C60248 in a BC3F2 population derived from two BC3F1 plants resistant to GRH. In a high-resolution mapping, the GRH6 region was delimited between the markers G6-c60k and 7L16f, and corresponded to an 31.2-kbp region of the 'Nipponbare' genome. Understanding the genetic basis of GRH resistance will facilitate the use of GRH resistance genes in marker-assisted breeding in rice.

Development of introgression lines of AA genome Oryza species, O. glaberrima, O. rufipogon, and O. nivara, in the genetic background of O. sativa L. cv. Taichung 65.

To evaluate and utilize potentially valuable quantitative trait loci or genes of wild relatives in the genetic background of domesticated crop species, chromosome segment substitution lines (CSSLs) are a valuable tool. CSSLs can be constructed through the exchange of chromosome segments of AA genome species of the genus Oryza with cultivated rice, Oryza sativa L. Here we report the development of three sets of CSSLs carrying segments of AA genome species closely related to Oryza sativa-O. glaberrima (IRGC 103777 from Mali), O. rufipogon (W1962 from China), and O. nivara (IRGC 105715 from Cambodia)-in the genetic background of ssp. japonica cultivar Taichung 65 through the use of 101 to 121 simple-sequence-repeat markers in whole-genome genotyping and marker-assisted selection. The materials are available via the National Bioresource Project (Rice) Oryzabase Web page.

Unanticipated benefits and potential ecological costs associated with pyramiding leafhopper resistance loci in rice.

We tested the hypotheses that increasing the number of anti-herbivore resistance loci in crop plants will increase resistance strength, increase the spectrum of resistance (the number of species affected), and increase resistance stability. We further examined the potential ecological costs of pyramiding resistance under benign environments. In our experiments, we used 14 near-isogenic rice lines with zero (T65: recurrent parent), one, two or three resistance loci introgressed through marker-assisted selection. Lines with two or more loci that were originally bred for resistance to the green rice leafhopper, Nephotettix cincticeps, significantly reduced egg-laying by the green leafhopper, N. virescens. Declines in egg-number and in nymph weight were correlated with the numbers of resistance loci in the rice lines. To test the spectrum of resistance, we challenged the lines with a range of phloem feeders including the zig-zag leafhopper, Recilia dorsalis, brown planthopper, Nilaparvata lugens, and whitebacked planthopper, Sogatella furcifera. There was an increase in the number of tested species showing significant declines in egg-laying and nymph survival on lines with increasing numbers of loci. In a screen house trial that varied rates of nitrogenous fertilizer, a line with three loci had stable resistance against the green leafhopper and the grain yields of infested plants were maintained or increased (overcompensation). Under benign conditions, plant growth and grain yields declined with increasing numbers of resistance loci. However, under field conditions with natural exposure to herbivores, there were no significant differences in final yields. Our results clearly indicate the benefits, including unanticipated benefits such as providing resistance against multiple herbivore species, of pyramiding anti-herbivore resistance genes/loci in crop plants. We discuss our results as part of a review of existing research on pyramided resistance against leafhoppers and planthoppers in rice. We suggest that potential ecological costs may be overcome by the careful selection of gene combinations for pyramiding, avoidance of high (potentially redundant) loci numbers, and introgression of loci into robust plant types such as hybrid rice varieties.

Four resistance alleles derived from Oryza longistaminata (A. Chev. & Roehrich) against green rice leafhopper, Nephotettix cincticeps (Uhler) identified using novel introgression lines.

The green rice leafhopper (GRH, Nephotettix cincticeps Uhler) is a serious insect pest of rice (Oryza sativa L.) in temperate regions of Asia. Wild Oryza species are the main source of resistance to insects. The W1413 accession of African wild rice (O. longistaminata A. Chev. & Roehrich) is resistant to GRH. To analyze its resistance, we developed 28 BC3F3 introgression lines carrying W1413 segments in the genetic background of Nipponbare, a susceptible rice cultivar, and evaluated their GRH resistance. Five BC3F3 populations were used for quantitative trait locus (QTL) analysis and seven BC3F4 populations for QTL validation. Four significant QTLs on the long arm of chromosome 2 (qGRH2), short arm of chromosome 4 (qGRH4), short arm of chromosome 5 (qGRH5), and long arm of chromosome 11 (qGRH11) were identified. The contribution of the W1413 allele at qGRH11 was the largest among the four QTLs; the other QTLs also contributed to GRH resistance. Chromosomal locations suggested that qGRH11 corresponds to the previously reported GRH resistance gene Grh2, qGRH4 to Grh6, and qGRH5 to Grh1. qGRH2 is a novel QTL for resistance to GRH. Thus, resistance of O. longistaminata to GRH can be explained by at least four QTLs.

Virulence adaptation in a rice leafhopper: Exposure to ineffective genes compromises pyramided resistance.

Pyramiding resistance genes is predicted to increase the durability of resistant rice varieties against phloem-feeding herbivores. We examined responses by the green leafhopper, Nephotettix virescens (Hemiptera: Cicadellidae), to near-isogenic rice lines with zero, one and two resistance genes. The recurrent parent (T65) and monogenic lines (GRH2-NIL and GRH4-NIL) with genes for resistance to the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), were susceptible to the green leafhopper, but the pyramided line (GRH2/GRH4-PYL) was highly resistant to the green leafhopper. We selected green leafhoppers, N. virescens, from five sites in the Philippines for over 20 generations on each of the four lines. Populations selected on GRH2/GRH4-PYL gained partial virulence (feeding and development equal to that on T65) to the pyramided line within 10 generations and complete virulence (egg-laying equal to that on T65) within 20 generations. After 20 generations of rearing on the susceptible monogenic lines, green leafhoppers were also capable of developing and laying eggs on GRH2/GRH4-PYL. Furthermore, green leafhoppers reared on the susceptible GRH4-NIL for 20 generations showed equal preferences for T65 and GRH2/GRH4-PYL in choice bioassays. Our results indicate that previous long-term exposure to ineffective genes (including unperceived resistance genes) could dramatically reduce the durability of pyramided resistance. We suggest that informed crop management and deployment strategies should be developed to accompany rice lines with pyramided resistance and avoid the build-up of virulent herbivore populations.

Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility.

BACKGROUND: To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood. RESULTS: Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S 1 and S 19 loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD. CONCLUSIONS: We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

eQTLs Regulating Transcript Variations Associated with Rapid Internode Elongation in Deepwater Rice.

To avoid low oxygen, oxygen deficiency or oxygen deprivation, deepwater rice cultivated in flood planes can develop elongated internodes in response to submergence. Knowledge of the gene regulatory networks underlying rapid internode elongation is important for an understanding of the evolution and adaptation of major crops in response to flooding. To elucidate the genetic and molecular basis controlling their deepwater response we used microarrays and performed expression quantitative trait loci (eQTL) and phenotypic QTL (phQTL) analyses of internode samples of 85 recombinant inbred line (RIL) populations of non-deepwater (Taichung 65)- and deepwater rice (Bhadua). After evaluating the phenotypic response of the RILs exposed to submergence, confirming the genotypes of the populations, and generating 188 genetic markers, we identified 10,047 significant eQTLs comprised of 2,902 cis-eQTLs and 7,145 trans-eQTLs and three significant eQTL hotspots on chromosomes 1, 4, and 12 that affect the expression of many genes. The hotspots on chromosomes 1 and 4 located at different position from phQTLs detected in this study and other previous studies. We then regarded the eQTL hotspots as key regulatory points to infer causal regulatory networks of deepwater response including rapid internode elongation. Our results suggest that the downstream regulation of the eQTL hotspots on chromosomes 1 and 4 is independent, and that the target genes are partially regulated by SNORKEL1 and SNORKEL2 genes (SK1/2), key ethylene response factors. Subsequent bioinformatic analyses, including gene ontology-based annotation and functional enrichment analysis and promoter enrichment analysis, contribute to enhance our understanding of SK1/2-dependent and independent pathways. One remarkable observation is that the functional categories related to photosynthesis and light signaling are significantly over-represented in the candidate target genes of SK1/2. The combined results of these investigations together with genetical genomics approaches using structured populations with a deepwater response are also discussed in the context of current molecular models concerning the rapid internode elongation in deepwater rice. This study provides new insights into the underlying genetic architecture of gene expression regulating the response to flooding in deepwater rice and will be an important community resource for analyses on the genetic basis of deepwater responses.

Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice.

Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1) on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras ) and the DGS2 allele from O. sativa (DGS2-T65s ) were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP) III subunit C4 (RPC4). RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

A hairy-leaf gene, BLANKET LEAF, of wild Oryza nivara increases photosynthetic water use efficiency in rice.

BACKGROUND: High water use efficiency is essential to water-saving cropping. Morphological traits that affect photosynthetic water use efficiency are not well known. We examined whether leaf hairiness improves photosynthetic water use efficiency in rice. RESULTS: A chromosome segment introgression line (IL-hairy) of wild Oryza nivara (Acc. IRGC105715) with the genetic background of Oryza sativa cultivar 'IR24' had high leaf pubescence (hair). The leaf hairs developed along small vascular bundles. Linkage analysis in BC5F2 and F3 populations showed that the trait was governed by a single gene, designated BLANKET LEAF (BKL), on chromosome 6. IL-hairy plants had a warmer leaf surface in sunlight, probably due to increased boundary layer resistance. They had a lower transpiration rate under moderate and high light intensities, resulting in higher photosynthetic water use efficiency. CONCLUSION: Introgression of BKL on chromosome 6 from O. nivara improved photosynthetic water use efficiency in the genetic background of IR24.