Drought tolerance genes in rice

Quantitative trait loci (QTLs) for drought tolerance (DT) can be readily identified in available databases and in this paper, these QTLs were summarized in the form of a consensus map. An in silico strategy was then deployed to mine for candidate genes associated with DT QTLs using rice dbEST and rice genome databases. DT QTLs on rice chromosomes 1, 2, 4, 8, and 9 were selected to test the method. The result showed candidate genes associated with DT could be readily identified.     

Genetic analysis for drought resistance of rice at reproductive stage in field with different types of soil

Drought resistance of rice is a complex trait and is mainly determined by mechanisms of drought avoidance and drought tolerance. The present study was conducted to characterize the genetic basis of drought resistance at reproductive stage in field by analyzing the QTLs for drought response index (DRI, normalized by potential yield and flowering time), relative yield, relative spikelet fertility, and four traits of plant water status and their relationships with root traits using a recombinant inbred population derived from a cross between an indica rice and upland rice. A total of 39 QTLs for these traits were detected with individual QTL explained 5.1–32.1% of phenotypic variation. Only two QTLs for plant water status were commonly detected in two environments, suggesting different mechanisms might exist in two types of soil conditions. DRI has no correlation with potential yield and flowering time under control, suggesting that it can be used as a good drought resistance index in field conditions. The co-location of QTLs for canopy temperature and delaying in flowering time suggested a usefulness of these two traits as indexes in drought resistance screening. Correlation and QTL congruence between root traits and putative drought tolerance traits revealed that drought avoidance (via thick and deep root traits) was the main genetic basis of drought resistance in sandy soil condition, while drought tolerance may play more role in the genetic basis of drought resistance in paddy soil condition. Therefore, both drought mechanisms and soil textures must be considered in the improvement of drought resistance at reproductive stage in rice.     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Screening of rice (Oryza sativa L.) genotypes for physiological characters contributing to salinity resistance,and their relationship to overall performance

Summary Phenotypic resistance of salinity is expressed as the ability to survive and grow in a salinised medium. Some subjective measure of overall performance has normally been used in plant breeding programmes aimed at increasing salinity resistance, not only to evaluate progeny, but to select parents. Salinity resistance has, at least implicitly, been treated as a single trait. Physiological studies of rice suggest that a range of characteristics (such as low shoot sodium concentration, compartmentation of salt in older rather than younger leaves, tolerance to salt within leaves and plant vigour) would increase the ability of the plant to cope with salinity. We describe the screening of a large number of rice genotypes for overall performance (using an objective measure based on survival) and for the aforementioned physiological traits. There was wide variation in all the characters studied, but only vigour was strongly correlated with survival. Shoot sodium concentration, which a priori is expected to be important, accounted for only a small proportion of the variability in the survival of salinity. Tissue tolerance (the cellular component of resistance reflecting the ability to compartmentalise salt within leaves) revealed a fivefold range between genotypes in the tolerance of their leaves to salt, but this was not correlated positively with survival. On the basis of such (lack of) correlation, these traits would be rejected in normal plant breeding practice, but we discuss the fallacies involved in attempting correlation between individual traits and the overall performance of a salt-sensitive species in saline conditions. We conclude that whilst overall performance (survival) can be used to evaluate the salt resistance of a genotype, it is not the basis on which parents should be selected to construct a complex character through breeding. It was the norm for varieties which had one good characteristic affecting salt resistance to be unexceptional or poor in the others. This constitutes experimental evidence that the potential for salt resistance present in the rice genome has not been realised in genotypes currently extant. The results are discussed in relation to the use of physiological traits in plant breeding, with particular reference to environmental stresses that do not affect a significant part of a species' ecological range.     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

RFLP tagging of a gene for aroma in rice

Summary We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F₃ segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.     

Influence of boric acid on somatic embryogenesis of a cytosterile line of indica rice

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22 μM to the embryo induction medium containing basal salts, with 9.84 μM 2,4-dichlorophenoxyacetic acid. Boric acid was found to exert different effects on scutellum, coleoptile and root tissues. Increased growth of embryogenic masses with increasing boric acid concentrations was observed on coleoptile tissues of mature seed. Embryogenic response was observed at 161.29 μM boric acid on coleoptile and 241.93 μM boric acid on root tissue. Scutellum tissue showed embryonic response in 161.29 and 241.93 μM boric acid. The results indicate that the boric acid used in standard Murashige and Skoog medium is not sufficient to support induction of somatic embryogenesis in IR-58025 A. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves

Polyethylene glycol (PEG) and sorbitol (ST) have each been used inosmotically induced water stress studies in plants, however, these osmotica maynot have equivalent effects in plants. The present study was designed to examinewhether antioxidant enzyme responses in rice leaves are different for PEG and STof osmotic potential –1.5 MPa. As judged by relative watercontent, PEG treatment resulted in a higher degree of water stress in riceleaves than ST treatment. PEG treatment markedly increased lipid peroxidation,judged by malondialdehyde content, in rice leaves. However, ST treatment had noeffect on lipid peroxidation. An increase in peroxidase (POX), ascorbateperoxidase (APX) and glutathione reductase (GR) activities was observed in riceleaves treated with ST. PEG treatment had no effect on POX and APX activitiesand decreased GR activity in rice leaves. The decrease in superoxide dismutaseactivity induced by PEG was more pronounced than by ST. Cycloheximide blockedthe enhanced activities of POX, APX and GR by ST, indicating de novo synthesisof the enzymes. Results suggest that ST but not PEG treatment can up-regulateantioxidant system in rice leaves.     

Fine linkage mapping enables dissection of closely linked quantitative trait loci for seed dormancy and heading in rice

Two quantitative trait loci (QTLs) for seed dormancy (tentatively designated Sdr1) and heading date (Hd8) have been mapped to approximately the same region on chromosome 3 by interval mapping of backcross inbred lines derived from crosses between the rice cultivars Nipponbare (japonica) and Kasalath (indica). To clarify whether Sdr1 and Hd8 could be dissected genetically, we carried out fine-scale mapping with an advanced backcross progeny. We selected a BC₄F₁ plant, in which a small chromosomal region including Sdr1 and Hd8, on the short arm of chromosome 3, remained heterozygous, whereas all the other chromosomal regions were homozygous for Nipponbare. Days-to-heading and seed germination rate in the BC₄F₂ plants showed continuous variation. Ten BC₄F₂ plants with recombination in the vicinity of Sdr1 and Hd8 were selected on the basis of the genotypes of the restriction fragment length polymorphism (RFLP) markers flanking both QTLs. Genotypes of those plants for Sdr1 and Hd8 were determined by advanced progeny testing of BC₄F₄ families. Sdr1 was mapped between the RFLP markers R10942 and C2045, and co-segregated with C1488. Hd8 was also mapped between C12534S and R10942. Six recombination events were detected between Sdr1 and Hd8. These results clearly demonstrate that Sdr1 and Hd8 were tightly linked. Nearly isogenic lines for Sdr1 and Hd8 were selected by marker-assisted selection.Communicated by D. Mackill     

QTLs for nutritional contents of rice seedlings (Oryza sativa L.) in solution cultures and its implication to tolerance to iron-toxicity

Iron toxicity is one of the major constraints for lowland rice production in highly weathered soils, which are widely distributed in tropics and subtropics and often lack macronutrients such as potassium (K) and phosphorus (P). To analyze the genetic factors for excess iron accumulation under K or P deficiency, a set of seedlings in F₃ and F₈ generations from an Oryza sativa cross between a japonica cultivar ‚Gimbozu’ and an indica cultivar ‚Kasalath’ were raised and exposed to nutritional stresses in a short period under nutritional solutions. In the F₈ lines, contents of K, P, Fe, and Mg in dried shoots were measured. Quantitative trait loci (QTL) for the iron accumulation and related mineral contents in each plant were analyzed with composite interval mapping. QTLs for the Fe, P and Mg content in shoots were compared in the maps of F₃ and F₈. The QTLs for the Fe content in shoots varied in three types of nutritional conditions, but consistently indicated two overlapping regions on chromosome 3 and 4. The obtained QTLs were crosschecked with those reported before. Some of these QTLs were indicative of iron excluding the power of the root, which was expressed under reduced P content in solution.     

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

Bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most serious diseases of rice. The recessive gene xa-13 confers resistance to Philippine race 6 of Xoo. To tag xa-13 with molecular markers, RAPD analysis was conducted with the combined use of near-isogenic lines and bulked segregant analysis. From the survey of 260 arbitrary 10-nucleotide primers, one primer (OPAC05) was detected to amplify specifically a 0.9-kb band from the DNA of susceptible plants. The distance between the RAPD marker OPAC05-900 and xa-13 was estimated to be 5.3 cM. The RAPD marker was then mapped on chromosome 8 using a mapping population of doubled haploid lines derived from the cross of IR64/Azucena. The linkage between RFLP markers and the RAPD marker was analyzed using an F₂ population of 135 plants derived from a cross between a near-isogenic line for xa-13, IR66699-5-5-4-2, and IR24. No recombinants were found between RZ28 and CDO116 and their distance from xa-13 was estimated to be 4.8 cM. RG136 was located at 3.7 cM on the other side of xa-13. The mapping of xa-13 with closely linked DNA markers provides the basis for marker-aided selection for rice improvement.Department of Agronomy, South China Agricultural University, Guangzhou, China     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Transgene flow to hybrid rice and its male-sterile lines

Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640m² in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037–0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1–2 m; (3) The maximum distance of transgene flow was 30–40 m to rice cultivars and 40–150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used. Shirong Jia, Feng Wang and Lei Shi contributed equally to this investigation.