Cytological and molecular characterization of a novel monogenic dominant GMS in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5′-CTATGCCGAC-3′) gave a repeatable 1500-bp DNA polymorphic segment S243₁₅₀₀ between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243₁₅₀₀ is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.     

Distinguishing among male sterile and fertile lines of tomatoes (Lycopersicon esculentum Mill.) by means of electrophoretic protein patterns

Vertical block SDS-PAAG electrophoresis of individual tomato seeds was used to examine the potential of seed proteins as gene markers for distinguishing male sterile from fertile lines and cultivars. Seeds of three genic male sterile lines and four fertile cultivars and lines were investigated. The line 6944 is carrier of the gene ms 10 ³⁵for pollen male sterility and the lines P-1a and PPS-3 carry the gene ps-2 for positional sterility. A modified extract solution which includes 1M tris — buffer pH 7.0, 7 % SDS, dimethylformamide and distilled water was applied. Qualitative variation in the A protein locus which could be used as a marker for distinguishing male sterile from fertile tomato lines was established.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Development of SCAR markers linked to male sterility and very high linoleic acid content in safflower

Practically no molecular tools have been developed so far for safflower (Carthamus tinctorius L.) breeding. The objective of the present research was to develop molecular markers for the closely linked genes Li, controlling very high linoleic acid content, and Ms, controlling nuclear male sterility (NMS). A mapping population of 162 individuals was developed from the NMS line CL1 (64–79% linoleic acid) and the line CR-142 (84–90%), and phenotyped in the F₂ and F₃ generations. Bulked segregant analysis with random amplified polymorphic (RAPD) markers revealed linkage of five RAPD bands to the Li and Ms genes. RAPD fragments were converted into sequence-characterized amplified region (SCAR) markers. A linkage map including the five SCAR markers and the Li and Ms genes was constructed. SCAR markers flanked both loci at minimum distances of 15.7 cM from the Li locus and 3.7 cM from the Ms locus. These are the first molecular markers developed for trait selection in safflower.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Identification of RAPD markers linked to a major rust resistance gene block in common bean

Rust in bean (Phaseolus vulgaris L.), caused byUromyces appendiculatus (Pers.) Unger var.appendiculatus [ =U. phaseoli (Reben) Wint.], is a major disease problem and production constraint in many parts of the world. The predominant form of genetic control of the pathogen is a series of major genes which necessitate the development of efficient selection strategies. Our objective was focused on the identification of RAPD (random amplified polymorphic DNA) markers linked to a major bean rust resistance gene block enabling marker-based selection and facilitating resistance gene pyramiding into susceptible bean germplasm. Using pooled DNA samples of genotyped individuals from two segregating populations, we identified two RAPD markers linked to the gene block of interest. One such RAPD, OF10₉₇₀ (generated by a 5-GGAAGCTTGG-3 decamer), was found to be closely linked (2.15±1.50 centi Morgans) in coupling with the resistance gene block. The other identified RAPD, OI19₄₆₀ (generated by a 5-AATGCGGGAG-3 decamer), was shown to be more tightly linked (also in coupling) than OF10₉₇₀ as no recombinants were detected among 97 BC₆F₂ segregating individuals in the mapping population. Analysis of a collection of resistant and susceptible cultivars and experimental lines, of both Mesoamerican and Andean origin, revealed that: (1) recombination between OF10₉₇₀ and the gene block has occurred as evidenced by the presence of the DNA fragment in several susceptible genotypes, (2) recombination between OI19₄₆₀ and the gene block has also occurred indicating that the marker is not located within the gene block itself, and (3) marker-facilitated selection using these RAPD markers, and another previously identified, will enable gene pyramiding in Andean germplasm and certain Mesoamerican bean races in which the resistance gene block does not traditionally exist. Observations of variable recombination among Mesoamerican bean races suggested suppression of recombination between introgressed segments and divergent recurrent backgrounds.Research supported by the Michigan Agricultural Research Station and the USDA-ARS. Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.)

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence

Plant mitochondria include gamma-type carbonic anhydrases (γCAs) of unknown function. In Arabidopsis, the γCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2–3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H₂O₂ dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility. Gene bank accession number: AY085025 (At1g47260).     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat

 RAPD (random amplified polymorphic DNA) analysis was used to identify molecular markers linked to the Dn2 gene conferring resistance to the Russian wheat aphid (Diuraphis noxia Mordvilko). A set of near-isogenic lines (NILs) was screened with 300 RAPD primers for polymorphisms linked to the Dn2 gene. A total of 2700 RAPD loci were screened for linkage to the resistance locus. Four polymorphic RAPD fragments, two in coupling phase and two in repulsion phase, were identified as putative RAPD markers for the Dn2 gene. Segregation analysis of these markers in an F₂ population segregating for the resistance gene revealed that all four markers were closely linked to the Dn2 locus. Linkage distances ranged from 3.3 cM to 4.4 cM. Southern analysis of the RAPD products using the cloned RAPD markers as probes confirmed the homology of the RAPD amplification products. The coupling-phase marker OPB10_880c and the repulsion-phase marker OPN1_400r were converted to sequence characterized amplified region (SCAR) markers. SCAR analysis of the F₂ population and other resistant and susceptible South African wheat cultivars corroborated the observed linkage of the RAPD markers to the Dn2 resistance locus. These markers will be useful for marker-assisted selection of the Dn2 gene for resistance breeding and gene pyramiding. Received: 1 July 1997 / Accepted: 20 October 1997     

Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques

The discovery and application of the thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid seed production technology in rice. Use of the TGMS system in two-line breeding is simple, inexpensive, efficient, and eliminates the limitations associated with the cytoplasmic-genetic male sterility (CMS) system. An F₂ population developed from a cross between a TGMS indica mutant, TGMS–VN1, and a fertile indica line, CH1, was used to identify molecular markers linked to the TGMS gene and to subsequently determine its chromosomal location on the linkage map of rice. Bulk segregant analysis was performed using the AFLP technique. From the survey of 200 AFLP primer combinations, four AFLP markers (E2/M5–600, E3/M16–400, E5/M12–600, and E5/M12–200) linked to the TGMS gene were identified. All the markers were linked to the gene in the coupling phase. All except E2/M5–200 were found to be low-copy sequences. However, the marker E5/M12–600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64×Azucena and CT9993×IR62666, available at IRRI, Philippines, and Texas Tech University, respectively. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12–600, was sequenced so that a PCR marker can be developed for the marker-assisted transfer of this gene to different genetic backgrounds. The new TGMS gene is tentatively designated as tms4(t). Received: 13 July 1999 / Accepted: 27 July 1999     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Inheritance of inter-simple-sequence-repeat polymorphisms and linkage with a fusarium wilt resistance gene in chickpea

 The inheritance of an inter-simple-sequence-repeat (ISSR) polymorphism was studied in a cross of cultivated chickpea (Cicer arietinum L.) and a closely related wild species (C. reticulatum Lad.) using primers that anneal to a simple repeat of various lengths, sequences and non-repetitive motifs. Dinucleotides were the majority of those tested, and provided all of the useful banding patterns. The ISSR loci showed virtually complete agreement with expected Mendelian ratios. Twenty two primers were used for analysis and yielded a total of 31 segregating loci. Primers based on (GA)_n repeats were the most abundant while primers with a (TG)_n repeat gave the largest number of polymorphic loci. Nucleotides at the 5′ and 3′ end of the primers played an important role in detecting polymorphism. All the markers showed dominance. We found an ISSR marker linked to the gene for resistance to fusarium wilt race 4. The marker concerned, UBC-855₅₀₀, was found to be linked in repulsion with the fusarium wilt resistance gene at a distance of 5.2 cM. It co-segregated with CS-27₇₀₀, a RAPD marker previously shown to be linked to the gene for resistance to fusarium wilt race 1, and was mapped to linkage group 6 of the Cicer genome. This indicated that genes for resistance to fusarium wilt races 1 and 4 are closely linked. The marker UBC-855₅₀₀ is located 0.6 cM from CS-27₇₀₀ and is present on the same side of the wilt resistance gene. To our knowledge this is the first report of the utility of an ISSR marker in gene tagging. These markers may provide valuable information for the development of sequence-tagged microsatellite sites (STMS) at a desired locus. Received: 10 August 1997 / Accepted: 6 October 1997     

Assessing hybrid sterility in <Emphasis Type="Italic">Oryza glaberrima</Emphasis> × <Emphasis Type="Italic">O. sativa</Emphasis> hybrid progenies by PCR marker analysis and crossing with wide compatibility varieties

Interspecific crossing of the African indigenous rice Oryza glaberrima with Oryza sativa cultivars is hindered by crossing barriers causing 100% spikelet sterility in F₁ hybrids. Since hybrids are partially female fertile, fertility can be restored by back crossing (BC) to a recurrent male parent. Distinct genetic models on spikelet sterility have been developed predicting, e.g., the existence of a gamete eliminator and/or a pollen killer. Linkage of sterility to the waxy starch synthase gene and the chromogen gene C, both located on chromosome 6, have been demonstrated. We selected a segregating BC₂F₃ population of semi-sterile O. glaberrima × O. sativa indica hybrid progenies for analyses with PCR markers located at the respective chromosome-6 region. These analyses revealed that semi-sterile plants were heterozygous for a marker (OSR25) located in the waxy promoter, whereas fertile progenies were homozygous for the O. glaberrima allele. Adjacent markers showed no linkage to spikelet sterility. Semi-sterility of hybrid progenies was maintained at least until the F₄ progeny generation, suggesting the existence of a pollen killer in this plant material. Monitoring of reproductive plant development showed that spikelet sterility was at least partially due to an arrest of pollen development at the microspore stage. In order to address the question whether genes responsible for F₁ sterility in intraspecific hybrids (O. sativa indica × japonica) also cause spikelet sterility in interspecific hybrids, crossings with wide compatibility varieties (WCV) were performed. WCV accessions possess "neutral" S-loci (Sⁿ) improving fertility in intraspecific hybrids. This experiment showed that the tested Sⁿ-loci had no fertility restoring effect in F₁ interspecific hybrids. Pollen development was completely arrested at the microspore stage and grains were never obtained after selfing. This suggests that distinct or additional S-loci are responsible for sterility of O. glaberrima × O. sativa hybrids.Communicated by H.C. Becker