Copy number variation of the restorer Rf4 underlies human selection of three-line hybrid rice breeding.

Cytoplasmic male sterility (CMS) lines are important for breeding hybrid crops, and utilization of CMS lines requires strong fertility restorer (Rf) genes. Rf4, a major Rf for Wild-Abortive CMS (CMS-WA), has been cloned in rice. However, the Rf4 evolution and formation of CMS-WA/Rf system remain elusive. Here, we show that the Rf4 locus emerges earlier than the CMS-WA gene WA352 in wild rice, and 69 haplotypes of the Rf4 locus are generated in the Oryza genus through the copy number and sequence variations. Eight of these haplotypes of the Rf4 locus are enriched in modern rice cultivars during natural and human selections, whereas non-functional rf4i is preferentially selected for breeding current CMS-WA lines. We further verify that varieties carrying two-copy Rf4 haplotype have stronger fertility restoration ability and are widely used in three-line hybrid rice breeding. Our findings increase our understanding of CMS/Rf systems and will likely benefit crop breeding.

Changes in the expression pattern of OsWUS negatively regulate plant stature and panicle development in rice.

WUSCHEL (WUS) and WUSCHEL-RELATED HOMEOBOX (WOX) encode transcription factors and play important roles in regulating the formation and maintenance of shoot and floral meristems. OsWUS have distinct functions in meristem development with slightly tuned expression. However, the mechanisms regulating the specific expression of OsWUS need to be further explored. In this study, an abnormal expression mutant of OsWUS, called Dwarf and aberrant panicle 1 (Dap1) was used. In order to identify the causal gene in Dap1, high-efficiency thermal asymmetric interlaced (hiTAIL)-PCR and co-segregation analysis were performed. We surveyed the growth and yield traits in Dap1 and wild type. Changes in gene expression between Dap1 and wild type were determined by RNA-seq. The Dap1 mutant is due to the T-DNA inserted at 3,628-bp upstream of the translation start codon of OsWUS. Plant height, tiller numbers, panicle length, the number of grains per main panicle, and the number of secondary branches was significantly reduced in the Dap1 mutant. The expression of OsWUS was markedly increased in Dap1 mutant plants compared to the wild type, which might be due to a disruption in the genomic sequence integrity. Simultaneously, the expression levels of gibberellic acid-related genes and genes involved in panicle development were significantly changed in the Dap1 mutant. Our results suggest that OsWUS is a precise regulatory element, its specific spatio-temporal expression pattern is critical for its function, and both loss-of-function and gain-of-function mutations lead to abnormal plant growth.

Natural variation of HTH5 from wild rice, Oryza rufipogon Griff., is involved in conferring high-temperature tolerance at the heading stage.

Global warming is a major abiotic stress factor, which limit rice production. Exploiting the genetic basis of the natural variation in heat resistance at different reproductive stages among diverse exotic Oryza germplasms can help breeding heat-resistant rice cultivars. Here, we identified a stable quantitative trait locus (QTL) for heat tolerance at the heading stage on chromosome 5 (qHTH5) in O. rufipogon Griff. The corresponding gene, HTH5, pertains to the pyridoxal phosphate-binding protein PLPBP (formerly called PROSC) family, which is predicted to encode pyridoxal phosphate homeostasis protein (PLPHP) localized to the mitochondrion. Overexpression of HTH5 increased the seed-setting rate of rice plants under heat stress at the heading stage, whereas suppression of HTH5 resulted in greater susceptibility to heat stress. Further investigation indicated that HTH5 reduces reactive oxygen species accumulation at high temperatures by increasing the heat-induced pyridoxal 5'-phosphate (PLP) content. Moreover, we found that two SNPs located in the HTH5 promoter region are involved with its expression level and associated with heat tolerance diversity. These findings suggest that the novel gene HTH5 might have great potential value for heightening rice tolerance to heat stress to the on-going threat of global warming.

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

Fine mapping of the qHTB1-1QTL, which confers heat tolerance at the booting stage, using an Oryza rufipogon Griff. introgression line.

KEY MESSAGE: The qHTB1-1 QTL, controlling heat tolerance at the booting stage in rice, was fine mapped to a 47.1 kb region containing eight candidate genes. Two positional candidate genes showed significant changes in expression levels under heat stress. High-temperature stress at the booting stage has the potential to significantly limit rice production. An interspecific advanced backcrossed population between the Oryza sativa L. cultivar R53 and the wild Oryza rufipogon Griff accession HHT4 was used as the source material to develop a set of chromosome segment introgression lines to elucidate the genetic mechanism of the qHTB1-1 QTL in heat tolerance. A single-chromosome-segment introgression line, IL01-15, was used to develop secondary populations for the mapping of qHTB1-1 on chromosome 1 for heat tolerance at the booting stage. Using the BC5F2, BC5F3, and BC5F4 populations, we first confirmed qHTB1-1 and validated the phenotypic effect. The qHTB1-1 QTL explained 13.1%, 16.9%, and 17.8% of the phenotypic variance observed in the BC5F2, BC5F3, and BC5F4 generations, respectively. Using homozygous recombinants screened from larger BC6F2 and BC6F3 populations, qHTB1-1 was fine mapped within a 47.1 kb region between markers RM11633 and RM11642. Eight putative predicted genes were annotated in the region, and six genes were predicted to encode expressed proteins. The expression patterns of these six genes demonstrated that LOC_Os01g53160 and LOC_Os01g53220 were highly induced by heat stress in IL01-15 compared to R53. Sequence comparison of the gene-coding regions of LOC_Os01g53160 and LOC_Os01g53220 between R53 and IL01-15 revealed one synonymous and two nonsynonymous SNPs in exons, respectively. Our results provide a basis for identifying the genes underlying qHTB1-1 and indicate that markers linked to the qHTB1-1 locus can be used to improve the heat tolerance of rice at the booting stage by marker-assisted selection.

Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice.

Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (<2.5 cycle quantification) across tissues and developmental stages, and little fluctuation in expression in heatmaps from RiceXPro data were considered stable NRGs. To validate this strategy, we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs, and compared these genes with five established reference genes (ERGs). Only one of the ERGs (UBQ5) was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs. Therefore, public transcriptomic databases are useful for identifying NRGs. We selected two NRGs, UFC1 (Homolog of UFM1-Conjugating Enzyme 1) and FhaB (Homolog of Adhesin FhaB) for qRT-PCR analysis in rice; their homologs might be suitable for other monocot plants.

An asymmetric allelic interaction drives allele transmission bias in interspecific rice hybrids.

Hybrid sterility (HS) between Oryza sativa (Asian rice) and O. glaberrima (African rice) is mainly controlled by the S1 locus. However, our limited understanding of the HS mechanism hampers utilization of the strong interspecific heterosis. Here, we show that three closely linked genes (S1A4, S1TPR, and S1A6) in the African S1 allele (S1-g) constitute a killer-protector system that eliminates gametes carrying the Asian allele (S1-s). In Asian-African rice hybrids (S1-gS1-s), the S1TPR-S1A4-S1A6 interaction in sporophytic tissues generates an abortion signal to male and female gametes. However, S1TPR can rescue S1-g gametes, while the S1-s gametes selectively abort for lacking S1TPR. Knockout of any of the S1-g genes eliminates the HS. Evolutionary analysis suggests that S1 may have arisen from newly evolved genes, multi-step recombination, and nucleotide variations. Our findings will help to overcome the interspecific reproductive barrier and use Asian-African hybrids for increasing rice production.

Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice.

Hybrids between divergent populations commonly show hybrid sterility; this reproductive barrier hinders hybrid breeding of the japonica and indica rice (Oryza sativa L.) subspecies. Here we show that structural changes and copy number variation at the Sc locus confer japonica-indica hybrid male sterility. The japonica allele, Sc-j, contains a pollen-essential gene encoding a DUF1618-domain protein; the indica allele, Sc-i, contains two or three tandem-duplicated ~ 28-kb segments, each carrying an Sc-j-homolog with a distinct promoter. In Sc-j/Sc-i hybrids, the high-expression of Sc-i in sporophytic cells causes suppression of Sc-j expression in pollen and selective abortion of Sc-j-pollen, leading to transmission ratio distortion. Knocking out one or two of the three Sc-i copies by CRISPR/Cas9 rescues Sc-j expression and male fertility. Our results reveal the gene dosage-dependent allelic suppression as a mechanism of hybrid incompatibility, and provide an effective approach to overcome the reproductive barrier for hybrid breeding.

Advances in understanding the molecular mechanisms of cytoplasmic male sterility and restoration in rice.

Cytoplasmic male sterility (CMS) in plants is a male reproductive defect determined by mitochondrial genes and inherited maternally. CMS can be suppressed by nuclear restorer of fertility (Rf) genes. Therefore, CMS/Rf systems provide a classic model for the study of mitochondrial-nuclear interactions in plants. Moreover, CMS/Rf systems are economical, effective tools for the production of hybrid seeds. For example, CMS/Rf systems have been applied in over forty countries to breed hybrid rice (Oryza sativa L.) with improved yields due to hybrid vigor. The production of hybrid rice mainly depends on three types of CMS systems, namely Wild-Abortive type CMS (CMS-WA), Hong-Lian type CMS (CMS-HL) and Boro II type CMS (CMS-BT). Understanding the molecular mechanisms underlying these CMS/Rf systems will help us to understand mitochondrial-nuclear interactions, and accelerate the utilization of heterosis for improvement in yield. In the past decades, research benefitting from the availability of the high-quality, annotated mitochondrial and nuclear genome sequences of rice has isolated many CMS genes, identified the cognate nuclear Rf genes and studied the molecular mechanisms underlying CMS and restoration in rice. Here, we focus on recent advances in studies of the three major CMS/Rf systems in rice and discuss the key issues facing basic research and application of CMS/Rf systems in the future.

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes.

New gene origination is a major source of genomic innovations that confer phenotypic changes and biological diversity. Generation of new mitochondrial genes in plants may cause cytoplasmic male sterility (CMS), which can promote outcrossing and increase fitness. However, how mitochondrial genes originate and evolve in structure and function remains unclear. The rice Wild Abortive type of CMS is conferred by the mitochondrial gene WA352c (previously named WA352) and has been widely exploited in hybrid rice breeding. Here, we reconstruct the evolutionary trajectory of WA352c by the identification and analyses of 11 mitochondrial genomic recombinant structures related to WA352c in wild and cultivated rice. We deduce that these structures arose through multiple rearrangements among conserved mitochondrial sequences in the mitochondrial genome of the wild rice Oryza rufipogon, coupled with substoichiometric shifting and sequence variation. We identify two expressed but nonfunctional protogenes among these structures, and show that they could evolve into functional CMS genes via sequence variations that could relieve the self-inhibitory potential of the proteins. These sequence changes would endow the proteins the ability to interact with the nucleus-encoded mitochondrial protein COX11, resulting in premature programmed cell death in the anther tapetum and male sterility. Furthermore, we show that the sequences that encode the COX11-interaction domains in these WA352c-related genes have experienced purifying selection during evolution. We propose a model for the formation and evolution of new CMS genes via a "multi-recombination/protogene formation/functionalization" mechanism involving gradual variations in the structure, sequence, copy number, and function.

Genetic interactions between diverged alleles of Early heading date 1 (Ehd1) and Heading date 3a (Hd3a)/ RICE FLOWERING LOCUS T1 (RFT1) control differential heading and contribute to regional adaptation in rice (Oryza sativa).

Initiation of flowering, also called heading, in rice (Oryza sativa) is determined by the florigens encoded by Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1). Early heading date 1 (Ehd1) regulates Hd3a and RFT1. However, different rice varieties have diverged alleles of Ehd1 and Hd3a/RFT1 and their genetic interactions remain largely unclear. Here we generated three segregating populations for different combinations of diverged Ehd1 and Hd3a/RFT1 alleles, and analyzed their genetic interactions between these alleles. We demonstrated that, in an ehd1 mutant background, Hd3a was silenced, but RFT1 was expressed (although at lower levels than in plants with a functional Ehd1) under short-day (SD) and long-day (LD) conditions. We identified a nonfunctional RFT1 allele (rft1); the lines carrying homozygous ehd1 and Hd3a/rft1 failed to induce the floral transition under SD and LD conditions. Like Hd3a, RFT1 also interacted with 14-3-3 proteins, the florigen receptors, but a nonfunctional RFT1 with a crucial E105K mutation failed to interact with 14-3-3 proteins. Furthermore, analyses of sequence variation and geographic distribution suggested that functional RFT1 alleles were selected during rice adaptation to high-latitude regions. Our results demonstrate the important roles of RFT1 in rice flowering and regional adaptation.