Deep re-sequencing of a widely used maintainer line of hybrid rice for discovery of DNA polymorphisms and evaluation of genetic diversity

Genetic diversity within parental lines of hybrid rice is the foundation of heterosis utilization and yield improvement. Previous studies have suggested that genetic diversity was narrow in cytoplasmic male sterile (CMS/A line) and restorer lines (R line) for Three-line hybrid rice. However, the genetic diversity within maintainer lines (B line), especially at a genome-wide scale, remains largely unknown. In the present study, we performed deep re-sequencing of the elite maintainer line V20B (Oryza sativa L. ssp. indica). We then compared the V20B sequence with the 93-11 (Oryza sativa L. ssp. indica) genome sequence. 112.1 × 106 paired-end reads (PE reads) were generated with approximately 30-fold sequencing depth. The V20B PE reads uniquely covered 87.6 % of the 93-11 genome sequence. Overall, a total of 660,778 single-nucleotide polymorphism (SNPs) and 266,301 insertions and deletions (InDels) were identified, yielding an average of 2.1 SNPs/kb and 0.8 InDels/kb. Genome-wide distribution of the SNPs and InDels was non-random, and variation-rich and variation-poor regions were identified in all chromosomes. A total of 20,562 non-synonymous SNPs spanning 8,854 genes were annotated. Our results identified DNA polymorphisms at the genome-wide scale and uncovered the high level of genetic diversity between V20B and 93-11. Our results proved that next-generation sequencing technologies can be powerful tools to study genome-wide DNA polymorphisms, to query genetic diversity, and to enable molecular improvement efforts with Three-line hybrid rice. Further, our results also indicated that 93-11 could be used as core germplasm for the improvement of wild-abortive CMS lines and the maintainer lines.     

Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants

Background and aims

Whereas the expression patterns and kinetic properties of the rice (Oryza sativa) phosphate transporter gene OsPht1; 6 (OsPT6) are well documented, little is known about the biological functions of this gene. The aim of this study was to investigate the roles of OsPT6 in inorganic phosphate (Pi) acquisition and mobilization, and examine its potential to enhance agricultural production.

Methods

Here, we generated OsPT6 overexpression transgenic plants using Wuyujing 7, a widely cultivated variety of japonica rice, and then treated transgenic lines and wild type with different Pi supply in hydroponic and soil experiments to explore the functions of OsPT6 in rice.

Results

The OsPT6-overexpressing rice lines grew better and accumulated more biomass than wild-type plants, and exhibited significant increases in P accumulation in various tissues, including reproductive tissues under both hydroponic and soil culture conditions. Phosphate-uptake experiment using radiolabeled Pi (³³P) showed that the rate of Pi uptake was 75 % and 73 % greater in transgenic plants grown under Pi-sufficient and -deficient conditions, respectively, than the wild-type controls, and that the shoot/root ratio of ³³P was 104 % and 42 % greater, respectively. In addition, the grain yield per transgenic plant was much higher than that of the wild-type plants under field conditions.

Conclusions

Taken together, our results demonstrate that OsPT6 plays a vital role in Pi acquisition and mobilization in rice and suggest that this gene may be used for genetic engineering rice plants that require less Pi fertilizer.     

Evaluation of Pseudomonas fluorescens for Suppression of Sheath Rot Disease and for Enhancement of Grain Yields in Rice (Oryza sativa L.)

Pseudomonas fluorescens strains antagonistic to Sarocladium oryzae, the sheath rot (Sh-R) pathogen of rice (Oryza sativa L.), were evaluated in greenhouse and field tests for suppression of Sh-R severity and enhancement of grain yields of rice. Imprints of rice seedlings and a direct-observation technique of staining roots with fluorochromes confirmed the association of P. fluorescens with roots and the ability of the strain to move along shoot tips. In greenhouse tests, P. fluorescens-treated rice plants (cv. IR 20) showed a 54% reduction in the length of Sh-R lesions. In three field tests, treatment with P. fluorescens reduced the severity of Sh-R by 20 to 42% in five rice cultivars. Bacterization of rice cultivars with P. fluorescens enhanced plant height, number of tillers, and grain yields from 3 to 160%.     

Transgenic rice expressing the <Emphasis Type="Italic">cry</Emphasis>2AX1 gene confers resistance to multiple lepidopteran pests

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g^?1 leaf fresh weight and with 80–92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7–3.6 ng g^?1). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.     

The transfer RNA genes in Oryza sativa L. ssp. indica

The availability of the draft genome sequence of Oryza sativa L. ssp. indica has made it possible to study the rice tRNA genes. A total of 596 tRNA genes, including 3 selenocysteine tRNA genes and one suppressor tRNA gene are identified in 127551 rice contigs. There are 45 species of tRNA genes and the revised wobble hypothesis proposed by Guthrie and Abelson is perfectly obeyed. The relationship between codon usage and the number of corresponding tRNA genes is discussed. Redundancy may exist in the present list of tRNA genes and novel ones may be found in the future. A set of 33 tRNA genes is discovered in the complete chloroplast genome of Oryza sativa L. ssp. indica. These tRNA genes are identical to those in ssp. japonica identified by us independently from the origional annotation.     

Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis

A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)–induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice.     

Identification of QTLs associated with tissue culture response through sequencing-based genotyping of RILs derived from 93-11 × Nipponbare in rice (Oryza sativa)

Key message

The performance of callus induction and callus differentiation was evaluated by 9 indices for 140 RILs; 2 major QTLs associated with plant regeneration were identified.

Abstract

In order to investigate the genetic mechanisms of tissue culture response, 140 recombinant inbred lines (RILs) derived from 93-11 (Oryza sativa ssp. indica) × Nipponbare (Oryza sativa ssp. japonica) and a high quality genetic map based on the SNPs generated from deep sequencing of the RIL genomes, were used to identify the quantitative trait loci (QTLs) associated with in vitro tissue culture response (TCR) from mature seed in rice. The performance of callus induction was evaluated by indices of induced-callus color (ICC), induced-callus size (ICS), induced-callus friability (ICF) and callus induction rate (CIR), respectively, and the performance of callus differentiation was evaluated by indices of callus proliferation ability (CPA), callus browning tendency (CBT), callus greening ability (CGA), the average number of regenerated shoots per callus (NRS) and regeneration rate (%, RR), respectively. A total of 25 QTLs, 2 each for ICC, ICS, ICF, CIR and CBA, 3 for CPA, 4 each for CGA, NRS and RR, respectively, were detected and located on 8 rice chromosomes. Significant correlations were observed among the traits of CGA, NRS and RR, and QTLs identified for these three indices were co-located on chromosomes 3 and 7, and the additive effects came from both Nipponbare and 93-11, respectively. The results obtained from this study provide guidance for further fine mapping and gene cloning of the major QTL of TCR and the knowledge of the genes underlying the traits investigated would be very helpful for revealing the molecular bases of tissue culture response.     

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.     

The expression profile of genes in rice roots under low phosphorus stress

Phosphorus (P) is one of the most essential macronutrients required for plant growth. Although it is abundant in soil, P is often the limiting nutrient for crop yield potential because of the low concentration of soluble P that plants can absorb directly. The gene expression profile was investigated in rice roots at 6, 24 and 72 h under low P stress and compared with a control (normal P) profile, using a DNA chip of 60000 oligos (70 mer) that represented all putative genes of the rice genome. A total of 795 differentially expressed genes were identified in response to phosphate (Pi) starvation in at least one of the treatments. Based on the analysis, we found that: (i) The genes coding for the Pi transporter, acid phosphatase and RNase were up-regulated in rice roots; (ii) the genes involved in glycolysis were first up-regulated and then down-regulated; (iii) several genes involved in N metabolism and lipid metabolism changed their expression patterns; (iv) some genes involved in cell senescence and DNA or protein degradation were up-regulated; and (v) some transmembrane transporter genes were up-regulated. The results may provide useful information in the molecular process associated with Pi deficiency and thus facilitate research in improving Pi utilization in crop species.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.     

Differential expression of salt-responsive genes to salinity stress in salt-tolerant and salt-sensitive rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) at seedling stage

The understanding of physio-biochemical and molecular attributes along with morphological traits contributing to the salinity tolerance is important for developing salt-tolerant rice (Oryza sativa L.) varieties. To explore these facts, rice genotypes CSR10 and MI48 with contrasting salt tolerance were characterized under salt stress (control, 75 and 150 mM NaCl) conditions. CSR10 expressed higher rate of physio-biochemical parameters, maintained lower Na/K ratio in shoots, and restricted Na translocation from roots to shoots than MI48. The higher expression of genes related to the osmotic module (DREB2A and LEA3) and ionic module (HKT2;1 and SOS1) in roots of CSR10 suppresses the stress, enhances electrolyte leakage, promotes the higher compatible solute accumulation, and maintains cellular ionic homeostasis leading to better salt stress tolerance than MI48. This study further adds on the importance of these genes in salt tolerance by comparing their behaviour in contrasting rice genotypes and utilizing specific marker to identify salinity-tolerant accessions/donors among germplasm; overexpression of these genes which accelerate the selection procedure precisely has been shown.     

Application of T-DNA activation tagging to identify glutamate receptor-like genes that enhance drought tolerance in plants

Key message

A high-quality rice activation tagging population has been developed and screened for drought-tolerant lines using various water stress assays. One drought-tolerant line activated two rice glutamate receptor-like genes. Transgenic overexpression of the rice glutamate receptor-like genes conferred drought tolerance to rice and Arabidopsis.

Abstract

Rice (Oryza sativa) is a multi-billion dollar crop grown in more than one hundred countries, as well as a useful functional genetic tool for trait discovery. We have developed a population of more than 200,000 activation-tagged rice lines for use in forward genetic screens to identify genes that improve drought tolerance and other traits that improve yield and agronomic productivity. The population has an expected coverage of more than 90 % of rice genes. About 80 % of the lines have a single T-DNA insertion locus and this molecular feature simplifies gene identification. One of the lines identified in our screens, AH01486, exhibits improved drought tolerance. The AH01486 T-DNA locus is located in a region with two glutamate receptor-like genes. Constitutive overexpression of either glutamate receptor-like gene significantly enhances the drought tolerance of rice and Arabidopsis, thus revealing a novel function of this important gene family in plant biology.     

Differential responses of antioxidant enzymes to aluminum toxicity in two rice (Oryza sativa L.) cultivars with marked presence and elevated activity of Fe SOD and enhanced activities of Mn SOD and catalase in aluminum tolerant cultivar

Seedlings of two Indica rice (Oryza sativa L.) cvs. HUR-105 and Vandana, differing in Al-tolerance were used to identify the key mechanisms involved in their differential behaviour towards Al toxicity. Cv. HUR-105 appeared to be Al sensitive by showing significant reduction (p ≤ 0.01) in root/shoot length, fresh weight, dry weight and water content in presence of 421 μM Al³⁺ in growth medium whereas cv. Vandana appeared to be fairly Al³⁺ tolerant. A conspicuous and significant reduction in dry weight of root and shoot was observed in Al sensitive cv. HUR-105 with 178 μM Al³⁺ treatment for 3 days. Al was readily taken up by the roots and transported to shoots in both the rice cultivars. Localization of absorbed Al was always greater in roots than in shoots. Our results of the production of reactive oxygen species (ROS) H₂O₂ and O₂ ^.? and activities of major antioxidant enzymes such as total superoxide dismutase (SOD), Cu/Zn SOD, Mn SOD, Fe SOD, catalase (CAT) and guaiacol peroxidase revealed Al induced higher oxidative stress, greater production of ROS and lesser capacity to scavenge ROS in cv. HUR-105 than Vandana. With Al treatment, higher oxidative stress was noted in shoots than in roots. Greatly enhanced activities of SOD (especially Fe and Mn SOD) and CAT in Al treated seedlings of cv. Vandana suggest the role of these enzymes in Al tolerance. Furthermore, a marked presence of Fe SOD in roots and shoots of the seedlings of Al tolerant cv. Vandana and its significant (p ≤ 0.01) increase in activity due to Al-treatment, appears to be the unique feature of this cultivar and indicates a vital role of Fe SOD in Al-tolerance in rice.