Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

Differences in photosynthetic characterization of salt tolerance for two rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) cultivars

We photosynthetically characterized two rice cultivars - salt-sensitive ‘Annapurna’, and salt-tolerant ‘Dongjin’ - growing under NaCl stress. Both cultivars showed an increase in F_o/F_m (the ratio of initial to maximal chlorophyll fluorescence) and a decrease in F_v/F_m (an indicator of the photochemical efficiency of PS II). In particular, the F_v value for Annapurna significant declined while F_o/F_m was enhanced when plants were exposed to salt stress for 4 d. Annapurna also exhibited more rapid decreases in the coefficients for photochemical quenching (qQ) and non-photochemical quenching (qNP) than did Dongjin. In contrast, zeaxanthin formation was largely influenced by exposure to light rather than to high salinity, with Annapurna having a higher rate of production compared with Dongjin. When both cultivars were exposed to salt stress for 2 d, Annapurna had a much lower rate of photosynthetic oxygen evolution, corresponding to only 46% of the control; the rate for Dongjin was 90% of the control. Salt stress in both cultivars induced the accumulation of two osmoprotectants, glycinebetaine and proline, the rate being higher for the latter. These results indicate that Annapurna is more sensitive than Dongjin to salt stress, in terms of its deterioration in photosynthetic function.     

Fine mapping of the <Emphasis Type="Italic">qCTS4</Emphasis> locus associated with seedling cold tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Rice seedlings are sensitive to low temperatures (≤15°C) and under prolonged or repeated exposure, yellowing and stunting are commonly observed. Damage to seedlings results in poor stand establishment and delayed maturation, which can cause significant reductions in yield. In general, japonica rice varieties exhibit more cold tolerance than indica varieties. Earlier genetic analysis of the California rice variety M202 revealed several quantitative trait loci (QTL) that contribute to its tolerance to low temperatures in comparison to the indica rice variety IR50. Among these QTL, qCTS4 is associated with tolerance to yellowing and stunting of rice seedlings and accounts for 40% of the phenotypic variation. Here we report on the fine mapping of qCTS4 to a 128 kb region of chromosome 4, which is highly suppressed for recombination in our mapping populations. Our results provide the necessary foundation for identifying the gene(s) underlying qCTS4 and the markers developed here may be used to introgress this region into indica varieties to improve seedling tolerance to low temperatures. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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.     

Structural and histochemical studies on grain-filling in the caryopsis of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The endosperm and embryo that constitute the filial tissues of rice caryopsis are isolated from the maternal tissues by the absence of any symplastic continuity. Nutrients are transported to the endosperm through a single ovular vascular trace present on the ventral side of the ovary. Initially solute enters through the chalaza into the nucellar projection and then into the endosperm. At later stages transport occurs through the nucellar epidermis, centripetally towards the endosperm. The cell walls of the nucellar epidermis are provided with rib-like thickenings. A comparison of grain-filling in C₃ and C₄ cereals suggests that rice has structural features allied to C₃ cereals, such as wheat, but with significant differences.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Functional markers for <Emphasis Type="Italic">xa5</Emphasis>-mediated resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>, L.)

The recent cloning of several agronomically important genes has facilitated the development of functional markers. These markers reside within the target genes themselves and can be used with great reliability and efficiency to identify favorable alleles in a breeding program. Bacterial blight (BB) is a severe rice disease throughout the world that is controlled primarily through use of resistant cultivars. xa5 is a race-specific, recessive gene mediating resistance to BB. It is widely used in rice breeding programs throughout the tropics. Due to its recessive nature, phenotypic selection for xa5-mediated resistance is both slow and costly. Previously, marker assisted selection (MAS) for this resistance gene was not efficient because it involved markers that were only indirectly linked to xa5 and ran the risk of being separated from the trait by recombination. Recently, the cloning of the gene underlying this trait made it possible to develop functional markers. Here we present a set of CAPS markers for easy, quick and direct identification of cultivars or progeny carrying xa5-mediated resistance and provide evidence that these markers are 100% predictive of the presence of the xa5 allele. These markers are expected to enhance the reliability and cost-effectiveness of MAS for xa5-mediated resistance.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Microarray Analysis of Gene Expression Involved in Anther Development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA₃) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA₃ and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA₃- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA₃ or JA treatment, and 24 GA₃- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA₃. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA₃ signals in the control of anther development in rice.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.     

Assessment of genetic diversity of <Emphasis Type="Italic">Saltol</Emphasis> QTL among the rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Eight Saltol quantitative trait locus (QTL) linked simple sequence repeat (SSR) markers of rice (Oryza sativa L.) were used to study the polymorphism of this QTL in 142 diverse rice genotypes that comprised salt tolerant as well as sensitive genotypes. The SSR profiles of the eight markers generated 99 alleles including 20rare alleles and 16 null alleles. RM8094 showed the highest number (13) of alleles followed by RM3412 (12), RM562 (11), RM493 (9) and RM1287 (8) while as, RM10764 and RM10745 showed the lowest number (6) of alleles. Based on the highest number of alleles and PIC value (0.991), we identified RM8094 as suitable marker for discerning salt tolerant genotypes from the sensitive ones. Based upon the haplotype analysis using FL478 as a reference (salt tolerant genotypes containing Saltol QTL), we short listed 68 rice genotypes that may have at least one allele of FL478 haplotype. Further study may confirm that some of these genotypes might have Saltol QTL and can be used as alternative donors in salt tolerant rice breeding programmes.     

Identification of neutral alleles at pollen sterility gene loci of cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) from wild rice (<Emphasis Type="Italic">O. rufipogon</Emphasis> Griff.)

Oryza rufipogon Griff. is the ancestor of Asian cultivated rice (O. sativa L.) and possesses valuable genes for rice breeding. Pollen abortion is one of the major causes of indica–japonica hybrid sterility in rice and it happens due to allelic interaction at the pollen sterility gene loci. A total of six loci (Sa, Sb, Sc, Sd, Se, and Sf) have been found to be associated with F₁ pollen sterility between indica and japonica rice, and five of them (all except Sf) have been mapped. Neutral alleles (S ⁿ ) at each locus have the potential to overcome the pollen sterility associated with the respective locus. Therefore, exploitation and utilization of neutral alleles have significant importance in overcoming indica–japonica hybrid sterility. In this study, an accession (IRW28) of O. rufipogon, native to China, was selected as paternal to cross with typical japonica (Taichung 65) and indica (Guanglu’ai 4) tester lines, and two F₂ populations were developed. The simple sequence repeat markers tightly linked to five pollen sterility loci were applied for genotyping the F₂ populations. Chi-squared tests were applied to examine the normal segregation/distortion at each locus. The expected and observed pollen sterility for each locus were estimated. As a result, the genotypes at five pollen sterility gene loci for IRW28 were identified as: Sa ⁱ⁻¹/Sa ⁱ⁻¹, Sb ⁿ /Sb ⁿ , Sc ⁱ⁻²/Sc ⁱ⁻², Sd ⁿ /Sd ⁿ and Se ⁿ /Se ⁿ . Our results suggest that IRW28 (O. rufipogon) has the neutral alleles for pollen fertility at the Sb, Sd and Se loci, and these alleles have a good affinity with indica and japonica rice. These neutral alleles provide valuable gene resources to overcome the inter-subspecific hybrid pollen sterility in rice.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

A practical vector for efficient knockdown of gene expression in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene. These authors contributed equally to this work.     

Genomic paleontology provides evidence for two distinct origins of Asian rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The origin of rice domestication has been the subject of debate for several decades. We have compared the transpositional history of 110 LTR retrotransposons in the genomes of two rice varieties, Nipponbare (Japonica type) and 93-11 (Indica type) whose complete sequences have recently been released. Using a genomic paleontology approach, we estimate that these two genomes diverged from one another at least 200,000 years ago, i.e., at a time which is clearly older than the date of domestication of the crop (10,000 years ago, during the late Neolithic). In addition, we complement and confirm this first in silico analysis with a survey of insertion polymorphisms in a wide range of traditional rice varieties of both Indica and Japonica types. These experimental data provide additional evidence for the proposal that Indica and Japonica rice arose from two independent domestication events in Asia.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien