Inheritance and mapping of male sterility restoration gene in upland japonica restorer lines

Several upland Japonica breeding lines, WAB450-11-1-3-P40-HB (Abbreviated as WAB450-11), WAB450-11-1-2-P61-HB (WAB450-13), WAB450-l-B-P-91-HB (WAB450-14), IRAT216, IRAT359, and IRAT104, possessing restoring ability for the Dian 1 type cms (cms-D) line Dianyu 1A were recently identified at Food Crops Research Institute, Yunnan Academy of Agricultural Sciences, P. R. China. In this study, the inheritance of restoring ability in these lines was characterized through the production of backcross populations to the male-sterile and maintainer Dianyu 1 lines. Each of the restorer lines was used to pollinate Dianyu 1A to form a F₁ hybrid which was then backcrossed (1) with Dianyu 1B producing a BC₁F₁ population and (2) to the female parent Dianyu 1A producing a BC₅F₂ population. The lines were also crossed with the japonica restorer line C57, carrying the restorer gene Rf1 that was introgressed from indica, to form F₁ hybrids, these hybrids were then testcrossed with Dianyu 1A to study the allelic relationship of their restorer genes to Rf1. The inheritance in these testcross populations indicated that the complete restoring ability of WAB450-11, WAB450-13, WAB450-14, IRAT216, IRAT359, and the partial restoring ability of IRAT104 were controlled by dominant genes, and the gene in WAB450-13, WAB450-14, and IRAT216 was allelic or identical to Rf1. When 136 SSR markers were used to score 143 BC₁F₁ individuals from Dianyu 1A/WAB450-13//Dianyu 1B, the japonica Rf1 allele was found to be located between RM171 and RM6100 on the long arm of chromosome 10, an interval corresponding to that known for the indica Rf1 allele. The distance between RM171 and Rf1 is 2.8 cM, and that between Rf1 and RM6100 is 4.9 cM. Similar linkage results were obtained from mapping 89 individuals of the corresponding BC₅F₂ population (Dianyu 1A/6/Dianyu 1A/WAB450-13).     

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.     

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 a fertility restorer gene for cytoplasmic male sterility in soybean

In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F₂ population derived from a single F₁ plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.     

Interaction between the eui gene and thermo-sensitive genic male sterility in rice

Panicle enclosure is a typical phenotype of almost all male-sterile rice lines. An elongated uppermost internode (eui) mutant exhibited notably rapid elongation of the uppermost internode at the heading stage; this is considered as a potential mechanism to eliminate panicle enclosure. We developed thermo-sensitive genic male-sterile (TGMS) eui mutants that were characterized by notably elongated uppermost internodes. The elongation of the uppermost internode in the TGMS eui mutant Changxuan 3S (CX) is mainly attributed to an increase in cell number and cell elongation, the latter being the more significant process. Temperature treatments revealed that the effects of temperature on panicle exsertion were similar to those on fertility and that the most temperature-sensitive stage coincides with the period from the formation of the pollen mother cell to meiosis during panicle initiation. These results indicate that elongation increases as temperature decreases and that the expression of the eui gene is more efficient at low temperatures than at high temperatures. In hybrid rice seed production using the TGMS eui mutant, the temperature range should be optimized at 24–28°C in order to preserve the development of completely male-sterile pollen and to eliminate panicle enclosure. Consequently, by using TGMS eui rice lines, gibberellin application can be avoided, thereby reducing the cost of hybrid seed production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of male sterility in rice mutants with environmentally influenced levels of fertility

Summary Twenty-six male sterile plants grown in the field were recovered in the M7 generation from ethyl methane sulfonate-treated material of the rice cultivar M-201. Fertility increased five-fold when ratooned plants from the field were grown in a growth chamber with a 12 hour daylength. Crosses between mutant and normal fertile cultivars produced fertile F₁ plants. Female fertility was normal as judged by percent seed set from unbagged panicles of parental and recombinant lines. Transgressive segregation for fertility was observed for all crosses in the F₂ and F₃ generations. Five of 37 F₃ male sterile plants showed moderate levels of seed fertility under winter greenhouse conditions and reduced seed set when transplanted to summer field plots. Fertility data from reciprocal crosses suggested cytoplasmic factors had little or no effect on levels of male sterility in the mutant lines. Chi-squared analyses of F₂ and F₃ generation results indicated male sterility of the mutants is conditioned by two nuclear genes with epistatic effects.     

Expression of thermosensitive genic male sterility in rice under varying temperature situations

Response of thermosensitive genic male sterility in rice to varying temperature situations was studied by using four TGMS lines. In three sets of maximum, minimum and their combined temperatures, it was observed that maximum temperature played a predominant role in influencing sterility/fertility of TGMS lines under the combined regimes. Expression of a TGMS gene was found to be influenced by the genetic background of the recipient lines. Exposure for more than 8 hours of 32 ^°C was found necessary to induce complete male sterility in indica TGMS lines: IR68945-4-33-4-14 and IR68949-11-5-31 while, more than 4 hours of such exposure was enough to induce sterility in case of their japonica donor Norin PL 12. Sudden interruption with 27 ^°C even for 2 hours under the sterility inducing regime of 32/24 ^°C could induce partial fertility in the line IR68945-4-33-4-14. However, the line ID 24 remained completely sterile even with 10 hours of interruption with 27 ^°C. An interruption with lower temperature of 27 ^°C for 4 hours for even one day induced partial fertility in IR68945-4-33-4-14. The period of four to eight days after panicle initiation was the stage most sensitive to temperature variations. Hybrid rice breeders need to develop numerous genetically diverse TGMS lines, which possess critical sterility inducing temperature of 28 ^°C and are not affected by sudden interruptions with a lower temperature for few hours daily for a couple of days. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.     

Molecular mapping of a new gene for resistance to rice blast (Pyricularia grisea Sacc.)

A single dominant blast resistance gene conferring resistance to a Korean rice blast isolate was identified in rice variety `Suweon 365'. We report the chromosomal localization and molecular mapping of this blast resistance gene designated as Pi-18, which confers resistance to Korean isolate `KI-313' of the blast pathogen. To know whether there is a relationship among genes conditioning resistance to location-specific isolates of the blast pathogen and thereby to identify linked markers to resistance gene for isolate KI-313 collected in Korea, RFLP markers previously reported to be linked to major blast resistance genes in different rice germplasm and other markers mapped to nearby regions were surveyed for polymorphism between a resistant (`Suweon 365') and a susceptible (`Chucheongbyeo') parent. Linkage associations of the RFLP markers with the resistance gene were verified using an F2 and F3 segregating population of known blast reaction. RFLP analysis showed that Pi-18 was located near the end of chromosome 11, linked to a single copy clone RZ536 at a distance of 5.4 centiMorgans (cM) and that this gene was different from Pi-1(t). An allelism test revealed that this gene was also different from Pi-k. Currently, a combination of RAPD and microsatellite primers is being employed to find additional markers in this region. Tightly linked DNA markers will facilitate selection for resistant genotypes in breeding programs and provide the basis for map based cloning of this new blast resistance gene. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of near-isogenic lines: an innovative approach,validated for root and shoot morphological characters in a mapping population of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Near-isogenic lines (NILs) constitute valuable tools in genetic investigations and plant breeding programs. Conventional methods for developing these are time consuming and tedious. An innovative method for identifying NILs is proposed and validated. The method involves computation of simple correlation coefficients of all possible pairs of genotypes within a mapping population using molecular marker data, and phenotypic characterization of those pairs with very high positive correlation. The pairs having both genomic and phenotypic similarity except for a single trait are considered as NILs. This strategy was tested with a doubled haploid mapping population involving CT9993 and IR62266. This population was saturated with 315 markers and comprised 154 lines. The pairs showing very high correlation coefficients (0.70–0.97) and differing for less than 10% of the markers were considered as Genotypically Closely Related Pairs (GCRPs). Graphical genotyping was employed to visualize the genome of the closely related lines. A total of 39 such pairs were subjected to rigorous evaluation for root and shoot morphological traits in two contrasting moisture regimes. Four GCRPs under well-watered condition and ten GCRPs under low moisture stress condition are statistically significant for a single phenotypic trait and are considered as NILs for their respective traits and would be the valuable materials for genetic studies. Mapped QTLs and candidate genes were employed to explain the probable cause of phenotypic difference in NILs.     

Chromosomal location of fertility restoring genes for ‘wild abortive’ cytoplasmic male sterility using primary trisomics in rice

Summary Identification and location of fertility restoring genes facilitates their deployment in a hybrid breeding program involving cytoplasmic male sterility (CMS) system. The study aimed to locate fertility restorer genes of CMSWA system on specific chromosomes of rice using primary trisomics of IR36 (restorer), CMS (IR58025A) and maintainer (IR58025B) lines. Primary trisomic series (Triplo 1 to 12) was crossed as maternal parent with the maintainer line IR58025B. The selected trisomic and disomic F₁ plants were testcrossed as male parents with the CMS line IR58025A. Plants in testcross families derived from disomic F₁ plants (Group I crosses) were all diploid; however, in the testcross families derived from trisomic F₁ plants (Group II crosses), some trisomic plants were observed. Diploid plants in all testcross families were analyzed for pollen fertility using 1% IKI stain. All testeross families from Group I crosses segregated in the ratio of 2 fertile: 1 partially fertile+partially sterile: 1 sterile plants indicating that fertility restoration was controlled by two independent dominant genes: one of the genes was stronger than the other. Testcross families from Group II crosses segregated in 2 fertile: 1 partially fertile+ partially sterile: 1 sterile plants in crosses involving Triplo 1, 4, 5, 6, 8, 9, 11 and 12, but families involving triplo 7 and triplo 10 showed significantly higher X² values, indicating that the two fertility restorer genes were located on chromosome 7 and 10. Stronger restorer gene (Rf-WA-1) was located on chromosome 7 and weaker restorer gene (Rf-WA-2) was located on chromosome 10. These findings should facilitate tagging of these genes with molecular markers with the ultimate aim to practice marker-aided selection for fertility restoration ability.     

Characterization and fine mapping of thermo-sensitive chlorophyll deficit mutant1 in rice (Oryza sativa L.)

Chlorophyll content is one of the most important traits controlling crop biomass and economic yield in rice. Here, we isolated a spontaneous rice mutant named thermo-sensitive chlorophyll deficit 1 (tscd1) derived from a backcross recombinant inbred line population. tscd1 plants grown normally from the seedling to tiller stages showed yellow leaves with reduced chlorophyll content, but showed no significant differences after the booting stage. At temperatures below 22°C, the tscd1 mutant showed the most obvious yellowish phenotype. With increasing temperature, the yellowish leaves gradually turned green and approached a normal wild type color. Wild type and tscd1 mutant plants had obviously different chloroplast structures and photosynthetic pigment precursor contents, which resulted in underdevelopment of chloroplasts and a yellowish phenotype in tscd1. Genetic analysis indicated that the mutant character was controlled by a recessive nuclear gene. Through map-based cloning, we located the tscd1 gene in a 34.95 kb region on the long arm of chromosome 2, containing two BAC clones and eight predicted candidate genes. Further characterization of the tscd1 gene is underway. Because it has a chlorophyll deficit phenotype before the tiller stage and little influence on growth vigor, it may play a role in ensuring the purity of hybrids.     

Genetic analysis and fine mapping of LH1 and LH2, a set of complementary genes controlling late heading in rice (Oryza sativa L.)

Heading date in rice (Oryza sativa L.) is a critical agronomic trait with a complex inheritance. To investigate the genetic basis and mechanism of gene interaction in heading date, we conducted genetic analysis on segregation populations derived from crosses among the indica cultivars Bo B, Yuefeng B and Baoxuan 2. A set of dominant complementary genes controlling late heading, designated LH1 and LH2, were detected by molecular marker mapping. Genetic analysis revealed that Baoxuan 2 contains both dominant genes, while Bo B and Yuefeng B each possess either LH1 or LH2. Using larger populations with segregant ratios of 3 : 1, we fine-mapped LH1 to a 63-kb region near the centromere of chromosome 7 flanked by markers RM5436 and RM8034, and LH2 to a 177-kb region on the short arm of chromosome 8 between flanking markers Indel22468-3 and RM25. Some candidate genes were identified through sequencing of Bo B and Yuefeng B in these target regions. Our work provides a solid foundation for further study on gene interaction in heading date and has application in marker-assisted breeding of photosensitive hybrid rice in China.     

Yield of F1, F2 and F3 hybrids of rice (Oryza sativa L.)

Summary Yield and yield components of F₁ hybrids were studied in three experiments at 30×30 cm spacings and in one experiment at 15×15 cm spacings. In the 30×30 cm experiments, 10 of the 41 hybrids tested significantly outyielded their high parents. However, only 2 hybrids significantly outyielded the best cultivar: one hybrid yielded 23 % and the other 16 % more than their respective check cultivars. The four hybrids in the 15×15 cm experiment yielded only 59 to 92% as much as their high parents.In areas where rice is transplanted at relatively wide spacings, the observed levels of F₁ heterosis in selected hybrids may be sufficient to warrant production of hybrid rice, if enough hybrid seed can be produced. For direct-seeding at the high rates normal in the USA, the relatively small levels of heterosis and the difficulties of hybrid seed production preclude use of F₁ hybrid rice cultivars at present.None of 19 bulk F₂ and F₃ hybrids in two experiments yielded significantly more than its high parent. Similarly, none of the 12 mixtures included in one experiment yielded significantly more than its high parent. On the basis of yield alone, using bulk F₂ or F₃ or simple mixture populations is not merited.Contribution from the Western Region, Agricultural Research Service, U.S. Department of Agriculture, and the Department of Agronomy and Range Science, University of California, Davis, California 95616.     

Inheritance of resistance to rice stripe virus in rice line `BL 1'

Rice stripe is the most serious virus disease in temperate rice-growing countries. The most economical and environmentally safe practice for controlling this disease is virus-resistant cultivars. ‘BL 1’ is an elite germplasm line with the blast resistance gene Pib, and has been used as a differential line for testing the pathogenicity of the blast fungus. We found that certain progenies from BL 1 showed resistance to both blast and rice stripe virus (RSV). The objectives of this study were to evaluate the RSV resistance in the field and under artificial conditions, to assess the reaction to the insect vector(small brown plant hopper, SBPH), and to examine its inheritance and its relationship to blast resistance in BL 1.BL 1 was susceptible to SBPH, but resistant to RSV in field and artificial inoculation tests. The inheritance of RSV resistance in F₃ lines from the cross Nipponbare (NPB)/BL 1 was studied using artificial inoculation with a population of viruliferous SBPH. A serological assay for RSV infection using an enzyme-linked immunosorbent assay (ELISA) was used. RSV resistance in BL 1 was controlled by a single major gene with incomplete dominance. The locus responsible for RSV resistance was genetically independent of the blast resistance gene Pib. The resistance gene for RSV infection in BL 1 was also independent of Stvb-i, a gene widely distributed in resistant Japanese cultivars. Resistance to RSV must be diversified in rice cultivars considering the potential for future emergence of new RSV strains. The new resistance gene identified in BL 1, which has improved plant type and blast resistance, is considered useful for breeding RSV-resistant cultivars in japonica rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new gene for resistance in rice to Asian rice gall midge (Orseolia oryzae Wood Mason) biotype 1 population at Raipur, India

The Asian rice gall midge, Orseolia oryzae Wood Mason (Diptera: Cecidomyiidae), is a major pest of rice in several South and South East Asian countries. The maggots feed internally on the growing tips of the tillers and transform them into tubular galls, onion leaf-like structures called ‘silver shoots’ resulting into severe yield loss to the rice crop. We studied the mode of inheritance and allelic relationships of the resistance genes involved in resistant donor Line 9, a sib of a susceptible cultivar ‘Madhuri’. The segregation behaviour of F₁, F₂ and F₃ populations of the cross between Line 9 and susceptible cultivar MW10 confirmed the presence of a single dominant gene for resistance. Tests of allelism with all the known genes giving resistance to this population indicated that Line 9 possessed a new gene which was designated Gm 9 This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytological observations in some hybrids between the rice species Oryza sativa L. and O. glaberrima Steud.

Summary Several hybrids between Oryza sativa and O. glaberrima and their backcrosses with O. sativa were studied. Their seed sterility was very different; large differences were also observed in the level of pollen sterility and in the earliness of microspore failure. The proportion of aborted embryo sacs was much lower than the rate of sterile male gametophytes. The backcross populations were much more sterile than the corresponding F₁ hybrids. On the base of our observations and according to the literature, we may conclude that genic unbalance is the main cause of sterility of these hybrids, but that physiological factors may also be involved. Thus a restoration of fertility is generally possible by selection. On the other hand, male-sterile lines could be bred from some of these hybrids.     

RFLP/AFLP mapping of a brown planthopper (Nilaparvata lugens Stål) resistance gene Bph1 in rice

We have constructed a linkage map of the rice brown planthopper (BPH)resistance gene, Bph1. RFLP and AFLP markers were selected by thebulked segregant analysis and used in the mapping study of 262 F₂sthat were derived from a cross of `Tsukushibare', a susceptible japonica cultivar, and `Norin-PL3', an authentic japonicaBph1-introgression line. Twenty markers were mapped within a 28.9-cMregion containing the Bph1 locus on the long arm of rice chromosome12. Combining the result of segregation analysis of BPH resistance by themass seedling test and that of the markers, the Bph1 locus wasmapped within a 5.8-cM region between two flanking markers. The closestAFLP markers, em5814N and em2802N, was at 2.7 cM proximal to theBph1 locus. Together with the previously constructed high-resolutionmap of bph2 locating the locus at ca. 10 cM proximal to the Bph1 locus, this improved version of the linkage map would facilitatepyramiding these two important BPH resistance genes.