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.

Construction of rice chromosome segment substitution lines harboring Oryza barthii genome and evaluation of yield-related traits.

Rice (Oryza sativa L.) is one of the most important staple food in the world. To meet the increasing demand for food, a strategy for improving rice yield is needed. Alleles of wild relatives are useful because they confer adaptation to plants under diverse harsh environments and have the potential to improve rice. O. barthii is a wild rice species endemic to Africa and the known progenitor of the African cultivated rice, O. glaberrima. To explore the genetic potential of the O. barthii as a genetic resource, 40 chromosome segment substitution lines (CSSL) of O. barthii in the background of the elite japonica cultivar Koshihikari were developed and evaluated to identify QTLs associated with 10 traits related to flag leaf morphology, grain yield and other agronomic traits. More than 90% of the entire genome of the donor parent was represented in contiguous or overlapping chromosome segments in the CSSLs. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. These results suggest that alleles from O. barthii might be used as a novel genetic resource for improving the yield-related traits in cultivars of O. sativa.

Development of chromosome segment substitution lines harboring Oryza nivara genomic segments in Koshihikari and evaluation of yield-related traits.

Chromosome segment substitution lines (CSSLs) are rich genetic resources that can be mined for novel, agriculturally useful loci or that can be used directly as materials for breeding. To date, a number of rice CSSLs have been developed by crossing rice cultivars with its wild relatives as a means to tap into the potential of wild alleles in rice improvement. Oryza nivara is a wild relative of rice that is thought to be a progenitor of O. sativa spp. indica. In the present study, 26 CSSLs that covers the entire genome of O. nivara as contiguous, overlapping segments in the genomic background of a japonica cultivar, O. sativa cv. Koshihikari were developed. Evaluation of the CSSLs for several agriculturally important traits identified candidate chromosome segments that harbors QTLs associated with yield and yield-related traits. The results of the study revealed the potential of O. nivara as a source of novel alleles that can be used to improve the existing japonica cultivar.

Two Tightly Linked Genes at the hsa1 Locus Cause Both F1 and F2 Hybrid Sterility in Rice.

Molecular mechanisms of hybrid breakdown associated with sterility (F2 sterility) are poorly understood as compared with those of F1 hybrid sterility. Previously, we characterized three unlinked epistatic loci, hybrid sterility-a1 (hsa1), hsa2, and hsa3, responsible for the F2 sterility in a cross between Oryza sativa ssp. indica and japonica. In this study, we identified that the hsa1 locus contains two interacting genes, HSA1a and HSA1b, within a 30-kb region. HSA1a-j (japonica allele) encodes a highly conserved plant-specific domain of unknown function protein (DUF1618), whereas the indica allele (HSA1a-i(s)) has two deletion mutations that cause disruption of domain structure. The second gene, HSA1b-i(s), encodes an uncharacterized protein with some similarity to a nucleotide-binding protein. Homozygous introgression of indica HSA1a-i(s)-HSA1b-i(s) alleles into japonica showed female gamete abortion at an early mitotic stage. The fact that the recombinant haplotype HSA1a-j-HSA1b-i(s) caused semi-sterility in the heterozygous state with the HSA1a-i(s)-HSA1b-i(s) haplotype suggests that variation in the hsa1 locus is a possible cause of the wide-spectrum sterility barriers seen in F1 hybrids and successive generations in rice. We propose a simple genetic model to explain how a single causal mechanism can drive both F1 and F2 hybrid sterility.

Development and evaluation of chromosome segment substitution lines (CSSLs) carrying chromosome segments derived from Oryza rufipogon in the genetic background of Oryza sativa L.

The wild relatives of rice (Oryza sativa L.) are useful sources of alleles that have evolved to adapt in diverse environments around the world. Oryza rufipogon, the known progenitor of the cultivated rice, harbors genes that have been lost in cultivated varieties through domestication or evolution. This makes O. rufipogon an ideal source of value-added traits that can be utilized to improve the existing rice cultivars. To explore the potential of the rice progenitor as a genetic resource for improving O. sativa, 33 chromosome segment substitution lines (CSSLs) of O. rufipogon (W0106) in the background of the elite japonica cultivar Koshihikari were developed and evaluated for several agronomic traits. Over 90% of the entire genome was introgressed from the donor parent into the CSSLs. A total of 99 putative QTLs were detected, of which 15 were identified as major effective QTLs that have significantly large effects on the traits examined. Among the 15 major effective QTLs, a QTL on chromosome 10 showed a remarkable positive effect on the number of grains per panicle. Comparison of the putative QTLs identified in this study and previous studies indicated a wide genetic diversity between O. rufipogon accessions.

Efficacy of microarray profiling data combined with QTL mapping for the identification of a QTL gene controlling the initial growth rate in rice.

Seedling vigor, which is controlled by many quantitative trait loci (QTLs), is one of several important agronomic traits for direct-seedling rice systems. However, isolating these QTL genes is laborious and expensive. Here, we combined QTL mapping and microarray profiling to identify QTL genes for seedling vigor. By performing QTL mapping using 82 backcross inbred lines (BILs) of the Koshihikari (japonica) and Habataki (indica) cultivars for the rice initial growth, we identified two QTLs, early-stage plant development1/2 (qEPD1 and qEPD2), whose Koshihikari alleles promote plant height and/or leaf sheath length. Phenotypic analysis of the two substituted lines carrying the Habataki qEPD1 or qEPD2 allele revealed that qEPD2 functioned more dominantly for the initial growth of rice. From the microarray experiment, 55 and 45 candidate genes were found in the qEPD1 and qEPD2 genomic regions, which are expressed differentially between each substitution line (SL) and Koshihikari. Gibberellin 20 oxidase-2 (OsGA20ox2), which is identical to Semi Dwarf1 (SD1), was included among the 55 candidate genes of qEPD1, whereas its paralog, OsGA20ox1, was included among the 45 candidate genes of qEPD2. Consistently, introduction of the Koshihikari OsGA20ox1 allele into SL(qEPD2) increaseed its plant height and leaf sheath length significantly relative to the introduction of the Habataki OsGA20ox1 allele. Therefore, microarray profiling could be useful for rapidly screening QTL candidate genes. We concluded that OsGA20ox1 and OsGA20ox2 (SD1) function during the initial growth of rice, but OsGA20ox1 plays a dominant role in increasing plant height and leaf sheath length at the initial growth stage.

A WUSCHEL-related homeobox 3B gene, depilous (dep), confers glabrousness of rice leaves and glumes.

BACKGROUND: Glabrousness is an important agricultural trait for the practical breeding of rice. In this study, depilous (dep), the gene responsible for glabrous leaves and glumes of rice was identified by map-based cloning. RESULTS: The dep gene encodes a WUSCHEL-related homeobox 3B that was fine-mapped to a 22-kb region on the short arm of chromosome 5 using progenies derived from crosses between Koshihikari (pubescent) and GLSL15, an Oryza glaberrima chromosome segment substitution line (glabrous). Complementation tests confirmed the conditioning of the glabrous phenotype by the dep gene. Phylogenetic analysis showed that dep groups with the WOX3 family of plant-specific homeobox transcription factors that are involved in regulating lateral organ development. Localization of dep in the nucleus indicates the function of the gene as a transcription factor. Spatial expression of the gene was observed in the base of young shoots, the leaf sheath, midrib, young roots and nodal structures. CONCLUSION: The identification and cloning of dep will not only provide basis for future research on the elucidation of the molecular mechanisms underlying trichome formation in rice but will also aid in breeding programs for the development of glabrous varieties.

Gain of deleterious function causes an autoimmune response and Bateson-Dobzhansky-Muller incompatibility in rice.

Reproductive isolation plays an important role in speciation as it restricts gene flow and accelerates genetic divergence between formerly interbreeding population. In rice, hybrid breakdown is a common reproductive isolation observed in both intra and inter-specific crosses. It is a type of post-zygotic reproductive isolation in which sterility and weakness are manifested in the F(2) and later generations. In this study, the physiological and molecular basis of hybrid breakdown caused by two recessive genes, hbd2 and hbd3, in a cross between japonica variety, Koshihikari, and indica variety, Habataki, were investigated. Fine mapping of hbd2 resulted in the identification of the causal gene as casein kinase I (CKI1). Further analysis revealed that hbd2-CKI1 allele gains its deleterious function that causes the weakness phenotype by a change of one amino acid. As for the other gene, hbd3 was mapped to the NBS-LRR gene cluster region. It is the most common class of R-gene that triggers the immune signal in response to pathogen attack. Expression analysis of pathogen response marker genes suggested that weakness phenotype in this hybrid breakdown can be attributed to an autoimmune response. So far, this is the first evidence linking autoimmune response to post-zygotic isolation in rice. This finding provides a new insight in understanding the molecular and evolutionary mechanisms establishing post-zygotic isolation in plants.

Interaction of two recessive genes, hbd2 and hbd3, induces hybrid breakdown in rice.

Reproductive barriers are important for the maintenance of species identity. We discovered a reproductive barrier via hybrid breakdown among the progeny of a cross between the japonica rice cultivar Koshihikari and the indica rice cultivar Habataki. Genetic analysis indicated that the hybrid breakdown is regulated by the interaction of two recessive genes: hbd2 in Habataki and hbd3 in Koshihikari. Linkage mapping showed that hbd2 is located near the 100 cM region of chromosome 2 in Habataki, whereas hbd3 is located near the 60 cM region of chromosome 11 in Koshihikari. Construction of nearly isogenic lines for hbd2 and Hbd3 (NIL-hbd2 and NIL-Hbd3), as well as a pyramiding line (NIL-hbd2 + Hbd3), confirmed that the hybrid breakdown is induced by the interaction of these two recessive genes. Our results indicate that these genes are novel for the induction of hybrid breakdown in rice.

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems.

Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.

Cytokinin oxidase regulates rice grain production.

Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield. QTL pyramiding to combine loci for grain number and plant height in the same genetic background generated lines exhibiting both beneficial traits. These results provide a strategy for tailormade crop improvement.