Hybrid Rice Breeding Research Articles

Genetic and molecular mechanisms underlying the male sterility in rice.

Male reproductive development is a complex and highly ordered phenomenon which demands comprehensive understandings of underlying molecular mechanisms to expand its scope for crop improvement. Genetic manipulation of male fertility/sterility is critical for crop hybrid breeding. Although male sterility is not a good trait for the plant itself, its wider application in hybrid rice breeding has made it valuable. The currently widely used male sterile line breeding systems mainly include the following: three-line hybrid rice based on cytoplasmic male sterility and two-line hybrid rice based on environmentally sensitive gene male sterility. The study of male sterility is an excellent thoroughfare to critically understand the regulatory mechanisms essential for the complicated male reproductive developmental process. The unique trait of male sterility also provides valuable resources and convenience for the genetic improvement of rice hybrids. Therefore, deeper and broader understandings about the genetic causes of male sterility are necessary for both basic studies and rice genetic improvement.

Development and Trait-Based Molecular Characterization of Thermosensitive Genic Male-Sterile Rice (Oryza sativa L.) Lines at Texas A&M AgriLife Research

This study aimed to develop and genetically characterize thermosensitive genic male-sterility (TGMS) lines for use in hybrid rice (Oryza sativa L.) breeding. Male-sterile F2 to F4 generation lines were screened during the high-temperature summer season, and ratoon crops of selected male-sterile rows were harvested for pure seed. Sixty-six F5 TGMS lines were genotyped using DNA markers controlling 16 traits from the LSU80 QA/QC Rice PlexSeq SNP Panel. Ten TGMS lines with desirable traits that included semidwarf, glabrous, non-aromatic, long-grain, narrow brown leaf spot resistance, and blast resistance genes were selected for further genotypic characterization using markers for low chalkiness (chalk5), wide compatibility (S5-n), cold tolerance (qSCT-11 and qCST-12), and anaerobic germination (AG1 and AG2). TGMS lines TIL21051S and TIL21052S possess favorable alleles for each of the genes evaluated in this study and are desirable parents for two-line hybrid breeding in the southeast United States. TIL21044S, TIL21095S, TIL21060S, and TIL21066S each contain three blast resistance genes and have potential as parental lines. TIL21014S-2, TIL21015S, and TIL21016S-1 include the fgr allele for aroma and can also be used as parental lines for aromatic two-line hybrids.

Impaired 2',3'-cyclic phosphate tRNA repair causes thermo-sensitive genic male sterility in rice.

Hybrid rice, widely planted in Asia, is pathogen resistant and has superior yields, making it a major contributor to global food security. The two-line hybrid rice system, which utilizes mutants exhibiting photo-/thermo-sensitive genic male sterility (P/TGMS), is the leading hybrid rice breeding technology. Mutations in THERMO-SENSITIVE GENIC MALE STERILE 5 (TMS5) accounts for over 95% of current TGMS lines. We previously found that tms5 carries a mutation in ribonuclease ZS1. Despite its importance for breeding robust rice lines, the mechanism underlying tms5-mediated TGMS remains elusive. Here, we demonstrate that TMS5 is a tRNA 2',3'-cyclic phosphatase. The tms5 mutation leads to accumulation of 2',3'-cyclic phosphate (cP)-ΔCCA-tRNAs (tRNAs without 3'CCA ended with cP), which is exacerbated by high temperatures, and reduction in the abundance of mature tRNAs, particularly alanine tRNAs (tRNA-Alas). Overexpression of tRNA-Alas in the tms5 mutant restores male fertility to 70%. Remarkably, male fertility of tms5 mutant is completely restored at high temperatures by knocking out OsVms1 which encodes the enzyme for cP-ΔCCA-tRNA generation. Our study reveals the mechanism underlying tms5-mediated TGMS in rice and provides mechanistic insight into the further improvement of TGMS in hybrid crop development.

Improvement of Flowering Stage in Japonica Rice Variety Jiahe212 by Using CRISPR/Cas9 System.

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2, Ghd7, and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2. This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T0 plants with various effective mutations. Among the 17 mutant T1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica-japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica-japonica interspecific hybrid rice breeding with wider adaptability.

The OsMYC2-JA feedback loop regulates diurnal flower-opening time via cell wall loosening in rice.

Diurnal flower-opening time (DFOT), the time of spikelet opening during the day, is an important trait for hybrid rice (Oryza sativa L.) seed production. Hybrids between indica and japonica rice varieties have strong heterosis, but the parental lines usually have different, nonoverlapping DFOTs. This reduces the success of hybrid seed production in crosses between indica and japonica subspecies, thus hindering the utilization of indica and japonica inter-subspecies heterosis. However, little is known about the molecular mechanisms regulating DFOT in rice. Here, we obtained japonica rice lines with a DFOT 1.5 h earlier than the wild type by overexpressing OsMYC2, a gene encoding a key transcription factor in the jasmonate (JA) signaling pathway. OsMYC2 is activated by JA signaling and directly regulates the transcription of genes related to JA biosynthesis and cell wall metabolism. Overexpressing OsMYC2 led to significantly increased JA contents and decreased cellulose and hemicellulose contents in lodicule cells, as well as the softening of lodicule cell walls. This may facilitate the swelling of lodicules, resulting in early diurnal flower-opening. These results suggest that the OsMYC2-JA feedback loop regulates DFOT in rice via cell wall remodeling. These findings shed light on the understanding of regulatory mechanism of the DFOT of plants, which should promote the development of indica and japonica varieties suitable for hybrid rice breeding.

Characterization of an Excellent Hybrid Rice Restorer Line R382 with Enhanced Lodging Resistance

Hybrid rice needs to prevent lodging due to excessive grain weight during maturity, which can lead to yield loss, grain quality deterioration and difficulty in harvesting. Breeding excellent parents (restorer and male-sterile lines) is the foundation for cultivating lodging-resistant hybrid rice. However, there is still limited information about restorers with improved lodging resistance. To identify a new restorer with significantly enhanced resistance to lodging, the restorer line Huazhan, which has been used for many years in hybrid rice breeding in China, was used as the control, to evaluate the lodging resistance of the new restorer R382. A two-year field experiment was executed to investigate the difference on lodging-related traits of the basal two internodes above-ground, such as out diameters of stems with or without leaf sheath (DWS, DWOS), culm thickness (CT), the section modulus (SM), bending stress (BS), and bending moment at break (M). The results indicated that DWS, CT, BS, M of R382 was at least 19.9%, 13.3%, 36.5% and 52.6% higher than that of Huazhan. However, the negative indicator lodging index of R382 was over 17.5% lower. The number and area of vascular bundles, and the contents of the chemical components of the cell wall including cellulose, lignin, and hemicellulose, together with the accumulation of starch granules, were significantly greater in R382. Furthermore, in the basal stems during the jointing stage, a series of genes that promote cell wall formation exhibited significantly higher expression levels in R382. Meanwhile, there was no significant difference in the yield of R382 and Huazhan. Thus, this new restorer could be a valuable donor in the future hybrid rice breeding programs for improving lodging resistance.

Ideal phenotypic characteristics and parental lines for hybrid rice breeding to cope with global dimming

The global food crisis has intensified with climate change, and hybrid rice (Oryza sativa L.) is expected to play a crucial role in ensuring world food security. This study analyzed the parental origins and variety characteristics of 930 hybrid rice varieties bred in Sichuan Province, a typical low-light region and one of the main production areas for hybrid rice, from 2002 to 2022. The results indicate that these varieties were hybridized using 329 direct female and 539 direct male parents. Notably, the female parent Peiai 64 and the male parents Yuehesimiao and Fenghuazhan demonstrated a higher probability of breeding rice varieties with high yield and good quality. An ideal panicle type should possess more grains, a lower 1000-grain weight, a higher seed setting percentage, and greater grain density. It is recommended to increase the utilization of excellent parents in Sichuan Province, such as parents with ideal panicle type to improve resistance to climate change, particularly global dimming.

Genetic editing of grain size genes enables fully mechanized hybrid rice breeding.

Genetic editing of grain size genes enables fully mechanized hybrid rice breeding.

Modulation of histone acetylation enables fully mechanized hybrid rice breeding.

Hybrid rice has achieved high grain yield and greatly contributes to food security, but the manual-labour-intensive hybrid seed production process limits fully mechanized hybrid rice breeding. For next-generation hybrid seed production, the use of small-grain male sterile lines to mechanically separate small hybrid seeds from mixed harvest is promising. However, it is difficult to find ideal grain-size genes for breeding ideal small-grain male sterile lines without penalties in the number of hybrid seeds and hybrid rice yield. Here we report that the use of small-grain alleles of the ideal grain-size gene GSE3 in male sterile lines enables fully mechanized hybrid seed production and dramatically increases hybrid seed number in three-line and two-line hybrid rice systems. The GSE3 gene encodes a histone acetyltransferase that binds histones and influences histone acetylation levels. GSE3 is recruited by the transcription factor GS2 to the promoters of their co-regulated grain-size genes and influences the histone acetylation status of their co-regulated genes. Field trials demonstrate that genome editing of GSE3 can be used to immediately improve current elite male sterile lines of hybrid rice for fully mechanized hybrid rice breeding, providing a new perspective for mechanized hybrid breeding in other crops.

Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

Dominance complementation of parental heading date alleles of Hd1, Ghd7, DTH8, and PRR37 confers transgressive late maturation in hybrid rice.

Rice (Oryza sativa L.) is a short-day plant whose heading date is largely determined by photoperiod sensitivity (PS). Many parental lines used in hybrid rice breeding have weak PS, but their F1 progenies have strong PS and exhibit an undesirable transgressive late-maturing phenotype. However, the genetic basis for this phenomenon is unclear. Therefore, effective methods are needed for selecting parents to create F1 hybrid varieties with the desired PS. In this study, we used bulked segregant analysis with F1 Ningyou 1179 (strong PS) and its F2 population, and through analyzing both parental haplotypes and PS data for 918 hybrid rice varieties, to identify the genetic basis of transgressive late maturation which is dependent on dominance complementation effects of Hd1, Ghd7, DTH8, and PRR37 from both parents rather than from a single parental genotype. We designed a molecular marker-assisted selection system to identify the genotypes of Hd1, Ghd7, DTH8, and PRR37 in parental lines to predict PS in F1 plants prior to crossing. Furthermore, we used CRISPR/Cas9 technique to knock out Hd1 in Ning A (sterile line) and Ning B (maintainer line) and obtained an hd1-NY material with weak PS while retaining the elite agronomic traits of NY. Our findings clarified the genetic basis of transgressive late maturation in hybrid rice and developed effective methods for parental selection and gene editing to facilitate the breeding of hybrid varieties with the desired PS for improving their adaptability.

Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

MORE FLORET1 controls anther development by negatively regulating key tapetal genes in both diploid and tetraploid rice.

Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well studied in polyploid rice than in diploid rice. We previously identified an MYB transcription factor, MORE FLORET1 (MOF1), whose mutation causes male sterility in neo-tetraploid rice. MOF1 expression in anthers peaks at anther Stage 7 (S7) and progressively decreases to low levels at S10. However, it remains unclear how the dynamics of MOF1 expression contribute to male fertility. Here, we carefully examined anther development in both diploid and tetraploid mof1 rice mutants, as well as lines ectopically expressing MOF1 in a temporal manner. MOF1 mutations caused delayed degeneration of the tapetum and middle layer of anthers and aberrant pollen wall organization. Ectopic MOF1 expression at later stages of anther development led to retarded cytoplasmic reorganization of tapetal cells. In both cases, pollen grains were aborted and seed production was abolished, indicating that precise control of MOF1 expression is essential for male reproduction. We demonstrated that 5 key tapetal genes, CYP703A3 (CYTOCHROME P450 HYDROXYLASE 703A3), OsABCG26 (O. sativa ATP BINDING CASSETTE G26), PTC1 (PERSISTENT TAPETAL CELL1), PKS2 (POLYKETIDE SYNTHASE 2), and OsABCG15 (O. sativa ATP BINDING CASSETTE G15), exhibit expression patterns opposite to those of MOF1 and are negatively regulated by MOF1. Moreover, DNA affinity purification sequencing (DAP-seq), luciferase activity assays, and electrophoretic mobility shift assays indicated that MOF1 binds directly to the PKS2 promoter for transcriptional repression. Our results provide a mechanistic basis for the regulation of male reproduction by MOF1 in both diploid and tetraploid rice. This study will facilitate the development of polyploid male sterile lines, which are useful for breeding of polyploid hybrid rice.

Temperature and light reverse the fertility of rice P/TGMS line ostms19 via reactive oxygen species homeostasis.

P/TGMS (Photo/thermo-sensitive genic male sterile) lines are crucial resources for two-line hybrid rice breeding. Previous studies revealed that slow development is a general mechanism for sterility-fertility conversion of P/TGMS in Arabidopsis. However, the difference in P/TGMS genes between rice and Arabidopsis suggests the presence of a distinct P/TGMS mechanism in rice. In this study, we isolated a novel P/TGMS line, ostms19, which shows sterility under high-temperature conditions and fertility under low-temperature conditions. OsTMS19 encodes a novel pentatricopeptide repeat (PPR) protein essential for pollen formation, in which a point mutation GTA(Val) to GCA(Ala) leads to ostms19 P/TGMS phenotype. It is highly expressed in the tapetum and localized to mitochondria. Under high temperature or long-day photoperiod conditions, excessive ROS accumulation in ostms19 anthers during pollen mitosis disrupts gene expression and intine formation, causing male sterility. Conversely, under low temperature or short-day photoperiod conditions, ROS can be effectively scavenged in anthers, resulting in fertility restoration. This indicates that ROS homeostasis is critical for fertility conversion. This relationship between ROS homeostasis and fertility conversion has also been observed in other tested rice P/TGMS lines. Therefore, we propose that ROS homeostasis is a general mechanism for the sterility-fertility conversion of rice P/TGMS lines.

Grain Yield and Yield Attributes of Currently Popular Hybrid Rice Varieties Compared to Representative Super Hybrid Rice Varieties in Subtropical Environments

Understanding the yield attributes of the popular rice (Oryza sativa L.) hybrids can provide useful information for developing new hybrid rice varieties. Field experiments were conducted at two subtropical sites (Hengyang and Xingyi) in two years (2021 and 2022) to compare grain yield and yield attributes of three currently popular hybrid rice varieties (Jingliangyouhuazhan, Jingliangyou 534, and Longliangyouhuazhan) with three representative super hybrid rice varieties (Y-liangyou 1, Y-liangyou 2, and Y-liangyou 900). No significant differences in grain yield were observed between the group of popular hybrids and the group of super hybrids at Hengyang and Xingyi in 2021 or at Xingyi in 2022, but at Hengyang in 2022, the group of popular hybrids produced a 15% higher grain yield. The grain yield at Hengyang in 2022 was lower than that at Hengyang and Xingyi in 2021 and at Xingyi in 2022. At Hengyang in 2022, the group of popular hybrids had 9–15% higher panicles per m2, spikelet filling percentage, and harvest index but similar spikelets per panicle and total biomass production and 12% lower grain weight compared to the group of super hybrids. Correlation plot analysis showed that grain yield was significantly related to panicles per m2 but not other yield attributes across six varieties at Hengyang in 2022. These results indicated that the currently popular hybrid rice varieties had higher yield performance than the representative super hybrid rice varieties under the condition of relatively lower productivity, and the key factor for this higher yield performance in the popular hybrids was the higher panicles per m2. This finding highlights that more attention should focus on the yield performance under medium- and low-productivity conditions in hybrid rice breeding programs, and an effective breeding strategy is to select the varieties with high panicle numbers.

A historical review of hybrid rice breeding.

The development of germplasm resources and advances in breeding methods have led to steady increases in yield and quality of rice (Oryza sativa L.). Three milestones in the recent history of rice breeding have contributed to these increases: dwarf rice breeding, hybrid rice breeding, and super rice breeding. On the 50th anniversary of the success of three-line hybrid rice, we highlight important scientific discoveries in rice breeding that were made by Chinese scientists and summarize the broader history of the field. We discuss the strategies that could be used in the future to optimize rice breeding further in the hope that China will continue to play a leading role in international rice breeding.

Unveiling a half-century mystery of molecular bases for three-line hybrid rice breeding system.

Hybrid rice derived from Wild Abortive (WA) cytoplasmic male sterility type three-line systems is widely used in production. The fertility restoration gene Rf4 can reduce WA352c transcript levels in a dose-dependent manner. A two-copy haplotype of Rf4 (H1) is identified as the most valuable haplotype for future hybrid rice breeding.

Improving hybrid rice breeding programs via stochastic simulations: number of parents, number of hybrids, tester update, and genomic prediction of hybrid performance

Key messageSchemes that use genomic prediction outperform others, updating testers increases hybrid genetic gain, and larger population sizes tend to have higher genetic gain and less depletion of genetic varianceOne of the most common methods to improve hybrid performance is reciprocal recurrent selection (RRS). Genomic prediction (GP) can be used to increase genetic gain in RRS by reducing cycle length, but it is also possible to use GP to predict single-cross hybrid performance. The impact of the latter method on genetic gain has yet to be previously reported. Therefore, we compared via stochastic simulations various phenotypic and genomics-assisted RRS breeding schemes which used GP to predict hybrid performance rather than reducing cycle length, which allows minimal changes to traditional breeding schemes. We also compared three breeding sizes scenarios that varied the number of genotypes crossed within heterotic pools, the number of genotypes crossed between heterotic pools, the number of hybrids evaluated, and the number of genomic predicted hybrids. Our results demonstrated that schemes that used genomic prediction of hybrid performance outperformed the others for the average interpopulation hybrid population and the best hybrid performance. Furthermore, updating the testers increased hybrid genetic gain with phenotypic RRS. As expected, the largest breeding size tested had the highest rates of genetic improvement and the lowest decrease in additive genetic variance due to the drift. Therefore, this study demonstrates the usefulness of single-cross prediction, which may be easier to implement than rapid-cycling RRS and cyclical updating of testers. We also reiterate that larger population sizes tend to have higher genetic gain and less depletion of genetic variance.

MRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

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.