Improving bridge effect to overcome interspecific hybrid sterility by pyramiding hybrid sterile loci from Oryza glaberrima.

In order to evaluate the genetic effect caused by hybrid sterile loci, NILs with O. glaberrima fragment at six hybrid sterile loci under O. sativa genetic background (single-locus-NILs) were developed; two lines harboring two hybrid sterile loci, one line harboring three hybrid sterile loci were further developed. A total of nine NILs were used to test cross with O. sativa recurrent parent, and O. glaberrima accessions respectively. The results showed that the sterility of pollen grains in F1 hybrids deepened with the increase of the number of hybrid sterile loci, when the nine lines test crossed with O. sativa recurrent parent. The F1 hybrids were almost completely sterile when three hybrid sterile loci were heterozygeous. On the other hand, the single-locus-NILs had limited bridge effect on improving pollen grain fertility of interspecific hybrids. Compared single-locus-NILs, the multiple-loci-NILs showed increasing effect on pollen fertility when test crossing with O. glaberrima accessions. Further backcrossing can improve the fertility of pollen grain and spikelet of interspecific hybrids. The optimal solution to improve the fertility of interspecific hybrid can be utilization of pyramiding bridge parent plus backcrossing. This report has potential for understanding the nature of interspecific hybrid sterility, and overcoming the interspecific hybrid F1 pollen grain sterility between O. sativa and O. glaberrima.

Solving the mystery of Obake rice in Africa: population structure analyses of Oryza longistaminata reveal three genetic groups and evidence of both recent and ancient introgression with O. sativa.

The undomesticated rice relative Oryza longistaminata is a valuable genetic resource for the improvement of the domesticated Asian rice, Oryza sativa. To facilitate the conservation, management, and use of O. longistaminata germplasm, we sought to quantify the population structure and diversity of this species across its geographic range, which includes most of sub-Saharan Africa, and to determine phylogenetic relationships to other AA-genome species of rice present in Africa, including the prevalence of interspecific hybridization between O. longistaminata and O. sativa. Though past plant breeding efforts to introgress genes from O. longistaminata have improved biotic stress resistance, ratooning ability, and yield in O. sativa, progress has been limited by substantial breeding barriers. Nevertheless, despite the strong breeding barriers observed by plant breeders who have attempted this interspecific cross, there have been multiple reports of spontaneous hybrids of O. sativa and O. longistaminata (aka "Obake") obtained from natural populations in Africa. However, the frequency and extent of such natural introgressions and their effect on the evolution of O. longistaminata had not been previously investigated. We studied 190 O. longistaminata accessions, primarily from the International Rice Research Institute genebank collection, along with 309 O. sativa, 25 Oryza barthii, and 83 Oryza glaberrima control outgroups, and 17 control interspecific O. sativa/O. longistaminata hybrids. We analyzed the materials using 178,651 single-nucleotide polymorphisms (SNPs) and seven plastid microsatellite markers. This study identified three genetic subpopulations of O. longistaminata, which correspond geographically to Northwestern Africa, Pan-Africa, and Southern Africa. We confirmed that O. longistaminata is, perhaps counterintuitively, more closely related to the Asian species, O. sativa, than the African species O. barthii and O. glaberrima. We identified 19 recent spontaneous interspecific hybrid individuals between O. sativa and O. longistaminata in the germplasm sampled. Notably, the recent introgression between O. sativa and O. longistaminata has been bidirectional. Moreover, low levels of O. sativa alleles admixed in many predominantly O. longistaminata accessions suggest that introgression also occurred in the distant past, but only in Southern Africa.

A toxin-antidote system contributes to interspecific reproductive isolation in rice.

Breakdown of reproductive isolation facilitates flow of useful trait genes into crop plants from their wild relatives. Hybrid sterility, a major form of reproductive isolation exists between cultivated rice (Oryza sativa) and wild rice (O. meridionalis, Mer). Here, we report the cloning of qHMS1, a quantitative trait locus controlling hybrid male sterility between these two species. Like qHMS7, another locus we cloned previously, qHMS1 encodes a toxin-antidote system, but differs in the encoded proteins, their evolutionary origin, and action time point during pollen development. In plants heterozygous at qHMS1, ~ 50% of pollens carrying qHMS1-D (an allele from cultivated rice) are selectively killed. In plants heterozygous at both qHMS1 and qHMS7, ~ 75% pollens without co-presence of qHMS1-Mer and qHMS7-D are selectively killed, indicating that the antidotes function in a toxin-dependent manner. Our results indicate that different toxin-antidote systems provide stacked reproductive isolation for maintaining species identity and shed light on breakdown of hybrid male sterility.

A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Three QTL from Oryza meridionalis Could Improve Panicle Architecture in Asian Cultivated Rice.

Rice panicle architecture is directly associated with grain yield and is also the key target in high-yield rice breeding program. In this study, three BC6F2 segregation populations derived from the crosses between two accessions of Oryza meridionalis and a O. sativa spp. japonica cultivar Dianjingyou 1, were employed to map QTL for panicle architecture. Three QTL, EP4.2, DEP7 and DEP8 were identified and validated using substitution mapping strategy on chromosome 4, 9 and 8, respectively. The three QTL showed pleiotropic phenotype on panicle length (PL), grain number per panicle (GNPP), number of primary branches (NPB), number of secondary branches (NSB), and grain width. DEP7 and DEP8 showed yield-enhancing potential by increasing GNPP, NPB and NSB, while EP4.2 exhibited wide grain, short stalk and panicle which can improve plant and panicle architecture, too. Moreover, epistatic interaction for PL was detected between EP4.2 and DEP7, and epistatic analysis between DEP7 and DEP8 for GNPP and NPB also revealed significant two QTL interactions. The result would help us understand the molecular basis of panicle architecture and lay the foundation for using these three QTL in rice breeding.

A Rapid and Simple Method for DNA Preparation of Magnaporthe oryzae from Single Rice Blast Lesions for PCR-Based Molecular Analysis.

Rice blast is one of the most destructive diseases of rice worldwide, and the causative agent is the filamentous ascomycete Magnaporthe oryzae. With the successful cloning of more and more avirulence genes from M. oryzae, the direct extraction of M. oryzae genomic DNA from infected rice tissue would be useful alternative for rapid monitoring of changes of avirulence genes without isolation and cultivation of the pathogen. In this study, a fast, low-cost and reliable method for DNA preparation of M. oryzae from a small piece of infected single rice leaf or neck lesion was established. This single step method only required 10 min for DNA preparation and conventional chemical reagents commonly found in the laboratory. The AvrPik and AvrPi9 genes were successfully amplified with the prepared DNA. The expected DNA fragments from 570 bp to 1,139 bp could be amplified even three months after DNA preparation. This method was also suitable for DNA preparation from M. oryzae strains stored on the filter paper. All together these results indicate that the DNA preparation method established in this study is reliable, and could meet the basic needs for polymerase chain reaction-based analysis of M. oryzae.

Understanding the Nature of Hybrid Sterility and Divergence of Asian Cultivated Rice.

Intraspecific hybrid sterility is a common form of postzygotic reproductive isolation in Asian cultivated rice, which is also the major obstacle to utilize the strong heterosis in the rice breeding program. Here, we review recent progress in classification and hybrid sterility in Asian cultivated rice. A genome-wide analysis of numerous wild relatives of rice and Asian cultivated rice has provided insights into the origin and differentiation of Asian cultivated rice, and divided Asian cultivated rice into five subgroups. More than 40 conserved and specific loci were identified to be responsible for the hybrid sterility between subgroup crosses by genetic mapping, which also contributed to the divergence of Asian cultivated rice. Most of the studies are focused on the sterile barriers between indica and japonica crosses, ignoring hybrid sterility among other subgroups, leading to neither a systematical understanding of the nature of hybrid sterility and subgroup divergence, nor effectively utilizing strong heterosis between the subgroups in Asian cultivated rice. Future studies will aim at identifying and characterizing genes for hybrid sterility and segregation distortion, comparing and understanding the molecular mechanism of hybrid sterility, and drawing a blueprint for intraspecific hybrid sterility loci derived from cross combinations among the five subgroups. These studies would provide scientific and accurate guidelines to overcome the intraspecific hybrid sterility according to the parent subgroup type identification, allowing the utilization of heterosis among subgroups, also helping us unlock the mysterious relationship between hybrid sterility and Asian cultivated rice divergence.

Interspecific Hybridization Is an Important Driving Force for Origin and Diversification of Asian Cultivated Rice Oryza sativa L.

As one of the most important crops, Asian cultivated rice has evolved into a complex group including several subgroups adapting various eco-climate-systems around the globe. Here, we pictured a comprehensive view of its original domestication, divergences, and the origin of different subgroups by integrating agriculture, archeology, genetics, nuclear, and cytoplasm genome results. Then, it was highlighted that interspecific hybridization-introgression has played important role in improving the genetic diversity and adaptation of Oryza sativa during its evolution process. Natural hybridization-introgression led to the origin of indica, aus, and basmatic subgroups, which adapted to changing cultivated environments, and produced feral weedy rice coexisting and competing with cultivars under production management. Artificial interspecific hybridization-introgression gained several breakthroughs in rice breeding, such as developing three-line hybrid rice, new rice for Africa (NERICA), and some important pest and disease resistance genes in rice genetic improvement, contributing to the stable increase of rice production to meet the expanding human population. We proposed a series to exploit the virtues of hybridization-introgression in the genetic improvement of Asian cultivated rice. But some key issues such as reproductive barriers especially hybrid sterility should be investigated further, which are conducive to gene exchange between cultivated rice and its relatives, and even is beneficial to exploiting interspecific hybrid vigor. New technologies help introduce favorable genes from distant wild species to Asian cultivated rice, such as transgenic and genome editing systems. Rising introgression lines in a wider range with multi-donor benefits allele mining, understanding genetic network of rice growth and development, yield formation, and environmental adaptation. Then, integration of new tools and interspecific hybridization can be a future direction to develop more usable breeding populations which can make Asian cultivated rice more resilient to the changing climate and world.

A Genetic Resource for Rice Improvement: Introgression Library of Agronomic Traits for All AA Genome Oryza Species.

Rice improvement depends on the availability of genetic variation, and AA genome Oryza species are the natural reservoir of favorable alleles that are useful for rice breeding. To systematically evaluate and utilize potentially valuable traits of new QTLs or genes for the Asian cultivated rice improvement from all AA genome Oryza species, 6,372 agronomic trait introgression lines (ILs) from BC2 to BC6 were screened and raised based on the variations in agronomic traits by crossing 170 accessions of 7 AA genome species and 160 upland rice accessions of O. sativa as the donor parents, with three elite cultivars of O. sativa, Dianjingyou 1 (a japonica variety), Yundao 1 (a japonica variety), and RD23 (an indica variety) as the recurrent parents, respectively. The agronomic traits, such as spreading panicle, erect panicle, dense panicle, lax panicle, awn, prostrate growth, plant height, pericarp color, kernel color, glabrous hull, grain size, 1,000-grain weight, drought resistance and aerobic adaption, and blast resistance, were derived from more than one species. Further, 1,401 agronomic trait ILs in the Dianjingyou 1 background were genotyped using 168 SSR markers distributed on the whole genome. A total of twenty-two novel allelic variations were identified to be highly related to the traits of grain length (GL) and grain width (GW), respectively. In addition, allelic variations for the same locus were detected from the different donor species, which suggest that these QTLs or genes were conserved and the different haplotypes of a QTL (gene) were valuable resources for broadening the genetic basis in Asian cultivated rice. Thus, this agronomic trait introgression library from multiple species and accessions provided a powerful resource for future rice improvement and genetic dissection of agronomic traits.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Genetic characterization and fine mapping of qHMS4 responsible for pollen sterility in hybrids between Oryza sativa L. and Oryza glaberrima Steud.

African cultivated rice (Oryza glaberrima Steud) contains many favorable genes for tolerance to biotic and abiotic stresses and F1 hybrids between Asian cultivated rice (Oryza sativa L.) show strong heterosis. However, the hybrids of two species often exhibit hybrid sterility. Here, we identified a male sterility locus qHMS4 on chromosome 4 (Chr.4), which induces pollen semi-sterility in F1 hybrids of japonica rice variety Dianjingyou1 (DJY1) and a near-isogenic line (NIL) carrying a Chr.4 segment from Oryza glaberrima accession IRGC101854. Cytological observations indicated that non-functional pollen grains produced by the hybrids and lacking starch accumulation abort at the late bicellular stage. Molecular genetic analysis revealed distorted segregation in male gametogenesis carrying qHMS4 allele from DJY1. Fine-mapping of qHMS4 using an F2 population of 22,500 plants delimited qHMS4 to a region of 110-kb on the short arm of Chr.4. Sequence analysis showed that the corresponding sequence region in DJY1 and Oryza glaberrima were 114-kb and 323-kb, respectively, and that the sequence homology was very poor. Gene prediction analysis identified 16 and 46 open reading frames (ORFs) based on the sequences of DJY1 and O. glaberrima, respectively, among which 3 ORFs were shared by both. Future map-based cloning of qHMS4 will help to understand the underlying molecular mechanism of hybrid sterility between the two cultivated rice species. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01306-8.

A hybrid sterile locus leads to the linkage drag of interspecific hybrid progenies.

Interspecific hybridization plays an important role in rice breeding by broadening access to desirable traits such as disease resistance and improving yields. However, interspecific hybridization is often hindered by hybrid sterility, linkage drag, and distorted segregation. To mine for favorable genes from Oryza glaberrima, we cultivated a series of BC4 introgression lines (ILs) of O. glaberrima in the japonica rice variety background (Dianjingyou 1) in which the IL-2769 (BC4F10) showed longer sterile lemmas, wider grains and spreading panicles compared with its receptor parent, suggesting that linkage drag may have occurred. Based on the BC5F2 population, a hybrid sterility locus, S20, a long sterile lemma locus, G1-g, and a new grain width quantitative trait locus (QTL), qGW7, were mapped in the linkage region about 15 centimorgan (cM) from the end of the short arm of chromosome 7. The hybrid sterility locus S20 from O. glaberrima eliminated male gametes of Oryza sativa, and male gametes carrying the alleles of O. sativa in the heterozygotes were aborted completely. In addition, the homozygotes presented a genotype of O. glaberrima, and homozygous O. sativa were not produced. Surprisingly, the linked traits G1-g and qGW7 showed similar segregation distortion. These results indicate that S20 was responsible for the linkage drag. As a large number of detected hybrid sterility loci are widely distributed on rice chromosomes, we suggest that hybrid sterility loci are the critical factors for the linkage drag in interspecific and subspecific hybridization of rice.

New Insights Into the Nature of Interspecific Hybrid Sterility in Rice.

Interspecific and intraspecific hybrid sterility is a typical and common phenomenon of postzygotic reproductive barrier in rice. This is an indicator of speciation involved in the formation of new species or subspecies, and it significantly hampers the utilization of favorable genes from distant parents for rice improvement. The Oryza genus includes eight species with the same AA genome and is a model plant for studying the nature of hybrid sterility and its relationship with speciation. Hybrid sterility in rice is mostly controlled by nuclear genes, with more than 50 sterility loci genetically identified to date, of which 10 hybrid sterility loci or pairs were cloned and characterized at the molecular level. Comparing the mapping results for all sterility loci reported indicated that some of these loci from different species should be allelic to each other. Further research revealed that interactions between the multiple alleles at the hybrid sterility locus caused various genetic effect. One hypothesis for this important phenomenon is that the hybrid sterility loci are orthologous loci, which existed in ancient ancestors of rice. When one or more ancestors drifted to different continents, genetic divergence occurred because of adaptation, selection, and isolation among them such that various alleles from orthologous loci emerged over evolutionary time; hence, interspecific hybrid sterility would be mainly controlled by a few orthologous loci with different alleles. This hypothesis was tested and supported by the molecular characterization of hybrid sterility loci from S1, S5, Sa, qHMS7, and S27. From this, we may further deduce that both allelic and non-allelic interactions among different loci are the major genetic basis for the interspecific hybrid sterility between O. sativa and its AA genome relatives, and the same is true for intraspecific hybrid sterility in O. sativa. Therefore, it is necessary to raise the near-isogenic lines with various alleles/haplotypes and pyramided different alleles/haplotypes from sterile loci in the same genetic background aiming to study allelic and non-allelic interaction among different hybrid sterility loci in the AA genome species. Furthermore, the pyramiding lines ought to be used as bridge parents to overcome hybrid sterility for rice breeding purposes.

Identification and Fine Mapping of Pi69(t), a New Gene Conferring Broad-Spectrum Resistance Against Magnaporthe oryzae From Oryza glaberrima Steud.

The discovery and deployment of new broad-spectrum resistance (R) genes from cultivated rice and its wild relatives is a strategy to broaden the genetic basis of modern rice cultivars to combat rice blast disease. Oryza glaberrima possessing many valuable traits for tolerance to biotic and abiotic stresses, is an elite gene pool for improvement of Asian cultivated rice. An introgression line IL106 derived from O. glaberrima (Acc. IRGC100137) confers complete resistance to Magnaporthe oryzae in blast nursery. Genetic analysis using 2185 BC6F2 progenies derived from a cross between IL106 and the recurrent parent Dianjingyou 1 showed that IL106 harbors a single dominant resistance gene against M. oryzae strain 09BSH-10-5A. This gene was preliminarily mapped on the long arm of chromosome 6 of rice in a region of ca. 0.9 cM delimited by two SSR markers (RM20650 and RM20701). In order to finely map this gene, 17,100 additional progenies were further analyzed. As a result, this gene was further narrowed down to a region flanked by two molecular markers STS69-15 and STS69-7, and co-segregated with 3 molecular markers, RM20676, STS69-21 and STS69-22 on the long arm of chromosome 6. Based on reference genome sequences, this R gene was mapped in silico in 76.1-Kb and 67.7-Kb physical intervals, and containing 4 and 3 NBS-LRR candidate genes in O. sativa cultivar Nipponbare and O. glaberrima cultivar CG14, respectively. Because no blast resistance gene was finely mapped in this physical interval before, this R gene was considered as not described yet and designated as Pi69(t), which is the first identified and finely mapped blast R gene from O. glaberrima, as far as we know. Evaluation of IL106 with 151 blast strains collected from 6 countries in Asia showed that 148 strains are avirulent on IL106, suggesting that Pi69(t) is a broad-spectrum blast R gene, and a promising resistant resource for improvement of Asian cultivated rice.

Identification and Validation of Aerobic Adaptation QTLs in Upland Rice.

The aerobic adaptation of upland rice is considered as the key genetic difference between upland rice and lowland rice. Genetic dissection of the aerobic adaptation is important as the basis for improving drought tolerance and terrestrial adaptation by using the upland rice. We raised BC1-BC3 introgression lines (ILs) in lowland rice Minghui 63 (MH63) background. The QTLs of yield and yield-related traits were detected based on ILs under the aerobic and lowland environments, and then the yield-related QTLs were identified in a backcrossed inbred population of BC4F5 under aerobic condition. We further verified phenotypes of QTL near-isogenic lines. Finally, three QTLs responsible for increasing yield in aerobic environment were detected by multiple locations and generations, which were designated as qAER1, qAER3, and qAER9 (QTL of aerobic adaptation). The qAER1 and qAER9 were fine-mapped. We found that qAER1 and qAER9 controlled plant height and heading date, respectively; while both of them increased yields simultaneously by suitable plant height and heading date without delay in the aerobic environment. The phenotypic differences between lowland rice and upland rice in the aerobic environment further supported the above results. We pyramided the two QTLs as corresponding molecular modules in the irrigated lowland rice MH63 background, and successfully developed a new upland rice variety named as Zhongkexilu 2. This study will lay the foundation for using aerobic adaptation QTLs in rice breeding programs and for further cloning the key genes involved in aerobic adaptation.