Indica Genotypes Research Articles

Effect of NiR gene on in vitro regeneration protocol of indica, japonica, aromatic and wild rice genotypes

Effect of <i>NiR</i> gene on <i>in vitro</i> regeneration protocol of <i>indica</i>, <i>japonica</i>, aromatic and wild rice genotypes

Influence of silicon treatment on antimony uptake and translocation in rice genotypes with different radial oxygen loss

Antimony (Sb) pollution in soil may have a negative impact on the health of people consuming rice. This study investigated the effect of silicon (Si) application on rice biomass, iron plaque formation, and Sb uptake and speciation in rice plants with different radial oxygen loss (ROL) using pot experiments. The results demonstrated that Si addition increased the biomass of straw and grain, but had no obvious impact on the root biomass. Indica genotypes with higher ROL underwent greater iron plaque formation and exhibited more Sb sequestration in iron plaque. Silicon treatments increased iron levels in iron plaque from the different genotypes but decreased the total Sb concentration in root, straw, husk, and grain. In addition, Si treatment reduced the inorganic Sb concentrations but slightly increased the trimethylantimony (TMSb) concentrations in rice straw. Moreover, rice straw from hybrid genotypes accumulated higher concentrations of TMSb and inorganic Sb than that from indica genotypes. The conclusions from this study indicate that Sb contamination in rice can be efficiently reduced by applying Si treatment and selecting genotypes with high ROL.

Genetic Control of Plasticity in Root Morphology and Anatomy of Rice in Response to Water Deficit.

Elucidating the genetic control of rooting behavior under water-deficit stress is essential to breed climate-robust rice (Oryza sativa) cultivars. Using a diverse panel of 274 indica genotypes grown under control and water-deficit conditions during vegetative growth, we phenotyped 35 traits, mostly related to root morphology and anatomy, involving 45,000 root-scanning images and nearly 25,000 cross sections from the root-shoot junction. The phenotypic plasticity of these traits was quantified as the relative change in trait value under water-deficit compared with control conditions. We then carried out a genome-wide association analysis on these traits and their plasticity, using 45,608 high-quality single-nucleotide polymorphisms. One hundred four significant loci were detected for these traits under control conditions, 106 were detected under water-deficit stress, and 76 were detected for trait plasticity. We predicted 296 (control), 284 (water-deficit stress), and 233 (plasticity) a priori candidate genes within linkage disequilibrium blocks for these loci. We identified key a priori candidate genes regulating root growth and development and relevant alleles that, upon validation, can help improve rice adaptation to water-deficit stress.

Genetic factors underlying boron toxicity tolerance in rice: genome-wide association study and transcriptomic analysis.

Boron (B) toxicity is a nutritional disorder affecting crop production in many parts of the world. This study explored genetic factors associated with B tolerance in rice (Oryza sativa L.) through an integrated genome mapping and transcriptomic approach. Variation in B tolerance was first evaluated by screening a panel of 137 indica genotypes in B toxic conditions (+2 mM B), followed by genome-wide association study (GWAS). Leaf bronzing and greenness were significantly correlated with shoot and root dry weight, but B uptake was not correlated with any stress phenotype. Single nucleotide polymorphism (SNP) markers exceeding a significance value of –log10P>4.0 were obtained for four traits, namely leaf bronzing, shoot dry weight, root dry weight, and root length. Linkage disequilibrium block analysis of the corresponding chromosomal regions revealed candidate loci containing 75 gene models. Two contrasting genotypes from the panel were selected for transcriptomic analysis, which included gene ontology enrichment analysis of differentially regulated genes and investigating transcriptional responses of GWAS candidate genes. Characteristic expression patterns associated with tolerance or sensitivity were seen in genes related to biochemical binding, transport, transcriptional regulation, and redox homeostasis. These results advance the understanding of genetic and physiological factors associated with B tolerance in rice.

Effect of silicate on arsenic fractionation in soils and its accumulation in rice plants

Four rice genotypes, two hybrid and two indica, were selected to investigate the effects of silicate (Si) application on arsenic (As) accumulation and speciation in rice and As fractionation in soil. There were significant differences in root, straw and grain biomass among genotypes (p < 0.05), and Si application significantly increased root (p < 0.05) and grain biomass (p < 0.001). Silicate addition reduced the proportion of As associated with well-crystallized hydrous oxides of Fe and Al and residual phases, whilst increasing the proportions of specifically-sorbed As and As associated with amorphous and poorly-crystalline Fe and Al hydrous oxides. Furthermore, the results indicated that the fraction proportions of non-specifically sorbed, specifically-sorbed, and associated with amorphous and poorly-crystalline hydrous oxides of Fe and Al in rhizosphere soils, were greater than non-rhizosphere soils. Silicate application had a significant effect decreasing total As concentrations in root (p < 0.005), straw (p < 0.05) and husk (p < 0.001) of rice plants. The effect of Si on reducing As accumulation in rice leaves was revealed by SXRF. Indica genotypes transported and accumulated less As than hybrid genotypes. Both percentage and concentration of iAs were lower in indica genotype XFY-9 than in hybrid genotype XWX-12. Silicate reduced iAs and DMA by 21% and 58% in grain (polished) respectively. DMA may have a greater translocation capacity from straw to grain (polished) than inorganic As. The study provides the potential for understanding As uptake mechanisms in rice and mitigating the health risks posed by As contamination in paddy fields.

Identification and physiological characterization of two sister lines of indica rice (Oryza sativa L.) with contrasting levels of cold tolerance

Exposure to low temperature during germination and vegetative growth is a limiting factor to the establishment and development of rice seedlings. Higher cold tolerance of japonica than indica subspecies is well documented. However, reports of cold tolerance in indica genotypes are rare. We screened a large number of indica rice genotypes for cold tolerance during germination and initial vegetative growth. The indica genotypes IRGA 959-1-2-2F-4-1-4-A and IRGA 959-1-2-2F-4-1-4-D-1-CA-1, derived from the same cross, were characterized, respectively, as tolerant and sensitive to low temperature. Indexes of photosynthetic performance during light absorption were heavily affected by cold in both genotypes, but recovered after cold exposure only in the tolerant genotype. Activities of the antioxidant enzymes SOD and CAT (at the vegetative stage) and CAT and APX (at the germination stage) were higher in the tolerant than in the sensitive genotype. Expression of 20 genes previously related to cold response in rice was evaluated. Expression of OsLIP9 and OsWCOR413 were higher in the tolerant genotype upon or prior to cold exposure, respectively. The two sister lines show different molecular and physiological responses to low temperature stress. Further in-depth studies with these lines may help to identify new cold tolerance mechanisms in rice.

The effect of silicon on iron plaque formation and arsenic accumulation in rice genotypes with different radial oxygen loss (ROL)

Rice is one of the major pathways of arsenic (As) exposure in human food chain, threatening over half of the global population. Greenhouse pot experiments were conducted to examine the effects of Si application on iron (Fe) plaque formation, As uptake and rice grain As speciation in indica and hybrid rice genotypes with different radial oxygen loss (ROL) ability. The results demonstrated that Si significantly increased root and grain biomass. Indica genotypes with higher ROL induced greater Fe plaque formation, compared to hybrid genotypes and sequestered more As in Fe plaque. Silicon applications significantly increased Fe concentrations in iron plaque of different genotypes, but it decreased As concentrations in the roots, straws and husks by 28–35%, 15–35% and 32–57% respectively. In addition, it significantly reduced DMA accumulation in rice grains but not inorganic As accumulation. Rice of indica genotypes with higher ROL accumulated lower concentrations of inorganic As in grains than hybrid genotypes with lower ROL.

Analysis of Stress-Responsive Gene Expression in Cultivated and Weedy Rice Differing in Cold Stress Tolerance.

Rice (Oryza sativa L.) cultivars show impairment of growth in response to environmental stresses such as cold at the early seedling stage. Locally adapted weedy rice is able to survive under adverse environmental conditions, and can emerge in fields from greater soil depth. Cold-tolerant weedy rice can be a good genetic source for developing cold-tolerant, weed-competitive rice cultivars. An in-depth analysis is presented here of diverse indica and japonica rice genotypes, mostly weedy rice, for cold stress response to provide an understanding of different stress adaptive mechanisms towards improvement of the rice crop performance in the field. We have tested a collection of weedy rice genotypes to: 1) classify the subspecies (ssp.) grouping (japonica or indica) of 21 accessions; 2) evaluate their sensitivity to cold stress; and 3) analyze the expression of stress-responsive genes under cold stress and a combination of cold and depth stress. Seeds were germinated at 25°C at 1.5- and 10-cm sowing depth for 10d. Seedlings were then exposed to cold stress at 10°C for 6, 24 and 96h, and the expression of cold-, anoxia-, and submergence-inducible genes was analyzed. Control plants were seeded at 1.5cm depth and kept at 25°C. The analysis revealed that cold stress signaling in indica genotypes is more complex than that of japonica as it operates via both the CBF-dependent and CBF-independent pathways, implicated through induction of transcription factors including OsNAC2, OsMYB46 and OsF-BOX28. When plants were exposed to cold + sowing depth stress, a complex signaling network was induced that involved cross talk between stresses mediated by CBF-dependent and CBF-independent pathways to circumvent the detrimental effects of stresses. The experiments revealed the importance of the CBF regulon for tolerance to both stresses in japonica and indica ssp. The mechanisms for cold tolerance differed among weedy indica genotypes and also between weedy indica and cultivated japonica ssp. as indicated by the up/downregulation of various stress-responsive pathways identified from gene expression analysis. The cold-stress response is described in relation to the stress signaling pathways, showing complex adaptive mechanisms in different genotypes.

Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes

Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13°C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca2+-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

Arsenic accumulation and speciation in rice grains influenced by arsenic phytotoxicity and rice genotypes grown in arsenic-elevated paddy soils

Rice consumption is a major route of As exposure to human for the population of worldwide. This study investigates the effect of phytotoxicity and rice genotypes on the content and speciation of As in rice grains grown in different levels of As-elevated paddy soils from Taiwan. Three levels of As-elevated soils and six rice genotypes commonly planted in Taiwan were used for this study. The results indicate that As contents in grains of rice is not proportional to soil As concentrations and they were equal or higher in indica genotypes than japonica genotypes used in this study. It was also found that the As phytotoxicity not only reducing the grain yields but also the As concentrations in grain of rice. The predominant As species found in rice grains were dimethylarsinic acid (DMA) and arsenite. The concentrations of DMA increased with total As concentrations, wherggeas the arsenite remained in a narrow range from 0.1 to 0.3mgkg−1. Because of the lower toxicity of DMA than inorganic As species, the health risks may not be increased through consumption of rice even when total As content in the grains is increased.

Identification of QTLs for root and shoot traits in RIL population of rice (Oryza sativaL.)

In present study, quantitative trait loci (QTL) analysis was carried out using 93 SSR and 11 SNP markers in F7 recombinant inbred lines derived from a cross between two potential indica genotypes, Danteshwari and Dagad Deshi. Fourteen putative QTLs identified for 7 root and shoot traits on chromosomes 1, 2, 4, 5, 6 and 8, explained 3.77%-29.24% phenotypic variance at LOD score of > 2.5. The number of putative QTLs was 2 for each root volume, root dry weight, shoot length, shoot fresh weight and total plant length, 1 for root/shoot fresh weight ratio and 3 for root/shoot dry weight ratio. Identified putative QTLs for root and shoot traits in this study provide insight into the mode of drought tolerance, which will accelerate the rice molecular breeding.

Agro-Morpho and Molecular Characterization of World Mini core Collections using Hypervariable Microsatellite Markers in Rice (Oryza sativaL.)

Mini core collection plays an important role in evaluating genetic resources hence, an attempt was made to characterize world mini core collections on the basis of 35 uniformly distributed hyper variable SSR molecular markers. Thirty five markers amplified a total of 131 allelic fragments with an average of 3.28 alleles per locus indicating high level of polymorphism among the analyzed rice lines. The two major clusters shown by PPS SRS technique indicated no difference for grouping japonica and indica genotypes. Therefore the allele specific amplification pattern was adapted for identification of particular genotypes. The similarity coefficients among the genotypes ranged from 0.58 to 0.83 with an average similarity value of 0.36. Maximum similarity value of 88% was observed between PR33319-9-1-1-5-3-5-4-1 and PR-114. Whereas minimum similarity value of 54% was found between TJ 50836 and TJ 35724 followed by TJ 39050 & TJ 35724 and TJ 50836 and NDR359 genotypes with 55% of genetic similarity. Breeders worldwide could use these findings to choice the germplasm for transferring characters

Cold Tolerance in Rice Plants: Why, How and When?

Low temperature stress is one of the major abiotic factors which reduce rice yield in several countries [1]. Losses can range from 0.5 to 2.5 t/ha [2] and grain yields can drop by up to 26% [3], mostly due to low temperature during the reproductive stage, even though cold temperature can be harmful during the entire developmental stage of rice plants, from germination to grain filling [4,5]. During germination, the most common symptoms of cold temperature damage are delayed and lower percentage of germination [6]. During early growth stages, it can severely affect seedling establishment, showing yellowing of the leaves, growth retardation, and decreased tillering [5]. When cold coincides with the reproductive stage of the rice plant, sterility of the spikelets is the most common symptom of injury, but incomplete panicle exsertion and spikelet abortion may also occur, along with delayed and incomplete grain maturation [7]. Irrespective of the developmental stage, the duration of low temperature is an essential element determining the extent of cold damage; however, the critical air temperature that induces cold damage depends on the cultivar [8]. It is already known that some rice cultivars can tolerate stressful conditions and are able to grow under low temperature [9]. These cold tolerant rice cultivars normally belong to japonica subspecies [10], which are more adapted to temperate climates. Therefore, the challenge still remains to develop cold tolerant indica genotypes suitable for high-latitude regions.

Mapping quantitative trait loci (QTL) for grain size in rice using a RIL population from Basmati × indica cross showing high segregation distortion

Grain size is one of the three productivity related traits in rice and hence a major target for genetic improvement. Since understanding genetic variation in grain size between Basmati and indica genotypes is important for rice improvement, a recombinant inbred population was developed from a traditional aromatic cultivar ‘Basmati 370’ and a non-aromatic indica genotype ‘IRBB60’. This population was phenotyped in two locations for grain length (GL), grain breadth (GB), GL/GB ratio (LBR) and grain weight (GW). Though the RIL population reported in the current study exhibited segregation distortion (SD) for 54 % of the markers, they were utilized in analysis using an appropriate statistical package, PROC QTL in the SAS environment. Interval mapping revealed a total of 15 QTLs for GL, seven for GB, 15 for LBR and two for GW. Among them 13 were not reported earlier and thus novel. For a known major QTL identified in the study, GW8 for GB, a PCR based functional marker was designed and validated. This is the first report wherein a very high proportion of markers (>50 %) exhibiting SD have been successfully used for QTL mapping.

Characterization of an Italian rice germplasm collection with genetic markers useful for breeding to improve eating and cooking quality

Gelatinization temperature and apparent amylose content are key parameters used to describe the eating and cooking qualities of rice. Sequence variants of SSIIa and Waxy genes are important determinants of gelatinization temperature and apparent amylose content, respectively. A collection of Italian non-glutinous japonica rice accessions was characterized for sequence polymorphisms in SSIIa and Waxy genes, in comparison with non-Italian japonica and indica genotypes. For SSIIa two markers, SNP3 and SNP4, were used. A PCR amplification of multiple specific alleles protocol was developed for the identification of G/T polymorphism in 5′ splice site of first intron and A/C polymorphism in exon 6 of the Waxy gene. Based on simple allele-specific PCR, it can be proposed as a user-friendly, cost-effective tool for marker-assisted selection of amylose content. The collection was characterized also for the (CT)n repeats in exon 1 of the Waxy gene. The results showed that while SSIIa haplotypes were rather similar between Italian and non-Italian japonica rice, the Waxy gene haplotype T/A/(CT)18 was largely predominant in Italian accessions, other haplotypes, well represented in non-Italian japonica [T/A/(CT)19] and indica [e.g. G/C/(CT)20] genotypes, were present at lower frequency. Grain starch quality traits as apparent amylose content and RVA profile were also analysed. The In1/Ex6 SNP haplotypes of Wx gene were found to explain 79 % of variation in apparent amylose content, and 36, 22 and 25 %, of variation in the RVA parameters peak viscosity, breakdown and setback, respectively. The additional use of (CT)n repeats marker further improved the association of haplotypes with RVA parameters.

High-quality reference genes for quantifying the transcriptional responses of Oryza sativa L. (ssp. indica and japonica) to abiotic stress conditions

Rice (Oryza sativa L.) is important to food security and is also an excellent model plant for numerous cereal crops. A functional genomics study in rice includes characterization of the expression dynamics of genes by quantitative real-time PCR (qPCR) analysis; this is a significant key for developing rice varieties that perform well in the face of adverse climate change. The qPCR analysis requires the use of appropriate reference genes in order to make any quantitative interpretations meaningful. Here, the new potential reference genes were selected from a huge public database of rice microarray experiments. The expression stability of 14 candidates and 4 conventional reference genes was validated by geNormPLUS and NormFinder software. Seven candidates are superior to the conventionally used reference genes in qPCR and three genes can be used reliably for quantitating the expression of genes involved in abiotic stress responses. These high-quality references EP (LOC_Os05g08980), HNR (LOC_Os01g71770), and TBC (LOC_Os09g34040) worked very well in three indica genotypes and one japonica genotype. One of indica genotypes including the Jasmine rice, KDML105 developed in Thailand for which no reference genes have been reported until now.

Haplotype structure in grain weight gene GW2 and its association with grain characteristics in rice

GW2, a grain weight quantitative trait locus (QTL) in rice encodes a ring type E-3 ubiquitin ligase. A single nucleotide deletion at the 346th nucleotide position in the ligase domain of GW2 was earlier reported to result in higher grain weight in rice. The present study aimed at validating the known functional polymorphism and identifying additional natural genetic variation if any, in the region that included the functional domain of GW2 in a set of indica and aromatic genotypes for which ninety three rice genotypes were phenotyped for grain length, grain width and 100 grain weight. A wide range of variation was observed for these traits. PCR amplification and sequencing of GW2 target region revealed absence of insertion/deletion (InDel) at the 346th position which suggested that the genetic variation in grain weight in Basmati and non-Basmati indica genotypes was not explained by this InDel. However, four new single nucleotide polymorphisms (SNPs) were discovered at nucleotide positions 406, 461, 466 and 501 in the fifth exon and one InDel each in second and fourth introns. Only two of these SNPs, at positions 461 and 501 led to amino acid substitutions. A total of 10 haplotypes were constructed based on these four SNPs which could be regrouped into four categories based on their amino acid substitutions. Association genetic analysis of these haplotypes with different grain traits revealed a moderate association with grain width (R2 = 0.18 at P < 0.05). Thirteen haplotypes constructed using both intronic and exonic polymorphisms did not have any association with grain traits.

Studies to Investigate the Interactions of Genotypes, Culture Media and Culture Temperatures on Androgenesis in Recalcitrant Indica Rice (Oryza Sativa L.)

Production of doubled haploids through androgenesis is well established in japonica rice and successfully used to develop high yielding varieties. However, indica rice is remaining recalcitrant to this in vitro technique. To understand the interactions of genotypes, culture media and culture temperature, two indica varieties (Pokkali and Nona Bokra) were cultured on three culture media under normal (25°C) and alternate temperatures (30°C/ 20°C, 14 hours/10 hours). The responses of the recalcitrant varieties were examined at callus induction and regeneration stages. Pokkali was found responsive to culture media as well as culture temperatures. Although there was an increase in albino shoot production but green shoot productivity was recorded nine fold under alternate temperatures. Interactions of recalcitrant indica genotypes, culture media and culture temperatures were found significant at callus induction and regeneration stages. It is therefore reflected that to activate the recalcitrant genes for callus induction and regeneration of green shoots, interaction of genotypes, culture media and culture temperatures could play significant role. Pokkali responded well to SKI and SK II rather than others, indicated that alteration of culture media composition would be an important aspect in future investigation.

Contribution of leaf nitrogen to photosynthetic gas exchange in contrasting rice (Oryza sativa L.) cultivars during the grain-filling period

Photosynthetic parameters and leaf carbon isotope composition (&delta;13C) in contrasting rice genotypes in relation to supplemental nitrogen (N) application and water management during the grain-filling period were compared. The changes in stomatal conductance (g s) and ratio of intercellular to ambient CO2 mole fraction (C i/C a) depended on the leaf nitrogen concentration (leaf N) in both 'Hinohikari' (temperate japonica genotype) and 'IR36' (indica genotype). In 'Hinohikari', &delta;13C reflects photosynthetic gas exchange during the grain-filling period, which is indicated by the significant response of &delta;13C to leaf N. In contrast, in 'IR36' &delta;13C did not depend on leaf N. This varietal difference in &delta;13C to leaf N can be attributed to a difference in the timing of leaf senescence. In 'IR36', leaf N and photosynthetic parameters decreased more rapidly, indicating earlier senescence and a shorter grain-filling period in comparison with 'Hinohikari'. The significant increase in shoot dry mass in 'Hinohikari' resulting from supplemental N application, compared with nonsignificant effect observed in 'IR36', suggests that the timing of senescence in relation to the grainfilling period has a preponderant influence on productivity.

Molecular evaluation of genetic diversity and association studies in rice (Oryza sativa L.)

In the present study, we tested rice genotypes that included un(der)exploited landraces of Tamil Nadu along with indica and japonica test cultivars to ascertain their genetic diversity structure. Highly polymorphic microsatellite markers were used for generating marker segregation data. A novel measure, allele discrimination index, was used to determine subpopulation differentiation power of each marker. Phenotypic data were collected for yield and component traits. Pattern of molecular differentiation separated indica and japonica genotypes; indica genotypes had two subpopulations within. Landraces were found to have indica genome, but formed a separate subgroup with low linkage disequilibrium. The landraces further separated into distinct group in both hierarchical clustering analysis using neighbour-joining method as well as in the model based population structure analysis. Japonica and the remaining indica cultivars formed two other distinct groups. Linkage disequilibrium observed in the whole population was considerably reduced in subpopulations. Low linkage disequilibrium of landforms suggests their narrow adaptation in local geographical niche. Many population specific alleles could be identified particularly for japonica cultivars and landraces. Association analysis revealed nine marker-trait associations with three agronomic traits, of which 67% were previously reported. Although the testing landraces together with known cultivars had permitted genomewide association mapping, the experiment offers scope to study more landraces collected from the entire geographical region for drawing more reliable information.