Developing rice lines possessing neutral alleles at sterility loci to improve the width of compatibility

To improve the width of compatibility for overcoming various sterilities in inter‐subspecific hybrid rice, some elite lines combining several sterility‐neutral genes were developed and the effects on mitigating various hybrid sterilities were tested. From Akihikari// IR36/Dular, neutral genes at ga11 and six sterility loci, S5, S7, S8, S9, S15 and S16, were combined and elite lines were obtained in their successive progeny. Four of the lines tested were confirmed to combine the neutral alleles S5‐n, S7‐n, S8‐n, S9‐n, S15‐n and S16‐n at the sterility loci and, among them, two harboured an additional gamete abortion‐neutral allele, ga11‐n. F₁s, which used the lines and various testers as parents, mitigated the spikelet sterilities by six sterility loci and gamete abortion by a gametophyte gene, ga11. These lines could be selectively used as parents or donors to increase the width of compatibility of rice varieties for improving fertility in inter‐subspecific hybrid rice breeding.     

Genic analysis of hybrid sterility caused by anther indehiscence between distantly related rice varieties

Hybrid sterility of the cross between Silewah, an Indonesian native variety and Hayakogane, a Hokkaido rice variety in Japan was confirmed to be caused by anther indehiscence, based on a lot of spikelets with fewer numbers than 3 of dehiscent anthers and fewer numbers of pollens poured onto stigma per spikelet in Silewah/Hayakogane F1. In Silewah/Hayakogane//Hayakogane BC₁F₁, spikelet fertility was correlated with mean number of dehiscent anthers per spikelet. So, genic analyses for hybrid sterility by anther indehiscence were conducted by scoring spikelet sterility in F₂, BC₁F₁s and triple cross. As a result, it was concluded that the hybrid sterility was controlled by complementary action of three dominant genes. Silewah has one of the three genes and Hayakogane has the other two. It was presumed that the two genes which Hayakogane possesses were derived from those of Aikoku, an old native variety in Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of two types of differentiation in cultivated rice (Oryza sativa L.) detected by polymorphism of isozymes and hybrid sterility

Magnitudes of genetic variation within each of major varietal groups of cultivated rice were surveyed in terms of isozyme polymorphism and allelic differentiation of hybrid sterility loci, both of which are considered to have litt le selective value. Allelic differences for 20 isozyme loci were examined in a total of 337 accessions, including Indica and Japonica rices. Aus cultivars from India, Javanica cultivars and both landraces and cultivars from China. Eleven out of the 20 isozyme loci were polymorphic. The Aus cultivars contained more alleles per locus than others. The hybrid sterilities in the crosses among Chinese cultivars, Indica and Japonica cultivars were mainly controlled by locus S-5, whereas the hybrid sterilities of Aus cultivars, when they were crossed to Indica, Japonica or Javanica cultivars, were found to be controlled by allelic interaction at hybrid sterility loci S-5, S-7, S-9 and S-15. Also in terms of the number of alleles at S-5 and S-7, Aus cultivars contained more alleles than others. While the Aus group showed an extreme diversity for both hybrid sterility alleles and isozymes, modern cultivars from the south of YangZi River in China were classified into Indica type and those from the north were into Japonica, which were almost the same as those in Japan. On the basis of the measured polymorphism the Indica-Japonica differentiation was explained by founder effects, i.e., through selection and distribution of two original sources each with a unique set of markers. Contrastingly, the continuous and pronounced diversity in the Aus group was attributed to the absence of such a process. The intermediate groups in Yunnan province and Tai-hu Lake region of China are considered to be isolated from such founder effects, retaining an intermediate diversity in terms of isozymes and hybrid sterility genes. Since hybrid sterility hampers the exchange of genes between cultivars of different groups, the understanding of its genetic basis will be important in rice breeding, particularly in hybrid rice breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.     

Molecular mapping of a new genic male-sterility gene causing chalky endosperm in rice ( Oryza sativa L.)

A genic male-sterility gene newly induced by chemical mutagenesis, tentatively designated as ms-h(t), was located on the molecular map of rice and tested for its effect on chalky endosperm. Bulked segregant analysis was used to determine the chromosomal location of the ms-h(t) locus by screening four to five RFLP markers per chromosome. After confirming that the gene was located on chromosome 9, twenty-four RFLP markers from chromosome 9 were surveyed for polymorphism in the parents of the mapping population. Of these, eleven markers were mapped around the ms- h(t) locus. RG451 and RZ404 flanked the ms-h(t) gene, at 2.5cM and 3.3cM, respectively. Heterozygous F2 to F4 progenies were tested for co- segregation of male-sterility and chalky endosperm and it was revealed that ms-h(t) might have a pleiotropic effect on chalky endosperm. This mutant would be a good biological material to characterize the biochemical mechanism of male sterility and related pleiotropic effects. Further studies should be needed to know the usefulness of this mutant for hybrid seed production. This revised version was published online in July 2006 with corrections to the Cover Date.     

A genetic analysis of low-temperature-sensitive sterility in indica-japonica rice hybrids

Low-temperature-sensitive sterility has become one of the major obstacles in indica–japonica hybrid rice breeding. The objectives of this paper were to evaluate the extent of the fertility reduction and to determine the genetic basis of low-temperature-sensitive sterility. Seventeen varieties were crossed in various ways to produce 21 F₁s including 16 indica-japonica hybrids. Fertility of the F₁s and their parents was examined under both high and low temperature conditions. Considerable reduction in spikelet fertility was observed under low-temperature conditions in the majority of the indica–japonica hybrids having at least one wide compatibility parent. However, the extent of fertility reduction varied greatly, depending on the parental genotypes. Data from five pairs of reciprocal crosses indicated that the cytoplasm had no effect on fertility reduction. The more-or-less bimodal distribution of the fertility segregation of one BC₁F₁ and two F₂: populations under low-temperature conditions suggested that the low-temperature-sensitive sterility was controlled by only one or a few genes. It was also shown that the low-temperature-sensitivity is not related to wide compatibility. We conclude that it is possible to develop indica-japonica hybrids with wide compatibility and also insensitivity to the low-temperature conditions.     

An analysis of hybrid sterility in rice using a diallel cross of 21 parents involving indica, japonica and wide compatibility varieties

Summary There is much interest in the inter-varietal fertility of Asian cultivated rice. In this study, we analyzed the fertility of hybrids in a diallel set of 210 crosses involving 21 parents representing a broad range of the cultivated rice germplasm including landraces, primitive cultivars, modern elite cultivars and parents of hybrid rice. The materials were also carefully selected with respect to indica-japonica classification including typical indica/japonica, more or less intermediate and wide compatibility varieties. The level of hybrid fertility varied widely among the crosses from almost completely sterile to fully fertile. In general, hybrid fertility of intra-subspecific crosses, i.e., indica by indica (I×I) and japonica by japonica (J×J), is much higher than inter-subspecific crosses (I×J or J×I). The fertility varied widely in hybrids involving wide compatibility varieties and also in inter-subspecific crosses not involving wide compatibility varieties. An analysis of variance showed that both the main effects of indica and japonica parents and the interaction between the parents are highly significant in determining hybrid fertility. We speculate that, in addition to wide compatibility, the overall genetic difference between the indica and japonica parents, that may involve a series of minor mutations, play an important role in determining the hybrid fertility. There are also genes of sizable effect that influence fertility in hybrids resulting from specific combinations of the parents.Abbreviations I×I indica by indica cross - I×J indica by japonica cross - J×I japonica by indica cross - J×J japonica by japonica cross - WCV wide compatibility variety     

Identification and characterization of new sources of cytoplasmic male sterility in rice

Search for male sterility-inducing cytoplasm in wild species of the genus Oryza was attempted with a view to diversify the base of the cytoplasmic genetic male sterility system currently used in the development of commercial rice hybrids. A total of 132 interspecific crosses were made involving accessions of four wild and two cultivated species, all belonging to the A genome. Wild accessions possessing sterility-inducing cytoplasms were identified following reciprocal and sterile F₂ backcross methods. Sterile segregants were pursued through substitution backcrosses to develop cytoplasmic male-sterile (CMS) lines. CMS lines were developed with the cytoplasm of either O. rufipogon (VNI) or O. nivara (DRW 21018, DRW 21001, DRW 21039, DRW 21030 and RPW 21111). Based on shape, staining, and abortive pattern of pollen and also on type of interaction with a set of restorers and maintainers for known cytoplasmic male steriles of WA source (V 20A), the newly-developed CMS lines were grouped into four classes. Of these, RPMS 1 and RPMS 2 showed gametophytic male sterility with a restorer reaction different from WA cytoplasmic male-sterile stocks.     

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F₁ pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-c ^j, and its isogenic line TISL5, carrying alleleS-c ^j. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F₂ plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F₂ ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F₁ hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Wide compatibility in rice (Oryza sativa L.)

Summary Wide compatible varieties (WCVs) show normal spikelet fertility in crosses with Indica and Japonica rice varieties. Crosses of Indica and Japonica varieties frequently show high spikelet sterility which prevents exploitation of heterosis for grain yield. We screened 41 rice varieties for the wide compatibility trait by crossing each with three Indica and three Japonica testers. Varieties giving fertile F₁ hybrids with both groups of testers were classified as WCVs. Seven varieties viz., BPI-76 (Indica); N 22; Lambayeque-1 and Dular (Aus); Moroberekan, Palawan and Fossa HV (Japonicas), were identified as WCVs. The frequency of WCVs was higher among Aus and Japonicas. The wide compatibility trait in varieties: Dular and Moroberekan was controlled by a single dominant gene linked with the Est-2 and Amp-3 loci (mean recombination 32.0%). Est-2 and Amp-3 showed complete linkage. Pgi-2 was found to be linked with Est-2 and Amp-3 (mean recombination 16.1%). Est-2 and Amp-3, showed a tighter linkage with C ⁺ (mean recombination 4.1%). Pgi-2 showed a lower linkage with C ⁺ (mean recombination 17.3%). The recombination values between the WC gene in Dular and C ⁺ was much higher than those reported in Japan for the WC gene (S₅ ⁿ) from Ketan Nangka. It is possible that the WC gene from Dular is different from that in Ketan Nangka. Linkage intensities with the WC gene were not strong enough to be of use for indirect selection for the wide compatibility trait. A search for a more closely linked isozyme or DNA marker was proposed.     

Chromosomal location of Hwc2, one of the complementary hybrid weakness genes, in rice

A hybrid weakness phenomenon is controlled by a set of complementary genes, Hwc1 (hybrid weakness c) and Hwc2, in rice. The Hwc2 gene is prevalent among temperate Japonica but not among tropical Japonica or Indica. In this study, the chromosomal location of the Hwc2 locus was determined from the segregation in the F₁ hybrids made between 127 recombinant inbred lines and the cultivar ‘Jamaica’. Hwc2 was located between the two restriction fragment length polymorphism loci, XNpb264 and XNpb197 on chromosome 4. Further analysis indicated that Hwc2 was closely linked to Ph (phenol staining). The genetic and phylogenetic significance of the Hwc2 locus and the surrounding chromosomal region is discussed.     

Mapping and genetic analysis of two fertility restorer loci in the wild-abortive cytoplasmic male sterility system of rice (Oryza sativa L.)

Hybrid varieties developed by making use of the wild abortive cytoplasmic male sterility system account for 90% of hybrid rice produced. Previous inheritance studies have established that the fertility restoration in this system is controlled by two major loci, but the chromosomal locations of the fertility restorer (Rf) loci have yet to be resolved. In this study we determined the genomic locations of the two Rf loci by their linkage to molecular markers. The Rf gene containing regions were identified by surveying two bulks, made of 30 highly fertile and 46 highly sterile plants from a large F₂ population of the cross between Zhenshan 97A and Minghui 63, with RFLP markers covering the entire rice genome. The survey identified two likely Rf gene containing regions, located on chromosomes 1 and 10 respectively. This was confirmed by ANOVA using a large random sample from the same F₂ population and also with a genome-wide QTL analysis of a test-cross population. The results also showed that both loci have major effects of almost complete dominance on fertility restoration and the effect of the locus on chromosome 10 is larger than the one on chromosome 1. The two loci acted as a pair of classical duplicate genes; a single dominant allele at one of the two loci would suffice to restore the fertility to normal or nearly normal. Closely linked markers identified in this study may be used for marker assisted selection in hybrid rice breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetics of thermosensitive genic male sterility in rice

Summary Inheritance of thermosensitive genic male sterility (TGMS) in Norin PL12 and IR32364TGMS and their allelic relationship were studied from F₁, F₂ testcross (TC) and F₃ generations of the crosses made with the two mutants and several fertile tester parents. F₂, TC and F₃ segregation behavior for pollen and spikelet fertility indicated that the TGMS trait in the two mutants was controlled by a single recessive gene. Allelic relationship studies indicated that TGMS genes of the two mutants were different. Since TGMS gene in Norin PL12 has been designated as tms ₂ , the TGMS gene present in IR32364TGMS is tentatively designated as tms ₃ (t) until allelic test is done with another TGMS gene (tms ₁ ) reported from China in a line 5460S seeds of which were not available.     

QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

Genetic diversity among parental lines of Indica hybrid rice (Oryza sativa L.) in China based on coefficient of parentage

Genetic diversity constitutes the raw material for plant improvement, and provides protection against genetic vulnerability to biotic and abiotic stresses. Diversity of parental lines of indica hybrid rice in China is not well‐characterized. The major objective of this study was to quantify genetic diversity of Chinese parental lines of hybrid rice via coefficient of parentage (COP). All 100 parental lines of hybrid rice widely used in hybrid breeding and commercial production during 1976–2003 were studied by COP analysis. The mean COP for the 100 parental lines was low (0.056), indicating a potentially high degree of diversity in Chinese hybrid rice breeding. Forty‐nine percent of all pairs of parental lines were completely unrelated by pedigree data. The low mean COP for the parental lines was attributed to a continual incorporation of exotic germplasm (wild rice, japonica and javanica etc.) into the genetic base over time, to the introduction of foreign germplasm from the Philippines (International Rice Research Institute), Korea, the United States, Thailand, and Guyana as breeding stock. The mean COP from 1976 to 1990 was twice as much as that from 1990 to 2003. Cluster analysis was an effective method to discriminate diversity, ten clusters were identified, and maintainer lines, restorer lines and other parental lines with special genetic background were clearly grouped. In addition, restorer lines were further divided into 11 sub‐clusters, which basically was in agreement with hybrid rice breeding. Among ten provinces, Hunan, Sichuan and Fujian were outstanding for breeding 54 of 100 parental lines in hybrid rice production, and the genetic diversity of parental lines in Fujian, Sichuan,Guangxi, Hunan and Jiangsu were all narrower than that in Hubei, Guangdong, Zhejiang and Jiangxi. The result of coefficient of parentage analysis for 100 parental lines may promote the management of parental diversity and hybrid rice breeding in China.     

Relationship between molecular marker heterozygosity and hybrid performance in intra- and inter-subspecific crosses of rice

The objective of this study was to investigate the relationship between molecular marker diversity and heterosis in both intra-and inter-sub-specific hybrids of rice to evaluate the feasibility of predicting hybrid performance using molecular markers. Eleven elite lines were intermated resulting in a diallel set including 10 indica × indica, 15 japonica × japonica and 30 indica × japonica crosses. The F₁ hybrids and parents were evaluated for agronomic performance in a replicated field trial. The parental lines were tested for DNA polymorphisms with 113 restriction fragment length polymorphism (RFLP) probes covering the 12 rice chromosomes. Inter-subspecific crosses showed better performance and higher heterosis than intrasubspecific hybrids. Correlations of marker heterozygosity with hybrid performance and heterosis differed considerably between the two subspecies; they were higher in crosses within japonica subspecies than within indica subspecies. Very little correlation was detected in intersubspecific crosses. It was concluded that relationships between marker heterozygosity and hybrid performance were complex owing to germplasm diversity and the complexity of the genetic basis of heterosis. The implications of the results in predicting hybrid performance using molecular markers are discussed.     

Identification of a major blast resistance gene in the rice cultivar 'Tetep'

Analysis of near‐isogenic lines (NILs) indicated the presence of a novel resistance gene in the indica rice cultivar ‘Tetep’ which was highly resistant to the rice blast fungus Magnaporthe grisea.‘Tetep’ was crossed to the widely used susceptible cultivar ‘CO39’ to generate the mapping population. A Mendelian segregation ratio of 3 : 1 for resistant to susceptible F₂ plants further confirmed the presence of a major dominant locus, in ‘Tetep’, conferring resistance to the blast fungal isolate B157, corresponding to the international race IC9. Simple sequence length polymorphism (SSLP) was used for molecular genetic analysis. The analysis revealed that the SSLP marker RM 246 was linked to a novel blast resistance gene designated Pi‐tp(t) in ‘Tetep’.