Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

Molecular mapping and location of QTLs for drought-resistance traits in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) lines adapted to target environments

Drought is a major limitation for rice production in rainfed ecosystems. Identifying quantitative trait loci (QTLs) linked to drought resistance provides opportunity to breed high yielding rice varieties suitable for drought-prone areas. Although considerable efforts were made in mapping QTLs associated with drought-resistance traits in rice, most of the studies involved indica × japonica crosses and hence, the drought-resistance alleles were contributed mostly by japonica ecotypes. It is desirable to look for genetic variation within indica ecotypes adapted to target environment (TE) as the alleles from japonica ecotype may not be expressed under lowland conditions. A subset of 250 recombinant inbred lines (RILs) of F₈ generation derived from two indica rice lines (IR20 and Nootripathu) with contrasting drought-resistance traits were used to map the QTLs for morpho-physiological and plant production traits under drought stress in the field in TE. A genetic linkage map was constructed using 101 polymorphic PCR-based markers distributed over the 12 chromosomes covering a total length of 1,529 cM in 17 linkage groups with an average distance of 15.1 cM. Composite interval mapping analysis identified 22 QTLs, which individually explained 4.8–32.2% of the phenotypic variation. Consistent QTLs for drought-resistance traits were detected using locally adapted indica ecotypes, which may be useful for rainfed rice improvement.     

Fine mapping of <Emphasis Type="Italic">qSTV11</Emphasis><Superscript><Emphasis Type="Italic">KAS</Emphasis></Superscript>, a major QTL for rice stripe disease resistance

Rice stripe disease, caused by rice stripe virus (RSV), is one of the most serious diseases in temperate rice-growing areas. In the present study, we performed quantitative trait locus (QTL) analysis for RSV resistance using 98 backcross inbred lines derived from the cross between the highly resistant variety, Kasalath, and the highly susceptible variety, Nipponbare. Under artificial inoculation in the greenhouse, two QTLs for RSV resistance, designated qSTV7 and qSTV11 ^KAS , were detected on chromosomes 7 and 11 respectively, whereas only one QTL was detected in the same location of chromosome 11 under natural inoculation in the field. The stability of qSTV11 ^KAS was validated using 39 established chromosome segment substitution lines. Fine mapping of qSTV11 ^KAS was carried out using 372 BC₃F_2:3 recombinants and 399 BC₃F_3:4 lines selected from 7,018 BC₃F₂ plants of the cross SL-234/Koshihikari. The qSTV11 ^KAS was localized to a 39.2 kb region containing seven annotated genes. The most likely candidate gene, LOC_Os11g30910, is predicted to encode a sulfotransferase domain-containing protein. The predicted protein encoded by the Kasalath allele differs from Nipponbare by a single amino acid substitution and the deletion of two amino acids within the sulfotransferase domain. Marker-resistance association analysis revealed that the markers L104-155 bp and R48-194 bp were highly correlated with RSV resistance in the 148 landrace varieties. These results provide a basis for the cloning of qSTV11 ^KAS , and the markers may be used for molecular breeding of RSV resistant rice varieties.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Susceptibility to <Emphasis Type="Italic">Fusarium </Emphasis>head blight is associated with the <Emphasis Type="Italic">Rht-D1b</Emphasis> semi-dwarfing allele in wheat

Fusarium head blight (FHB) is an important disease of wheat worldwide. The cultivar Spark is more resistant than most other UK winter wheat varieties but the genetic basis for this is not known. A mapping population from a cross between Spark and the FHB susceptible variety Rialto was used to identify quantitative trait loci (QTL) associated with resistance. QTL analysis across environments revealed nine QTL for FHB resistance and four QTL for plant height (PH). One FHB QTL was coincident with the Rht-1D locus and accounted for up to 51% of the phenotypic variance. The enhanced FHB susceptibility associated with Rht-D1b is not an effect of PH per se as other QTL for height segregating in this population have no influence on susceptibility. Experiments with near-isogenic lines supported the association between susceptibility and the Rht-D1b allele conferring the semi-dwarf habit. Our results demonstrate that lines carrying the Rht-1Db semi-dwarfing allele are compromised in resistance to initial infection (type I resistance) while being unaffected in resistance to spread within the spike (type II resistance).     

Fine mapping of <Emphasis Type="Italic">S31</Emphasis>, a gene responsible for hybrid embryo-sac abortion in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Partial abortion of female gametes and the resulting semi-sterility of indica × japonica inter-subspecific rice hybrids have been ascribed to an allelic interaction, which can be avoided by the use of wide compatibility varieties. To further understand the genetic mechanism of hybrid sterility, we have constructed two sets of hybrids, using as male parent either the typical japonica variety Asominori, or the wide compatibility variety 02428; and as female, a set of 66 chromosome segment substitution lines in which various chromosomal segments from the indica variety IR24 have been introduced into a common genetic background of Asominori. Spikelet semi-sterility was observed in hybrid between CSSL34 and Asominori, which is known to carry the sterility gene S31 (Zhao et al. in Euphytica 151:331–337, 2006). Cytological analysis revealed that the semi-sterility of the CSSL34 × Asominori hybrid was caused primarily by partial abortion of the embryo sac at the stage of the mitosis of the functional megaspore. A population of 1,630 progeny of the three-way cross (CSSL34 × 02428) × Asominori was developed to map S31. Based on the physical location of linked molecular markers, S31 was thereby delimited to a 54-kb region on rice chromsome 5. This fragment contains eight predicted open reading frames, four of which encode known proteins and four putative proteins. These results are relevant to the map-based cloning of S31, and the development of marker-assisted transfer of non-sterility allele inducing alleles to breeding germplasm, to allow for a more efficient exploitation of heterosis in hybrid rice.     

Delimitation of the <Emphasis Type="Italic">fgr</Emphasis> gene for rice fragrance to a 28-kb DNA fragment

The fragrance gene plays an important role in high-quality rice varieties and has been widely used in breeding programs. Using a random sample of 370 individuals from an F₂ segregating population developed from a cross between a japonica rice variety 9407 with fragrant flavor and an indica variety IRBB60, the fgr locus was mapped on chromosome 8 between SSR markers, PSM465 and RM1109, with genetic distances of 0.3 cM and 0.1 cM to respective markers. These mapping efforts confirmed the previous mapping results. A large F₃ mapping population with 7300 individuals was then developed from F₂ plants, in which a small chromosomal region defined by the SSR markers, PSM465 and RM1109, was heterozygous. The analysis of recombinants in the fgr region anchored the gene locus to an interval of 28 kb flanked by the left marker NS9 and the right marker L06. Sequence analysis of this fragment predicted three open reading frames encoding putative 3-methylcrotonyl-CoA carboxylase, putative isoleucyl-tRNA synthetase, and betaine aldehyde dehydrogenase (BADH2). The latter was presumed to be the candidate gene for fragrance. This result will be very useful in molecular cloning of the fgr gene and marker-assisted transfer of the fgr gene in rice breeding programs. Published in Russian in Fiziologiya Rastenii, 2009, vol. 56, No. 4, pp. 587–595. This text was submitted by the authors in English.     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Development of Chromosome Segment Substitution Lines Derived from Backcross between Two Sequenced Rice Cultivars, <Emphasis Type="Italic">Indica</Emphasis> Recipient 93-11 and <Emphasis Type="Italic">Japonica</Emphasis> Donor Nipponbare

Chromosome segment substitution lines (CSSLs) are powerful tools for detecting and precisely mapping quantitative trait loci (QTLs) and evaluating gene action as a single factor. In this study, 103 CSSLs were produced using two sequenced rice cultivars: 93-11, an elite restorer indica cultivar as recipient, and Nipponbare, a japonica cultivar, as donor. Each CSSL carried a single chromosome substituted segment. The total length of the substituted segments in the CSSLs was 2,590.6 cM, which was 1.7 times of the rice genome. To evaluate the potential application of these CSSLs for QTL detection, phenotypic variations of seed shattering, grain length and grain width in 10 CSSLs were observed. Two QTLs for seed shattering and three for grain length and grain width were identified and mapped on rice chromosomes. The results demonstrate that CSSLs are excellent genetic materials for dissecting complex traits into a set of monogenic loci. These CSSLs are of great potential value for QTL mapping and plant marker-assisted breeding (MAB).     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.     

Fine Mapping QTL for Drought Resistance Traits in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Using Bulk Segregant Analysis

Drought stress is a major limitation to rice (Oryza sativa L.) yields and its stability, especially in rainfed conditions. Developing rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. Mapping quantitative trait loci (QTLs) linked to drought resistance traits will help to develop rice cultivars suitable for water-limited environments through molecular marker-assisted selection (MAS) strategy. However, QTL mapping is usually carried out by genotyping large number of progenies, which is labour-intensive, time-consuming and cost-ineffective. Bulk segregant analysis (BSA) serves as an affordable strategy for mapping large effect QTLs by genotyping only the extreme phenotypes instead of the entire mapping population. We have previously mapped a QTL linked to leaf rolling and leaf drying in recombinant inbred (RI) lines derived from two locally adapted indica rice ecotypes viz., IR20/Nootripathu using BSA. Fine mapping the QTL will facilitate its application in MAS. BSA was done by bulking DNA of 10 drought-resistant and 12 drought-sensitive RI lines. Out of 343 rice microsatellites markers genotyped, RM8085 co-segregated among the RI lines constituting the respective bulks. RM8085 was mapped in the middle of the QTL region on chromosome 1 previously identified in these RI lines thus reducing the QTL interval from 7.9 to 3.8 cM. Further, the study showed that the region, RM212–RM302–RM8085–RM3825 on chromosome 1, harbours large effect QTLs for drought-resistance traits across several genetic backgrounds in rice. Thus, the QTL may be useful for drought resistance improvement in rice through MAS and map-based cloning.     

SNP haplotypes of the <Emphasis Type="Italic">BADH1</Emphasis> gene and their association with aroma in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the synthesis of glycinebetaine—a powerful osmoprotectant against salt and drought stress in a large number of species. Rice is not known to accumulate glycinebetaine but it has two functional genes coding for the BADH enzyme. A non-functional allele of the BADH2 gene located on chromosome 8 is a major factor associated with rice aroma. However, similar information is not available regarding the BADH1 gene located on chromosome 4 despite the similar biochemical function of the two genes. Here we report on the discovery and validation of SNPs in the BADH1 gene by re-sequencing of diverse rice varieties differing in aroma and salt tolerance. There were 17 SNPs in introns with an average density of one per 171 bp, but only three SNPs in exons at a density of one per 505 bp. Each of the three exonic SNPs led to changes in amino acids with functional significance. Multiplex SNP assays were used for genotyping of 127 diverse rice varieties and landraces. In total 15 SNP haplotypes were identified but only four of these, corresponding to two protein haplotypes, were common, representing more than 85% of the cultivars. Determination of population structure using 54 random SNPs classified the varieties into two groups broadly corresponding to indica and japonica cultivar groups, aromatic varieties clustering with the japonica group. There was no association between salt tolerance and the common BADH1 haplotypes, but aromatic varieties showed specific association with a BADH1 protein haplotype (PH2) having lysine₁₄₄ to asparagine₁₄₄ and lysine₃₄₅ to glutamine₃₄₅ substitutions. Protein modeling and ligand docking studies show that these two substitutions lead to reduction in the substrate binding capacity of the BADH1 enzyme towards gamma-aminobutyraldehyde (GABald), which is a precursor of the major aroma compound 2-acetyl-1-pyrroline (2-AP). This association requires further validation in segregating populations for potential utilization in the rice breeding programs.     

Piercing and vacuum infiltration of the mature embryo: a simplified method for <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T ₀) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T ₀ plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T ₁ transformants.     

Fine mapping of QTL <Emphasis Type="Italic">qCTB10</Emphasis>-<Emphasis Type="Italic">2</Emphasis> that confers cold tolerance at the booting stage in rice

Key message

The QTL qCTB10 - 2 controlling cold tolerance at the booting stage in rice was delimited to a 132.5 kb region containing 17 candidate genes and 4 genes were cold-inducible.

Abstract

Low temperature at the booting stage is a major abiotic stress-limiting rice production. Although some QTL for cold tolerance in rice have been reported, fine mapping of those QTL effective at the booting stage is few. Here, the near-isogenic line ZL31-2, selected from a BC₇F₂ population derived from a cross between cold-tolerant variety Kunmingxiaobaigu (KMXBG) and the cold-sensitive variety Towada, was used to map a QTL on chromosome 10 for cold tolerance at the booting stage. Using BC₇F₃ and BC₇F₄ populations, we firstly confirmed qCTB10-2 and gained confidence that it could be fine mapped. QTL qCTB10-2 explained 13.9 and 15.9% of the phenotypic variances in those two generations, respectively. Using homozygous recombinants screened from larger BC₇F₄ and BC₇F₅ populations, qCTB10-2 was delimited to a 132.5 kb region between markers RM25121 and MM0568. 17 putative predicted genes were located in the region and only 5 were predicted to encode expressed proteins. Expression patterns of these five genes demonstrated that, except for constant expression of LOC_Os10g11820, LOC_Os10g11730, LOC_Os10g11770, and LOC_Os10g11810 were highly induced by cold stress in ZL31-2 compared to Towada, while LOC_Os10g11750 showed little difference. Our results provide a basis for identifying the genes underlying qCTB10-2 and indicate that markers linked to the qCTB10-2 locus can be used to improve the cold tolerance of rice at the booting stage by marker-assisted selection.

     

RICD: A rice <Emphasis Type="Italic">indica</Emphasis>cDNA database resource for rice functional genomics

Background  

The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones.     

Interaction of two recessive genes, <Emphasis Type="Italic">hbd2</Emphasis> and <Emphasis Type="Italic">hbd3</Emphasis>, induces hybrid breakdown in rice

Reproductive barriers are important for the maintenance of species identity. We discovered a reproductive barrier via hybrid breakdown among the progeny of a cross between the japonica rice cultivar Koshihikari and the indica rice cultivar Habataki. Genetic analysis indicated that the hybrid breakdown is regulated by the interaction of two recessive genes: hbd2 in Habataki and hbd3 in Koshihikari. Linkage mapping showed that hbd2 is located near the 100 cM region of chromosome 2 in Habataki, whereas hbd3 is located near the 60 cM region of chromosome 11 in Koshihikari. Construction of nearly isogenic lines for hbd2 and Hbd3 (NIL-hbd2 and NIL-Hbd3), as well as a pyramiding line (NIL-hbd2 + Hbd3), confirmed that the hybrid breakdown is induced by the interaction of these two recessive genes. Our results indicate that these genes are novel for the induction of hybrid breakdown in rice.     

Identification and fine mapping of <Emphasis Type="Italic">S-d</Emphasis>, a new locus conferring the partial pollen sterility of intersubspecific F<Subscript>1</Subscript> hybrids in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The partial pollen abortion of hybrids between the indica and japonica subspecies of Asian cultivated rice is one of the major barriers in utilizing intersubspecific heterosis in hybrid rice breeding. Although a single hybrid pollen sterility locus may have little impact on spikelet fertility, the cumulative effect of several loci usually leads to a serious decrease in spikelet fertility. Isolating of the genes conferring hybrid pollen sterility is necessary to understand this phenomenon and to overcome the resulting genetic barrier. In this study, a new locus for F₁ pollen sterility, S-d, was identified on the short arm of chromosome 1 by analyzing the genetic effect of substituted segments of the near-isogenic line E11-5 derived from the japonica variety Taichung 65 (recurrent parent) and the indica variety Dee-geo-woo-gen (donor parent). The S-d locus was first mapped to a 0.8 cM interval between SSR markers PSM46 and PSM80 using a F₂ population of 125 individuals. The flanking markers were then used to identify recombinants from a population of 2,160 plants derived from heterozygotes of the primary F₂ population. Simultaneously, additional markers were developed from genomic sequence divergence in this region. Analysis of the recombinants in the region resulted in the successful mapping of the S-d locus to a 67-kb fragment, containing 17 predicted genes. Positional cloning of this gene will contribute to our understanding of the molecular basis for partial pollen sterility of intersubspecific F₁ hybrids in rice.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

Development of genotype-independent regeneration system for transformation of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> ssp. <Emphasis Type="Italic">indica</Emphasis>)

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L⁻¹ thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L⁻¹ carbenicillin or 250 mg L⁻¹ cefotaxime to kill the rice shoot apical meristem was 50 mg L⁻¹ and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T₀ transformant plantlets.