Two new loci for hybrid sterility in cultivated rice (Oryza sativa L.)

Female gamete abortion in Indica-Japonica crosses of rice was earlier identified to be due to an allelic interaction at the S-5 locus on chromosome 6. Recently, in other crosses of rice, similar allelic interactions were found at loci designated as S-7 and S-8, located on chromosomes 7 and 6 respectively. All of them are independent of each other. At the S-5 locus, Indica and Japonica rice have S-5 ⁱ and S-5 ^j alleles respectively and Javanicas, such as Ketan Nangka, have a neutral allele S-5 ⁿ .The S-5 ⁱ /S-5 ^j genotype is semi-sterile due to partial abortion of female gametes carrying S-5 ^j , but both the S-5 ⁿ /S-5 ⁱ and S-5 ⁿ /S-5 ^j genotypes are fertile. The S-5 ⁿ allele is thus a wide-compatibility gene (WCG), and parents homozygous for this allele are called wide-compatible varieties (WCV). Such parents when crossed with Indica or Japonica varieties do not show F₁ hybrid sterility. Wide-compatible parents have been used to overcome sterility barriers in crosses between Indica and Japonica rice. However, a Javanica variety, Ketan Nangka (WCV), showed typical hybrid sterility when crossed to the Indian varieties N22 and Jaya. Further, Dular, another WCV from India, showed typical hybrid sterility when crossed to an IRRI line, IR2061-628-1-6-4-3(IR2061-628). By genetic analyses using isozyme markers, a new locus causing hybrid sterility in crosses between Ketan Nangka and the Indicas was located near isozyme loci Est-1 and Mal-1 on chromosome 4, and was designated as S-9. Another new locus for hybrid sterility in the crosses between Dular and the IR2061-628 was identified and was found linked to four isozyme loci, Sdh-1, Pox-2, Acp-1 and Acp-2, on chromosome 12. It was designated as S-15. On the basis of allelic interactions causing female-gamete abortion, two alleles were found at S-9, S-9 ^kn in Ketan Nangka and S-9 ⁱ in N22 and Jaya. In the heterozygote, S-9 ^kn /S-9 ⁱ , which was semisterile, female gametes carrying S-9 ^kn were aborted. The hybrid of Dular and IR2061-628, with a genetic constitution of S-15 ^Du /S-15 ⁱ , was semi-sterile and the female gametes carrying S-15 ^Du were aborted. A Japonica tester variety, Akihikari, and an Indica variety, IR36, were found to have neutral alleles, S-9 nand S-15 n, at these loci, in addition to S-7 nand at S-7. The accumulation of three neutral alleles into a breeding line should help solve the hybrid sterility problem in wide crosses of rice.     

Molecular analysis of the inheritance of the S-5 locus,conferring wide compatibility in Indica/Japonica hybrids of rice (O. sativa L.)

RFLP analysis was conducted on a population derived from a three-way cross to determine the location of the hybrid sterility locus, S-5, in relation to mapped molecular markers and to identify markers that would be useful for selection in breeding. S-5 is of interest to rice breeders because it is associated with spikelet sterility of F₁ hybrids in Indica/Japonica crosses. Identification of an S-5 allele which confers fertility in Indica/Japonica hybrids when introgressed into either the Indica or the Japonica parent has been reported. Varieties carrying this S-5 ⁿ allele are known as wide compatibility varieties (WCV). Our data suggests that RFLP marker RG213 on chromosome 6 is closely linked to the S-5 locus and can be efficiently used to identify wide compatibility (WC) lines. RG213 is a single-copy genomic clone that detects three bands of different molecular weights in DNA from Japonica (Akihikari) and Indica (IR36) varieties and WC line (Nekken 2). We demonstrate that the three alleles detected by this marker could be used to trace the inheritance of the wide compatible phenotype in breeders' material.     

具广亲和性的水稻隐性高秆细胞突变体

０２４２８ｈ系粳稻隐性高秆细胞突变体。来源于半矮秆广亲和种质０２４２８体细胞无性系变异。在１０３个Ｒ２代株系中,有１株系在抽穗后分离出高、矮秆两种类型,高秆比矮秆高６１．４％,系因例一和倒二节间伸长所致,以倒一节间伸长最为明显,占增长的６６．３％。在Ｒ３、Ｒ４代中矮秆类型继续分离出高秆和矮秆两在型,分离比为３：１,高秆类型未见分离。显示所分离的高秆是供体亲本０２４２８的单基因突变。暂名为０２４２８     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Variation in Anther Culture Response and Fertility of Backcrossed Hybrids between Indica and Japonica Rice (Oryza sativa)

This study was conducted to investigate the variations of fertility, callus induction and plant regeneration in backcrossed hybrids between a responsive japonica variety (Mankeumbyeo, recurrent parent) and a recalcitrant indica variety (Ranta Emas, donor parent) to anther culture. The mean spikelet fertility of inter-subspecific F₁ and BC₁F₁ hybrids was 33.5% and 67.2%, and the spikelet fertility of BC₁F₁ among backcrossed hybrids showed the most extensive variation (a low of 4.5% to a high of 90.6%). The mean fertility and distribution range in BC₅F₁ hybrids were almost the same as that of the japonica recurrent parent (94.6%). The mean callus induction of F₁ and BC₁F₁ hybrids was higher than that of donor parent, and the distribution range in BC₁F₁ hybrids varied from a low 0% to a high 18.7%. The mean callus induction and plant regeneration of BC₄F₁ hybrids was almost that of japonica recurrent parent, and there were no statistical differences between BC₄F₁ and BC₅F₁ hybrids. These results may help to accelerate the introgression of desirable traits from indica into japonica rice using anther culture of backcross hybrids as a breeding strategy.     

RFLP-based phylogenetic analysis of wide compatibility varieties in Oryza sativa L.

Twenty-one wide compatibility varieties (WCVs) of rice together with three indica and three japonica testers were assayed with 160 DNA probes that were selected to represent the entire RFLP map at an average interval of 11 cM. On the basis of four enzyme digestion 125 probes detected polymorphisms among the WCVs and subspecies' testers. Among these polymorphic probes there were 68 that could distinguish the indica from the japonica testers. Two dendrograms were constructed on the basis of 398 polymorphic fragments of 125 probes and 139 polymorphic fragments of 68 subspecies' differentiating probes in combination with single enzymes, respectively. The reliability and representativeness of the testers and the levels of DNA variations among WCVs were estimated. The potential of WCVs in the utilization of intersubspecific heterosis is discussed.     

Identification of simple sequence repeat markers for utilizing wide-compatibility genes in inter-subspecific hybrids in rice (Oryza sativa L.)

Although pronounced heterosis in inter-subspecific hybrids was known in rice for a long time, its utilization for hybrid rice breeding has been limited due to their hybrid sterility (HS). For the last two decades, however, a few inter-subspecific hybrids have been developed by incorporating wide-compatibility genes (WCG) that resolve HS, into parental lines of these inter-subspecific hybrids. For effective use of WCG, it is necessary to find convenient markers linked to WCG of practical importance. In this paper, initially a set of simple sequence repeat (SSR) markers in the vicinity of known WCG loci identified based on comparative linkage maps have been surveyed in a population derived from the three-way cross- IR36/Dular//Akihikari, where a known donor of WCG Dular was crossed to a representative indica and japonica cultivar. Of the five parental polymorphic markers, RM253 and RM276 were found to be closely linked to the WCG locus S5 at a distance of 3.0 and 2.8 cM, respectively. Later, loci for HS were examined in three F₂ populations derived from inter-subspecific crosses, with same set of SSR markers. The locus S8 was confirmed to have major influence on HS in the F₂ population derived from CHMRF-1/Taichung65 since two SSR markers in its vicinity, RM412 and RM141, co-segregated with HS at a map distance of 7.6 and 4.8 cM, respectively. In the F₂ population derived from the cross BPT5204/Taipei309, three SSR markers in the vicinity of S5, RM50, RM276 and RM136 co-segregated with HS at a map distance of 4.2, 3.2 and 7.8 cM, respectively. In the third F₂ population derived from Swarna/Taipei309, the SSR markers in the vicinity of S5, RM225, RM253, RM50, RM276 and RM136 were identified to co-segregate with HS at a map distance of 3.2, 2.6, 3.4, 2.6 and 6.6 cM, respectively. These results indicated a clear picture of WCG in Dular as well as the predominant role of HS alleles at S5 locus. The identified SSR markers are expected to be used for incorporation of WCG into parental lines in hybrid rice breeding to solve HS in inter-subspecific hybrids.S.P. Singh , R.M. Sundaram contributed equally     

Genetic variations in the hybrids of rice (Oryza sativa) and sorghum (Sorghum vulgare)

Summary Some of results from morphological and cytological observations and esterase-isozyme studies of a rice-sorghum hybrid are presented in this paper. There is a great diversity of morphological characters and some special characteristics in the progenies of the hybrids of rice with sorghum. The meiosis of pollen mother cells in the early generations of the hybrid was found to be abnormal. One main band coinciding with one found in sorghum but lacking in rice appeared in the majority of the hybrid lines. This band is characteristic of a are the specificities of the distant hybridization of rice and sorghum, and is rarely observed in the intervariety hybrids or hybrids between subspecies of Oryza sativa, indica and japonica. On the basis of these facts we concluded that the hybrids obtained are true hybrids of rice and sorghum.     

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts

Summary ⁶⁰Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile Fujiminori parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.     

Genetic and biochemical basis of scent in rice (Oryza sativa L.)

Summary The inheritance and biochemical basis of scent in rice was studied in the F₂ population along with the F₁ and its two parents, scented and non-scented Pokura rice strains. The F₁ plants were found to be nonscented while the F₂ plants seggregated into a 31 ratio (non-scented: scented). In scented F₂ seggregants and in the scented parental strain, a fast moving esterase isozyme, Rf 0.9, is missing whereas it is present in all nonscented F₂ seggregants, F₁s, and in the non-scented parent. This suggests that the absence of a specific esterase isozyme is associated with the scent character in rice.     

Inheritance of tolerance to rice tungro bacilliform virus (RTBV) in rice (Oryza sativa L.)

Summary From a large number of rice varieties tested, no variety was identified as resistant to tungro bacilliform virus (RTBV). Only in Utri Merah was the RTBV multiplication restrictive, whereas other varieties such as Kataribhog and Pankhari 203 were identified as tolerant. These varieties were crossed with a susceptible variety. TN1, to study the inheritance of restrictive multiplication and tolerance to RTBV. After 3 weeks of inoculation with RTBV, F₁; F₂, and F₃ progenies were assessed by enzyme-linked immunosorbent assay (ELISA). The RTBV concentration in all F₁ populations was intermediate between parents. The frequency distribution of F₂ seedlings with various levels of RTBV concentration indicated that the RTBV tolerance is controlled by multiple genes. The RTBV concentrations in F₁ and F₂ progenies from the Utri Merah x TN1 cross revealed that restrictive multiplication of RTBV in Utri Merah is a polygenic character. The continuous variation observed in F₂ populations from crosses between tolerant varieties and Utri merah indicated no allelic relationships between tolerant and restrictive multiplication traits.Part of PhD thesis submitted by senior author to University of Kebangsaan Malaysia, Bangi     

Genetic analysis of salinity tolerance in rice (Oryza sativa L.)

Summary The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant (Nona Bokra, Pokkali, and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron. Typical characteristics of salinity tolerance in rice were found to be Na⁺ exclusion and an increased absorption of K⁺ to maintain a good Na-K balance in the shoot. Genetic component analysis (GCA) revealed that a low Na-K ratio is governed by both additive and dominance gene effects. The trait exhibited overdominance, and two groups of genes were detected. Environmental effects were large, and the heritability of the trait was low. Our findings suggest that when breeding for salt tolerance, selection must be done in a later generation and under controlled conditions in order to minimize environmental effects. Modified bulk and single-seed descent would be the suitable breeding methods. Combining ability analysis revealed that both GCA and specific combining ability (SCA) effects were important in the genetics of salt tolerance. Moderately tolerant parents — e.g., IR4595-4-1-13 and IR9884-54-3-1E-P1 — were the best general combiners. Most of the best combinations had susceptible parents crossed either to moderate or tolerant parents. The presence of reciprocal effects among crosses necessitates the use of susceptible parents as males in hybridization programs. Large heterotic effects suggest the potential of hybrid rice for salt-affected lands.     

Chronopathological aspects of disease incidence in rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.)

Summary Two semidwarfism-related proteins, SRP-1 and SRP-2, were detected as major spots in a long-culm rice cultivar, Norin 29 and its semidwarf near-isogenic line, SC-TN1, respectively, by two-dimensional gel electrophoresis (2D-PAGE). The testcross showed that SRP-1 and SRP-2 are controlled by codominant alleles, Srp-1 and Srp-2, respectively, at a single locus Srp. This locus was considered to be closely linked with the semidwarfing gene locus sd-1. SRP-1 and SRP-2 were separated by 2D-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and sequenced by a gas-phase protein sequencer. The N-terminal amino acid sequences, however, could not be determined due to the blockage of the N-terminals of these proteins. After removal of the N-terminal residue with pyroglutamyl peptidase given to the membrane, the amino acid sequence in the N-terminal region was determined. The N-terminal and internal amino acid sequences of SRP-1 and SRP-2 were highly homologous with those of the glutelin -subunits of seed endosperm storage protein, which were deduced by the cDNA sequences. In the seed endosperms of Norin 29 and SC-TN1, a total of eight glutelin -subunits was identified by 2D-PAGE. The amino acid sequences in the N-terminal and internal regions of these proteins were determined. This experiment confirmed that SRP-1 and SRP-2 are almost identical in structure with the glutelin _5a- and _5b-subunits, respectively, which were identified in several organs such as endosperms, embryos, and leaves, unlike the other glutelin -subunits.     

Fine mapping of f5-Du, a gene conferring wide-compatibility for pollen fertility in inter-subspecific hybrids of rice (Oryza sativa L.)

Wide-compatibility varieties (WCVs), comprising a special class of rice germplasm, are able to produce fertile hybrids when crossed with both indica and japonica varieties. Dular, a landrace variety from India, has both a wide spectrum and high level of wide-compatibility when crossed with a range of indica and japonica varieties. In previous studies, an allele at the f5 locus from Dular (f5-Du) was identified as a neutral allele conferring wide-compatibility with a large effect on both pollen and spikelet fertility. Using a population of 1993 hybrid plants derived from a cross between ZS(f5-Du/f5-ZS) (F₁ of near isogenic line of Zhenshan 97 containing f5-Du) and Balilla (a japonica tester), f5-Du was genetically mapped to an interval of about 1.6 cM, with 0.8 cM from both SSR markers WFPM3 and WFPR1. Combined with bioinformatic analysis, a contig map was constructed for the f5 region, consisting of five bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones and spanning approximately 437 kb in length. By assaying the recombinant events in the region with markers developed using the sequence information, the f5 locus was further narrowed down to a 70 kb fragment containing nine predicted genes. The result will be very useful for cloning this gene and marker-assisted transferring of the neutral allele in rice breeding programs.     

Segregation distortion of isozyme loci in cherimoya (Annona cherimola Mill)

The allelic segregation of 13 isozyme loci in hand-fertilized heterozygous cherimoya trees (Annona cherimola Mill.) has been studied. We analyzed 63 locus x progeny combinations and found non-Mendelian segregation in 12 cases. The sequential Bonferroni method revealed only eight cases of non-Mendelian segregation; these have been investigated with several chi-square tests to discover what processes were involved. Gametic selection appears to be the main contributor, although zygotic selection seems also to play a part.     

Identification of mutable slender glume gene in rice (Oryza sativa L.).

The segregation pattern and chromosomal location of a slender glume mutation, induced by gamma-ray irradiation, was investigated. The mutation is genetically unstable: in the selfed progenies of slender glumed plants, not only plants with normal glumes but also plants that are chimeric for glume shape almost always appear at low frequency. The results showed that the mutation is controlled by a single recessive, mutable mutant gene slg. The frequency of reversion of slg to its wild-type state was little affected by crossing, backcrossing, genetic background or cytoplasmic factors. Conventional trisomic and linkage analyses revealed that the slg locus was located close to the rfs (rolled fine stripe leaf) locus on chromosome 7. In a subsequent RFLP analysis, slg was found to be located between the two RFLP loci XNpb20 and XNpb33, with recombination values of 3.0 and 3.2%, respectively. Southern analysis indicated that the mutability of slg is caused by none of the known transposable elements in rice. From these results, we infer that slg has a novel transposable DNA insert in its vicinity, which was possibly activated by gamma-ray irradiation. Received: 28 September 1998 / Accepted: 18 December 1998     

Genetic analysis of tolerance for phosphorous deficiency in rice (Oryza sativa L.)

The inheritance of phosphorous (P) — deficiency tolerance in rice was investigated by a sevenparent diallel. The parent materials involved were four P-efficient (IR20, IR54, IR28, and Mahsuri), one moderately P-efficient (TN1), and two P-inefficient (IR31406333-1 and IR34686-179-1-2-1), genotypes. Relative tilering ability (RTA) under P-deficient and P-supplemented soil conditions was the parameter used in determining the tolerance level of the different genotypes. Diallel graph analysis revealed that tolerant parents have an excess of recessive genes, while moderate and susceptible parents possess more dominant genes. Genetic-component analysis suggested that both additive and dominance gene effects are involved in the inheritance of P-deficiency tolerance in rice. The trait exhibited over doiminance as confirmed by the graphical analysis. Narrow-sense heritability of the trait was moderate (0.50) and environmental effects were low. Both the general combining ability (GCA) and the specific combining ability (SCA) were significant, but GCA was more prevalent than SCA. Tolerant parents exhibited a high GCA whereas susceptibles have a very poor GCA, suggesting that tolerant parents were mostly enriched in additive genes and susceptible parents in non-additive genes. Crosses involving two high general combiners showed low SCA effects whereas crosses between poor general combiners manifested highly-significant SCA values.     

Development of the endosperm in rice (Oryza sativa L.): Cellularization

The syncytial endosperm of rice undergoes cellularization according to a regular morphogenetic plan. At 3 days after pollination (dap) mitosis in the peripheral synctium ceases. Radial systems of microtubules emanating from interphase nuclei define nuclear-cytoplasmic domains (NCDs) which develop axes perpendicular, to the embryo sac wall. Free-growing anticlinal walls between adjacent NCDs compart-mentalize the cytoplasm into open-ended alveoli which are overtopped by syncytial cytoplasm adjacent to the central vacuole. At 4 dap, mitosis resumes as a wave originating adjacent to the vascular bundle. The spindles are oriented parallel to the alveolar walls and cell plates formed in association with interzonal phragmoplasts result in periclinal walls that cut off a peripheral layer of cells and an inner layer of alveoli displaced toward the center. Polarized growth of the newly formed alveoli and elongation of the anticlinal walls occurs during interphase. The next wave of cell division in the alveoli proceeds as the first and a second cylinder of cells is cut off inside the peripheral layer. The periods of polarized growth/anticlinal wall elongation alternating with periclinal cell division are repeated 3–4 times until the grain is filled by 5 dap.