Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryza sativa L.)

The characteristics of starch, such as gelatinization temperature (GT), apparent amylose content (AAC), pasting temperature (PT) and other physicochemical properties, determine the quality of various products of rice, e.g., eating, cooking and processing qualities. The GT of rice flour is controlled by the alk locus, which has been co-mapped to the starch synthase IIa (SSIIa) locus. In this study, we sequenced a 2,051 bp DNA fragment spanning part of intron 6, exon 7, intron 7, exon 8 and part of 3′ untranslated region of SSIIa for 30 rice varieties with diverse geographical distribution and variation in starch physicochemical properties. A total of 24 single nucleotide polymorphisms (SNPs) and one insertion/deletion (InDel) were identified, which could be classified into nine haplotypes. The mean pairwise nucleotide diversity π was 0.00292, and Watterson’s estimator θ was 0.00296 in this collection of rice germplasm. Tajima’s D test for selection showed no significant deviation from the neutral expectation (D = − 0.04612, P > 0.10). However, significant associations were found between seven of the SNPs and peak GT (T _p) at P < 0.05, of which two contiguous SNPs (GC/TT) showed a very strong association with T _p (P < 0.0001). With some rare exception, this GC/TT polymorphism alone can differentiate rice varieties with high or intermediate GT (possessing the GC allele) from those with low GT (possessing the TT allele). In contrast, none of these SNPs or InDel was significantly associated with amylose content. A further 509 rice varieties with known physicochemical properties (e.g., AAC and PT) and known alleles of other starch synthesizing genes were genotyped for the SSIIa GC/TT alleles. Association analysis indicated that 82% of the total variation of AAC in these samples could be explained by a (CT)n simple sequence repeat (SSR) and a G/T SNP of Waxy gene (Wx), and 62.4% of the total variation of PT could be explained by the GC/TT polymorphism. An additional association analysis was performed between these molecular markers and the thermal and retrogradation properties for a subset of 245 samples from the 509 rice varieties. The SSIIa GC/TT polymorphism explained more than 60% of the total variation in thermal properties, whereas the SSR and SNP of Wx gene explained as much as the SSIIa GC/TT of the total variation in retrogradation properties. Our study provides further support for the utilization of the GC/TT polymorphism in SSIIa. As shown in our study of 509 rice varieties, the GC/TT SNP could differentiate rice with high or intermediate GT from those with low GT in about 90% of cases. Using four primers in a single PCR reaction, the GC/TT polymorphism can be surveyed on a large scale. Thus, this SNP polymorphism can be very useful in marker-assisted selection for the improvement of GT and other physicochemical properties of rice.     

Association mapping for yield and grain quality traits in rice (Oryza sativa L.)

Association analysis was applied to a panel of accessions of Embrapa Rice Core Collection (ERiCC) with 86 SSR and field data from two experiments. A clear subdivision between lowland and upland accessions was apparent, thereby indicating the presence of population structure. Thirty-two accessions with admixed ancestry were identified through structure analysis, these being discarded from association analysis, thus leaving 210 accessions subdivided into two panels. The association of yield and grain-quality traits with SSR was undertaken with a mixed linear model, with markers and subpopulation as fixed factors, and kinship matrix as a random factor. Eight markers from the two appraised panels showed significant association with four different traits, although only one (RM190) maintained the marker-trait association across years and cultivation. The significant association detected between amylose content and RM190 was in agreement with previous QTL analyses in the literature. Herein, the feasibility of undertaking association analysis in conjunction with germplasm characterization was demonstrated, even when considering low marker density. The high linkage disequilibrium expected in rice lines and cultivars facilitates the detection of marker-trait associations for implementing marker assisted selection, and the mining of alleles related to important traits in germplasm.     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

Oxygen dynamics in submerged rice (Oryza sativa)

Complete submergence of plants prevents direct O(2) and CO(2) exchange with air. Underwater photosynthesis can result in marked diurnal changes in O(2) supply to submerged plants. Dynamics in pO(2) had not been measured directly for submerged rice (Oryza sativa), but in an earlier study, radial O(2) loss from roots showed an initial peak following shoot illumination. O(2) dynamics in shoots and roots of submerged rice were monitored during light and dark periods, using O(2) microelectrodes. Tissue sugar concentrations were also measured. On illumination of shoots of submerged rice, pO(2) increased rapidly and then declined slightly to a new quasi-steady state. An initial peak was evident first in the shoots and then in the roots, and was still observed when 20 mol m(-3) glucose was added to the medium to ensure substrate supply in roots. At the new quasi-steady state following illumination, sheath pO(2) was one order of magnitude higher than in darkness, enhancing also pO(2) in roots. The initial peak in pO(2) following illumination of submerged rice was likely to result from high initial rates of net photosynthesis, fuelled by CO(2) accumulated during the dark period. Nevertheless, since sugars decline with time in submerged rice, substrate limitation of respiration could also contribute to morning peaks in pO(2) after longer periods of submergence.     

Association of morphological and RFLP markers in rice (Oryza sativa L.).

Seventeen morphological marker genes were associated with restriction fragment length polymorphism markers in rice by using four F2 populations, each segregating for a few observable traits, and 14 near isogenic lines (NILs), each containing one morphological mutant gene. The location of five genes was confirmed on the basis of F2 analysis: Purple hull (Pr) (16.8 +/- 7.9 cM away from RG163 on chromosome 4); Phenol staining (Ph) (20.8 +/- 8.4 cM away from RG163 on chromosome 4); glabrous leaf and hull (gl-1) (14.3 +/- 7.4 cM away from RG182, and 20.9 +/- 8.3 cM from RG403 on chromosome 5); Brown pericarp (Rc) (12.5 +/- 7.2 cM away from RG30 on chromosome 7); and lazy growth habit (la) (28.8 +/- 9.4 cM away from RG118 on chromosome 11). In addition, 12 other morphological markers, including the agronomically important genes semi-dwarf (sd-1) and Pollen restoring factor (Rf-1) were tentatively associated with mapped DNA clones based on screening pairs of NILs.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

Double repression of soluble starch synthase genes SSIIa and SSIIIa in rice (Oryza sativa L.) uncovers interactive effects on the physicochemical properties of starch

Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in developing rice (Oryza sativa L.) endosperm. Despite extensive studies of SSs in various plant species including rice, the functional modes of action among multiple SS genes are still not clear. Here, we generated transgenic RNA interference (RNAi) repressed lines for seven of the eight members of the rice SS gene family and studied their effects on starch synthesis and grain formation. Consistent with their expression domains, RNAi repression of genes that encode isozymes SSI, SSIIa, and SSIIIa had strong effects on grain development, whereas no obvious phenotypic changes were observed in transgenic plants with the other SS genes being RNAi repressed, indicating functional redundancies among the genes. To study the potential functional interactions of SS genes, we generated SSIIa/SSIIIa double repression lines whose kernels displayed a chalky kernel appearance and had increased amylose levels, increased pasting temperatures, and decreased viscosities. The double mutation also reduced short (degree of polymerization (DP) 5-6) and long (DP 12-23) amylopectin chain contents in the grain and increased the medium long types (DP 7-11). The nonadditive nature of the double mutation line suggests that SSIIa and SSIIIa interact with each other during starch synthesis. Such interaction may be physical via starch phophorylase as indicated by our pair-wise yeast two-hybrid assays on major starch synthesis enzymes. Collectively, the data showed that SSIIa and SSIIIa play distinctive, but partially overlapping, roles during rice grain starch synthesis. The possibility of extensive redundancy or complementarity among SS isozymes is discussed.     

Association mapping of stigma and spikelet characteristics in rice (Oryza sativa L.)

Stigma and spikelet characteristics play an essential role in hybrid seed production. A mini-core of 90 accessions developed from USDA rice core collection was phenotyped in field grown for nine traits of stigma and spikelet and genotyped with 109 DNA markers, 108 SSRs plus an indel. Three major clusters were built upon Rogers’ genetic distance, indicative of indicas, and temperate and tropical japonicas. A mixed linear model combining PC-matrix and K-matrix was adapted for mapping marker-trait associations. Resulting associations were adjusted using false discovery rate technique. We identified 34 marker-trait associations involving 22 SSR markers for eight traits. Four markers were associated with single stigma exsertion (SStgE), six with dual exsertion (DStgE) and five with total exsertion. RM5_Chr1 played major role indicative of high regression with not only DStgE but also SStgE. Four markers were associated with spikelet length, three with width and seven with L/W ratio. Numerous markers were co-associated with multiple traits that were phenotypically correlated, i.e. RM12521_Chr2 associated with all three correlated spikelet traits. The co-association should improve breeding efficiency because single marker could be used to assist breeding for multiple traits. Indica entry 1032 (cultivar 50638) and japonica entry 671 (cultivar Linia 84 Icar) with 80.65 and 75.17% of TStgE, respectively are recommended to breeder for improving stigma exsertion.     

Salinity-induced enhancement of L-myo-inositol 1-phosphate synthase in rice (Oryza sativa L.)

The salt-tolerant varieties of rice (Oryza sativa L.) exhibit enhanced activity of the chloroplast form of L-myo-inositol 1-phosphate synthase (EC 5.5.4.1) under NaCl treatment either during the seedling stage or in fully grown plants during field growth. The salt-induced enhancement was noticeable only in chloroplasts from light-grown plants. The effects of these treatments on the cytosolic inositol synthase activity were less pronounced. While the effect of salt on the activity of the two forms was marginal in the salt-sensitive varieties during seedling growth, salinity affected the chloroplast inositol synthase activity adversely in these varieties during growth of the plants under field conditions. The salt-enhanced activities of inositol synthase(s) in the highly salt-tolerant varieties studied were found to be comparable to that observed in Porteresia coarctata, a halophytic wild rice species. The implications of these findings, which suggest a role of the inositol pathway in osmoregulation, are discussed.     

The mechanism of phloem loading in rice (Oryza sativa)

Carbohydrates, mainly sucrose, that are synthesized in source organs are transported to sink organs to support growth and development. Phloem loading of sucrose is a crucial step that drives long-distance transport by elevating hydrostatic pressure in the phloem. Three phloem loading strategies have been identified, two active mechanisms, apoplastic loading via sucrose transporters and symplastic polymer trapping, and one passive mechanism. The first two active loading mechanisms require metabolic energy, carbohydrate is loaded into the phloem against a concentration gradient. The passive process, diffusion, involves equilibration of sucrose and other metabolites between cells through plasmodesmata. Many higher plant species including Arabidopsis utilize the active loading mechanisms to increase carbohydrate in the phloem to higher concentrations than that in mesophyll cells. In contrast, recent data revealed that a large number of plants, especially woody species, load sucrose passively by maintaining a high concentration in mesophyll cells. However, it still remains to be determined how the worldwide important cereal crop, rice, loads sucrose into the phloem in source organs. Based on the literature and our results, we propose a potential strategy of phloem loading in rice. Elucidation of the phloem loading mechanism should improve our understanding of rice development and facilitate its manipulation towards the increase of crop productivity.     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

水稻水溶性环氧化合物水解酶的生物信息学分析

水溶性环氧化合物水解酶（Soluble Epoxide Hydrolase,SEH）是一组催化环氧化合物水解为相应邻位二醇的酶类,在哺乳动物、植物、昆虫和微生物体内广泛存在。通过BLAST对水稻基因组的蛋白质数据库进行搜索,获得10个水溶性环氧化物水解酶（Soluble Epoxide Hydrolase SEH）sEH蛋白的同源序列。经分析发现这些基因在水稻不同胁迫处理下各个部位都有所表达,而且不同成员之间的表达模式存在较大的差异。水稻sEH蛋白主要参与角质层形成,应激反应,以及病原防御等生理过程,特别在脱毒过程中扮演着重要的角色。对蛋白质多序列联配和三级结构预测结果表明：水溶性环氧化合物水解酶的核心结构域由3个催化残基Asp、His和Asp形成三位一体的催化活性构象。这类基因的表达受抗逆环境诱导,其功能与抗逆性有关,为基因工程抗逆育种提供了参考。     

Genetic studies of anther culture ability in rice (Oryza sativa)

Inheritance of three anther and culture characters, callus induction, green plant regeneration and culture efficiency was studied using incomplete diallel crosses with a gamete model. It was suggested that callus induction was mainly controlled by gametic additive effects and with less effect of the maternal effects. Green plant regeneration was mainly determined by maternal effects with less influence of gametic additive effects. Culture efficiency was controlled by gametic additive, maternal and cytoplasmic effects. Cultivar Lunhui 422 showed positive genetic effects for all three traits and was a very good parent for rice anther culture breeding. Significant positive heterosis was observed for callus induction. Both gametic additive and maternal correlations contributed to the significant genotypic and phenotypic correlations between callus induction and green plant regeneration suggesting these two traits to be linked.Abbreviations 2,4-d 2,4-dichlorphenoxyacetic acid - NAA -napthaleneacetic acid     

Characterization of starch produced by suspension cell cultures of Indica rice (Oryza sativa L.)

Suspension cultures of rice (Oryza sativa L.), initiated from seed, produced significant amounts of starch. Starch accumulated in the cultured cells throughout the growth phase and reached a maximum of 7% of the cell dry weight at stationary phase. Starch was present in compound granules which were birefringent under polarized light. Suspension culture starch had a higher amylose content and a lower gelatinization temperature than rice grain starch. Additionally, starch branching enzyme, an enzyme involved in starch biosynthesis, was characterized by anion exchange chromatography in culture cells and endosperm. Culture cells had at least one major form of starch branching enzyme which differed from the multiple enzyme forms present in endosperm.