Evaluation of Grain-Filling-Related Traits Using Taichung 65 x DV85 Chromosome Segment Substitution Lines (TD-CSSLs) of Rice.

Grain yield of rice consists of sink capacity and grain filling. There are some genes known to contribute to sink capacity, but few genes associated with grain filling are known. We conducted a genetic analysis on yield-related traits by using a chromosome segment substitution line population that have introgression from DV85, an aus variety of rice, in the background of T65, a japonica variety. Refined whole-genome genotypes of the 43 TD-CSSLs were obtained by genotyping-by-sequencing. The effects of previously detected quantitative trait loci (QTLs), qNSC1 and qNSC2, were confirmed by the amount of non-structural carbohydrate (NSC) at 5 days after heading (DAH). The CSSL for qSWTR11, the QTL for decrease in shoot weight during the maturity stage, showed the highest NSC at 5 DAH and lowest at 35 DAH. The brown rice yield of these lines were not stably significant. Most of the sink-related traits correlated between the 2 tested years, but most of the grain-filling traits did not show correlation between the 2 years. Correlation analysis revealed that the sink capacity is stable and primarily determines the yield, and grain filling is more affected by the environment. In addition, biomass production before heading and during the maturity stage contributes to higher yield in TD-CSSLs, and the amount of translocation of stem reserve does not affect much to the yield. We conclude that higher NSC at the heading stage and rapid decrease in shoot biomass during the maturity stage did not directly contribute to the yield formation in the japonica genetic background.

Genetic Variation of Blast (Pyricularia oryzae Cavara) Resistance in the Longistaminata Chromosome Segment Introgression Lines (LCSILs) and Potential for Breeding Use in Kenya.

In Kenya's rice-growing areas, Basmati varieties have been produced in monoculture since the late 1980s. This has resulted in the breakdown of the resistance (R) gene-mediated response of the local Basmati varieties to blast disease caused by Pyricularia oryzae. To improve blast resistance in Kenyan Basmati varieties, continuous identification of R genes and suitable breeding materials for Basmati are necessary. Longistaminata chromosome segment introgression lines (LCSILs) with the Kernel Basmati genetic background, developed using a rice line called potential low-input adaptable-1 (pLIA-1) derived from a cross between Taichung 65 (T65) (a rice variety in the Japonica Group) and O. longistaminata, are expected to contain useful blast R genes derived from O. longistaminata or T65. In this study, we investigated the genetic variation of blast R genes in LCSILs and their parents by using a new international differential system for designating blast races based on the gene-for-gene theory and molecular characterization using single nucleotide polymorphism (SNP) markers. LCSILs and their parents were classified into three groups-A, B1, and B2-based on reaction patterns to the standard differential blast isolates (SDBIs). Group A, including pLIA-1, showed the highest resistance in all groups, followed by groups B1 and B2. Kernel Basmati in group B1 was considered to possess Pik-p or Pi7(t), Pi19(t), and other unknown R genes. In addition to these R genes, LCSIL 6, 12, 27, 28, and 40, in group A, were determined to possess one of Pish, Piz-t, or both genes that confer resistance to the Kenyan blast races. These lines can be used for efficiently pyramiding blast R genes in the local Basmati varieties.

Utilization of Genotyping-by-Sequencing (GBS) for Rice Pre-Breeding and Improvement: A Review.

Molecular markers play a crucial role in the improvement of rice. To benefit from these markers, genotyping is carried out to identify the differences at a specific position in the genome of individuals. The advances in sequencing technologies have led to the development of different genotyping techniques such as genotyping-by-sequencing. Unlike PCR-fragment-based genotyping, genotyping-by-sequencing has enabled the parallel sequencing and genotyping of hundreds of samples in a single run, making it more cost-effective. Currently, GBS is being used in several pre-breeding programs of rice to identify beneficial genes and QTL from different rice genetic resources. In this review, we present the current advances in the utilization of genotyping-by-sequencing for the development of rice pre-breeding materials and the improvement of existing rice cultivars. The challenges and perspectives of using this approach are also highlighted.

QTL analysis for sodium removal ability in rice leaf sheaths under salinity using an IR-44595/318 F2 population.

Over-accumulation of salt in rice plants is an effect of salt stress which decreases growth and grain yield. Salt removal ability in leaf sheaths is a tolerance mechanism to decrease salt entry and accumulation in leaf blades and maintain photosynthesis under salinity. In this study, a QTL analysis of removal ability of sodium ions (Na+) in leaf sheaths and Na+ accumulation-related traits, was conducted using F2 population between two rice varieties, IR-44595 with superior Na+ removal ability, and 318 with contrasting Na+ removal ability in leaf sheaths under salinity. Suggestive QTLs for Na+ removal ability in leaf sheaths were found on chromosomes 4 and 11. The suggestive QTL on chromosome 11 overlapped with other significant QTLs for Na+ concentration in shoots, leaf blades and leaf sheaths, and Na+/K+ ratio in leaf blades. Correlation analysis indicated that Na+ removal ability in leaf sheaths is important in reducing Na+ accumulation in leaf blades. The varietal difference of Na+ removal ability in leaf sheaths at the whole plant level was greater at lower NaCl concentrations and became smaller as the treatment NaCl concentration increased. Although the Na+ removal ability in leaf sheath was comparable between IR-44595 and 318 under high salinity at the whole plant level, the younger leaves of IR-44595 still showed a higher Na+ sheath-blade ratio than 318, which implied the Na+ removal ability functions in the younger leaves in IR-44595 to reduce Na+ entry in young leaf blades even under high salinity.

A Novel Combination of Genes Causing Temperature-Sensitive Hybrid Weakness in Rice.

Reproductive isolation is an obstacle for plant breeding when a distant cross is demanded. It can be divided into two main types based on different growth stages: prezygotic isolation and postzygotic isolation. The hybrid weakness, which is a type of postzygotic isolation, can become a problem in crop breeding. In order to overcome reproductive isolation, it is necessary to elucidate its mechanism. In this study, genetic analysis for low temperature-dependent hybrid weakness was conducted in a rice F2 population derived from Taichung 65 (T65, Japonica) and Lijiangxintuanheigu (LTH, Japonica). The weak and severe weak plants in F2 showed shorter culm length, late heading, reduced panicle number, decreased grain numbers per panicle, and impaired root development in the field. Our result also showed that hybrid weakness was affected by temperature. It was observed that 24°C enhanced hybrid weakness, whereas 34°C showed recovery from hybrid weakness. In terms of the morphology of embryos, no difference was observed. Therefore, hybrid weakness affects postembryonic development and is independent of embryogenesis. The genotypes of 126 F2 plants were determined through genotyping-by-sequencing and a linkage map consisting of 862 single nucleotide polymorphism markers was obtained. Two major quantitative trait loci (QTLs) were detected on chromosomes 1 [hybrid weakness j 1 (hwj1)] and 11 [hybrid weakness j 2 (hwj2)]. Further genotyping indicated that the hybrid weakness was due to an incompatible interaction between the T65 allele of hwj1 and the LTH allele of hwj2. A large F2 populations consisting of 5,722 plants were used for fine mapping of hwj1 and hwj2. The two loci, hwj1 and hwj2, were mapped in regions of 65-kb on chromosome 1 and 145-kb on chromosome 11, respectively. For hwj1, the 65-kb region contained 11 predicted genes, while in the hwj2 region, 22 predicted genes were identified, two of which are disease resistance-related genes. The identified genes along these regions serve as preliminary information on the molecular networks associated with hybrid weakness in rice.

Chronic pulmonary aspergillosis in a patient with hyper-IgE syndrome.

Hyperimmunoglobulin E (IgE) syndrome (HIES) is a rare disease with an unclear prognosis. We report a case of HIES comorbid with chronic pulmonary aspergillosis (CPA). A 19-year-old male was referred to our department with a medical history of bacterial pneumonia and skin infection. Laboratory data showed an elevated eosinophil count and serum IgE level. Chest computed tomography (CT) showed a pneumatocele and bronchiectasis. On the basis of the clinical and laboratory findings and genetic mutation analysis, we diagnosed him as having HIES. Fourteen months later, he complained of blood-tinged sputum and haemoptysis. Chest CT showed pneumatocele wall thickening, fungus ball and consolidation. Serum Aspergillus precipitating antibody and serum galactomannan Aspergillus antigen were positive, and Aspergillus fumigatus was detected in the sputum. We diagnosed CPA and treated him using antifungal agents and bronchial artery embolization. CPA is a complication that requires attention in patients with HIES.

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.

Marker-Assisted Introgression and Stacking of Major QTLs Controlling Grain Number (Gn1a) and Number of Primary Branching (WFP) to NERICA Cultivars.

The era of the green revolution has significantly improved rice yield productivity. However, with the growing population and decreasing arable land, rice scientists must find new ways to improve rice productivity. Although hundreds of rice yield-related QTLs were already mapped and some of them were cloned, only a few were utilized for actual systematic introgression breeding programs. In this study, the major yield QTLs Grain Number 1a (Gn1a) and Wealthy Farmer's Panicle (WFP) were introgressed and stacked in selected NERICA cultivars by marker-assisted backcross breeding (MABB). The DNA markers RM3360, RM3452, and RM5493 were used for foreground selection. At BC3F4 and BC3F5 generation, a combination of marker-assisted selection and phenotypic evaluation were carried out to select lines with target alleles and traits. Further, genotyping-by-sequencing (GBS) was conducted to validate the introgression and determine the recurrent parent genome recovery (RPGR) of the selected lines. The Gn1a and/or WFP introgression lines showed significantly higher numbers of spikelets per panicle and primary branching compared to the recurrent parents. In addition, lines with Gn1a and/or WFP alleles were comparatively similar to the recurrent parents (RP) in most yield-related traits. This study demonstrates the success of utilizing yield QTLs and marker-assisted selection to develop and improve rice cultivars.

Diverse panicle architecture results from various combinations of Prl5/GA20ox4 and Pbl6/APO1 alleles.

Panicle architecture directly affects crop productivity and is a key target of high-yield rice breeding. Panicle length strongly affects panicle architecture, but the underlying regulatory mechanisms are largely unknown. Here, we show that two quantitative trait loci (QTLs), PANICLE RACHIS LENGTH5 (Prl5) and PRIMARY BRANCH LENGTH6 (Pbl6), independently regulate panicle length in rice. Prl5 encodes a gibberellin biosynthesis enzyme, OsGA20ox4. The expression of Prl5 was higher in young panicles resulting in panicle rachis elongation. Pbl6 is identical to ABERRANT PANICLE ORGANIZATION 1 (APO1), encoding an F-box-containing protein. We found a novel function that higher expression of Pbl6 is responsible for primary branch elongation. RNA-seq analysis revealed that these two genes independently regulate panicle length at the level of gene expression. QTL pyramiding of both genes increased panicle length and productivity. By combining these two genes in various combinations, we designed numerous panicle architecture without trade-off relationship.

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.

QTL analysis for carbon assimilate translocation-related traits during maturity in rice (Oryza sativa L.).

Problems with carbon assimilate translocation from source organs to sink (grains) during ripening cause yield losses in rice (Oryza sativa L.), especially in high-sink-capacity varieties. We conducted a genetic analysis of traits related to such translocation by using recombinant inbred lines. Shoot weight (SW) of T65, a japonica parent, was retained from heading to late maturity, whereas that of DV85, an aus parent, was greater than that of T65 at 5 days after heading (DAH) and then decreased until 20 DAH. This difference was observed clearly under standard-fertilizer but not low-fertilizer conditions. Non-structural carbohydrate (NSC) contents in the parents showed a tendency similar to that for SW. QTL analysis revealed pleiotropic QTLs on chromosomes 5 and 10, probably by associations with heading date QTLs. A QTL associated with harvest index and NSC at 5 DAH was detected on chromosome 1. By considering the temporal changes of the traits, we found a QTL for decrease in SW on chromosome 11; the DV85 allele of this QTL facilitated assimilate translocation and suppressed biomass growth. A suggestive QTL for NSC decrease was located on chromosome 2. These QTLs could represent potential targets for controlling carbon assimilate translocation in breeding programs.

Regional population differences of the brown planthopper (Nilaparvata lugens Stål) in Cambodia using genotyping-by-sequencing.

The brown planthopper Nilaparvata lugens Stål (BPH) can be found year-round in tropical region and causes severe damage to rice. Although there has been documented BPH damage to rice crops in the past decade in Cambodia, the extent of this epidemic is poorly understood. Here, we examined the time variation of BPH population in the abundance of morphotypes in 13 main rice-producing provinces (86 sites) by aspirator method and in the Takeo Province (five sites) by yellow sticky trap method. At least three generations were observed during the 3-month collection period in the rainy growing season. Regarding the occurrence of BPH morphotypes, in July the macropterous adults were restricted to south Cambodia and in August all morphotypes, adults (macropterous and brachypterous) and nymphs, appeared in all sampling sites. To explain the difference of regional distribution, the genetic differentiation was analyzed in south and northwest Cambodia (three sites) by using single nucleotide polymorphisms (SNP) analysis via genotyping-by-sequencing (GBS) using next-generation sequencing. The 2455 SNPs obtained by GBS clarified the three sub-populations and they corresponded to the expected dissemination patterns. These results provide a clue to understand the differentiation and epidemic of BPH in Cambodia.

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.

Adapting Genotyping-by-Sequencing for Rice F2 Populations.

Rapid and cost-effective genotyping of large mapping populations can be achieved by sequencing a reduced representation of the genome of every individual in a given population, and using that information to generate genetic markers. A customized genotyping-by-sequencing (GBS) pipeline was developed to genotype a rice F2 population from a cross of Oryza sativa ssp. japonica cv. Nipponbare and the African wild rice species O. longistaminata While most GBS pipelines aim to analyze mainly homozygous populations, we attempted to genotype a highly heterozygous F2 population. We show how species- and population-specific improvements of established protocols can drastically increase sample throughput and genotype quality. Using as few as 50,000 reads for some individuals (134,000 reads on average), we were able to generate up to 8154 informative SNP markers in 1081 F2 individuals. Additionally, the effects of enzyme choice, read coverage, and data postprocessing are evaluated. Using GBS-derived markers, we were able to assemble a genetic map of 1536 cM. To demonstrate the usefulness of our GBS pipeline, we determined quantitative trait loci (QTL) for the number of tillers. We were able to map four QTL to chromosomes 1, 3, 4, and 8, and partially confirm their effects using introgression lines. We provide an example of how to successfully use GBS with heterozygous F2 populations. By using the comparatively low-cost MiSeq platform, we show that the GBS method is flexible and cost-effective, even for smaller laboratories.

A single base change explains the independent origin of and selection for the nonshattering gene in African rice domestication.

Reduced seed shattering was a critical evolutionary step in crop domestication. Two cultivated rice species, Oryza sativa and Oryza glaberrima, were independently domesticated from the wild species Oryza rufipogon in Asia and Oryza barthii in Africa, respectively. A single nucleotide polymorphism (SNP) in the c gene, which encodes a trihelix transcription factor, causes nonshattering in O. sativa. However, the genetic mechanism of nonshattering in O. glaberrima is poorly understood. We conducted an association analysis for the coding sequences of SH3/SH4 in AA- genome rice species and the mutation suggested to cause nonshattering was demonstrated to do so using a positional-cloning approach in the O. sativa genetic background. We found that the loss of seed shattering in O. glaberrima was caused by an SNP resulting in a truncated SH3/SH4 protein. This mutation appears to be endemic and to have spread in the African gene pool by hybridization with some O. barthii accessions. We showed that interaction between the O. sativa and O. glaberrima domestication alleles of SH3 in heterozygotes induces a 'throwback' seed-shattering phenotype similar to that in the wild species. Identification of the causative SNP provides new insights into the molecular basis of seed shattering in crops and may facilitate investigation of the history of African rice domestication.

Loss of function at RAE2, a previously unidentified EPFL, is required for awnlessness in cultivated Asian rice.

Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

Identification of QTLs for yield-related traits in RILs derived from the cross between pLIA-1 carrying Oryza longistaminata chromosome segments and Norin 18 in rice.

To improve rice yield, a wide genetic pool is necessary. It is therefore important to explore wild rice relatives. Oryza longistaminata is a distantly related wild rice relative that carries the AA genome. Its potential for improving agronomic traits is not well studied. Introgression line (pLIA-1) that carries Oryza longistaminata's chromosome segments, showed high performance in yield-related traits under non-fertilized conditions. Therefore, to illustrate Oryza longistaminata's potential for improving yield-related traits, RILs from the F1 of a cross between pLIA-1 and Norin 18 were developed and QTL analysis was done using the RAD-Seq method. In total, 36 QTLs for yield-related traits were identified on chromosomes 1, 2, 3, 5, 6, 7, 8, 10, and 11. Clusters of QTLs for strongly correlated traits were also identified on chromosomes 1, 3, 6, and 8. Phenotypic data from recombinant plants for chromosomes 1 and 8 QTL clusters revealed that the pLIA-1 genotype on chromosome 1 region was more important for panicle-related traits and a combination of pLIA-1 genotypes on chromosomes 1 and 8 showed a favorable phenotype under non-fertilized conditions. These results suggest that Oryza longistaminata's chromosome segments carry important alleles that can be used to improve yield-related traits of rice.

Construction of a versatile SNP array for pyramiding useful genes of rice.

DNA marker-assisted selection (MAS) has become an indispensable component of breeding. Single nucleotide polymorphisms (SNP) are the most frequent polymorphism in the rice genome. However, SNP markers are not readily employed in MAS because of limitations in genotyping platforms. Here the authors report a Golden Gate SNP array that targets specific genes controlling yield-related traits and biotic stress resistance in rice. As a first step, the SNP genotypes were surveyed in 31 parental varieties using the Affymetrix Rice 44K SNP microarray. The haplotype information for 16 target genes was then converted to the Golden Gate platform with 143-plex markers. Haplotypes for the 14 useful allele are unique and can discriminate among all other varieties. The genotyping consistency between the Affymetrix microarray and the Golden Gate array was 92.8%, and the accuracy of the Golden Gate array was confirmed in 3 F2 segregating populations. The concept of the haplotype-based selection by using the constructed SNP array was proofed.

Convergent Loss of Awn in Two Cultivated Rice Species Oryza sativa and Oryza glaberrima Is Caused by Mutations in Different Loci.

A long awn is one of the distinct morphological features of wild rice species. This organ is thought to aid in seed dispersal and prevent predation by animals. Most cultivated varieties of Oryza sativa and Oryza glaberrima, however, have lost the ability to form long awns. The causal genetic factors responsible for the loss of awn in these two rice species remain largely unknown. Here, we evaluated three sets of chromosome segment substitution lines (CSSLs) in a common O. sativa genetic background (cv. Koshihikari) that harbor genomic fragments from Oryza nivara, Oryza rufipogon, and Oryza glaberrima donors. Phenotypic analyses of these libraries revealed the existence of three genes, Regulator of Awn Elongation 1 (RAE1), RAE2, and RAE3, involved in the loss of long awns in cultivated rice. Donor segments at two of these genes, RAE1 and RAE2, induced long awn formation in the CSSLs whereas an O. sativa segment at RAE3 induced long awn formation in O. glaberrima. These results suggest that the two cultivated rice species, O. sativa and O. glaberrima, have taken independent paths to become awnless.

Two linked genes on rice chromosome 2 for F1 pollen sterility in a hybrid between Oryza sativa and O. glumaepatula.

Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F1 pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glum (s) or S22B-glum (s) ) were sterile if produced on a heterozygous plant. No transmission of the S22A-glum (s) allele via pollen was observed, whereas a low frequency of transmission of S22B-glum (s) was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization.