Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice

We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease inhibitor, and a glutathione-S-transferase were expressed at significantly higher levels in CT9993 than in IR62266. Thus several genes involved in the regulation of DNA structure and mRNA translation were found to be drought-regulated, and may be implicated in drought resistance.Communicated by R. Hagemann     

Systematic analysis of <Emphasis Type="Italic">NPK1</Emphasis>-like genes in rice reveals a stress-inducible gene cluster co-localized with a quantitative trait locus of drought resistance

Phosphorylation by protein kinase is a ubiquitous key mechanism in translating external stimuli such as drought stress. NPK1 is a mitogen-activated protein kinase kinase kinase identified in Nicotiana tabacum and plays important roles in cytokinesis and auxin signaling transduction and responses to multiple stresses. Here we report the evolution, structure, and comprehensive expression profile of 21 NPK1-like genes in rice (Oryza sativa L.). Phylogenetic analysis of NPK1-like sequences in rice (OsNPKL), Arabidopsis, and other plants reveals that NPK1-like genes could be classified into three subgroups. Three OsNPKL gene clusters, located on chromosome 1 (OsNPKL1, 2, 3, and 4), 5 (OsNPKL14 and 15), and 10 (OsNPKL19 and 20), respectively, were identified in the rice genome. These clustered genes, which most likely evolved by tandem gene duplication, belong to the same phylogenetic subgroup, with similar genomic structures and conserved motifs in the kinase domain, which is unique to this subgroup. Expression analysis of OsNPKL genes under abiotic stresses suggests that the stress-responsive genes are mainly from the same subgroup. Especially interesting is that all the clustered genes are induced by drought, salt, or cold stress, and a few members are very strongly induced by drought. Some of the clustered genes are also induced by abscisic acid. The gene cluster on chromosome 1 is co-located with a quantitative trait locus (QTL) related to drought resistance. Although the drought-induced expression levels of the four genes in the cluster show no difference between the two parents used for QTL mapping, sequence variation in coding regions of the genes between the parents has provided some clues for further functional characterization of this gene cluster in abiotic stress tolerance in rice.     

A single segment substitution line population for identifying traits relevant to drought tolerance and avoidance

A population of chromosome segment substitution lines was developed using KDML105 as the recurrent parent and one of DH212 (IR68586-F2-CA-143) or DH103 (IR68586-F2-CA-31) as the donor parent. The donor parents are part of a doubled haploid population from a cross between CT9993, an upland japonica accession, and IR62266, a lowland indica accession. Multiple QTL that are relevant to drought avoidance, drought tolerance and yield traits under drought stress were mapped in this doubled haploid population and the segments selected for the chromosome segment substitution lines were chosen to capture these QTL. The chromosome segment substitution line population was phenotyped under irrigated and mild drought stress conditions, which identified that many yield traits under drought stress had been introduced into the chromosome segment substitution lines.     

Evaluation of near-isogenic lines for drought resistance QTL and fine mapping of a locus affecting flag leaf width, spikelet number, and root volume in rice

Drought stress is a major limiting factor for crop production and breeding for drought resistance is very challenging due to the complex nature of this trait. Previous studies in rice suggest that the upland japonica variety IRAT109 shows better drought resistance than the lowland indica variety Zhenshan 97. Numerous quantitative trait loci (QTL) have been previously mapped using a recombinant inbred line population derived from these two genotypes. In this study, near-isogenic lines (NILs) for 17 drought resistance-related QTL were constructed and phenotypic variations of these NILs were investigated under drought and normal conditions. Fourteen of these NILs showed significant phenotypic differences relative to the recurrent parent under at least one of the conditions and nine NILs showed significant differences under both conditions. After eliminating the effect of heading date on drought resistance, only four NILs carrying seven QTL (four for the same grain yield-related traits and three for the same or similar root traits QTL) showed differences consistent with the original QTL mapping results. One of these lines (N19) contains qFSR4, a QTL on chromosome 4 controlling root volume per tiller and co-segregating with flag leaf width and spikelet number per panicle. Using a population derived from N19, qFSR4 was mapped to a 38-kb region containing three open reading frames including the previously characterized NARROW LEAF 1 (NAL1) gene. NAL1, which controls leaf width and also affects vein patterning and polar auxin transport, is the most promising candidate genes for qFSR4. Our results underscore the importance of the development of NILs to confirm the identification of QTL affecting complex traits such as drought resistance.     

Meta-analysis of quantitative trait loci for grain yield and component traits under reproductive-stage drought stress in an upland rice population

A recombinant inbred population developed from a cross between high-yielding lowland rice (Oryza sativa L.) subspecies indica cv. IR64 and upland tropical rice subspecies japonica cv. Cabacu was used to identify quantitative trait loci (QTLs) for grain yield (GY) and component traits under reproductive-stage drought stress. One hundred fifty-four lines were grown in field trials in Indonesia under aerobic conditions by giving surface irrigation to field capacity every 4 days. Water stress was imposed for a period of 15 days during pre-flowering by withholding irrigation at 65 days after seeding. Leaf rolling was scored at the end of the stress period and eight agronomic traits were evaluated after recovery. The population was also evaluated for root pulling force, and a total of 201 single nucleotide polymorphism markers were used to construct the molecular genetic linkage map and QTL mapping. A QTL for GY under drought stress was identified in a region close to the sd1 locus on chromosome 1. QTL meta-analysis across diverse populations showed that this QTL was conserved across genetic backgrounds and co-localized with QTLs for leaf rolling and osmotic adjustment (OA). A QTL for percent seed set and grains per panicle under drought stress was identified on chromosome 8 in the same region as a QTL for OA previously identified in three different populations.     

Marker-assisted selection to introgress rice QTLs controlling root traits into an Indian upland rice variety

A marker-assisted back-crossing (MABC) breeding programme was conducted to improve the root morphological traits, and thereby drought tolerance, of the Indian upland rice variety, Kalinga III. This variety, the recurrent parent in the MABC, had not previously been used for quantitative trait locus (QTL) mapping. The donor parent was Azucena, an upland japonica variety from Philippines. Five segments on different chromosomes were targeted for introgression; four segments carried QTLs for improved root morphological traits (root length and thickness) and the fifth carried a recessive QTL for aroma. Some selection was made at non-target regions for recurrent parent alleles. We describe the selection made in three backcross (BC) generations and two further crosses between BC₃ lines to pyramid (stack) all five target segments. Pyramids with four root QTLs were obtained in eight generations, completed in 6 years using 3,000 marker assays in a total of 323 lines. Twenty-two near-isogenic lines (NILs) were evaluated for root traits in five field experiments in Bangalore, India. The target segment on chromosome 9 (RM242-RM201) significantly increased root length under both irrigated and drought stress treatments, confirming that this root length QTL from Azucena functions in a novel genetic background. No significant effects on root length were found at the other four targets. Azucena alleles at the locus RM248 (below the target root QTL on chromosome 7) delayed flowering. Selection for the recurrent parent allele at this locus produced early-flowering NILs that were suited for upland environments in eastern India.     

Quantitative trait loci associated with drought tolerance at reproductive stage in rice

Drought is a major constraint to rice (Oryza sativa) yield and its stability in rainfed and poorly irrigated environments. Identifying genomic regions influencing the response of yield and its components to water deficits will aid in our understanding of the genetics of drought tolerance and development of more drought tolerant cultivars. Quantitative trait loci (QTL) for grain yield and its components and other agronomic traits were identified using a subset of 154 doubled haploid lines derived from a cross between two rice cultivars, CT9993-510 to 1-M and IR62266-42 to 6-2. Drought stress treatments were managed by use of a line source sprinkler irrigation system, which provided a linearly decreasing level of irrigation coinciding with the sensitive reproductive growth stages. The research was conducted at the Ubon Rice Research Center, Ubon, Thailand. A total of 77 QTL were identified for grain yield and its components under varying levels of water stress. Out of the total of 77 QTL, the number of QTL per trait were: 7-grain yield (GY); 8-biological yield (BY); 6-harvest index (HI); 5-d to flowering after initiation of irrigation gradient (DFAIG); 10-total spikelet number (TSN); 7-percent spikelet sterility (PSS); 23-panicle number (PN); and 11-plant height (PH). The phenotypic variation explained by individual QTL ranged from 7.5% to 55.7%. Under well-watered conditions, we observed a high genetic association for BY, HI, DFAIG, PSS, TSN, PH, and GY. However, only BY and HI were found to be significantly associated with GY under drought treatments. QTL flanked by markers RG104 to RM231, EMP2_2 to RM127, and G2132 to RZ598 on chromosomes 3, 4, and 8 were associated with GY, HI, DFAIG, BY, PSS, and PN under drought treatments. The aggregate effects of these QTL on chromosomes 3, 4, and 8 resulted in higher grain yield. These QTL will be useful for rainfed rice improvement, and will also contribute to our understanding of the genetic control of GY under drought conditions at the sensitive reproductive stage. Close linkage or pleiotropy may be responsible for the coincidence of QTL detected in this experiment. Digenic interactions between QTL main effects for GY, BY, HI, and PSS were observed under irrigation treatments. Most (but not all) DH lines have the same response in measure of productivity when the intensity of water deficit was increased, but no QTL by irrigation treatment interaction was detected. The identification of genomic regions associated with GY and its components under drought stress will be useful for marker-based approaches to improve GY and its stability for farmers in drought-prone rice environments.     

QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress

Cell-membrane stability (CMS) is considered to be one of the major selection indices of drought tolerance in cereals. In order to determine which genomic region is responsible for CMS, 104 rice (Oryza sativa L.) doubled haploid (DH) lines derived from a cross between CT9993–5-10–1-M and IR62266-42–6-2 were studied in the greenhouse in a slowly developed drought stress environment. Drought stress was induced on 50-day-old plants by withholding water. The intensity of stress was assessed daily by visual scoring of leaf wilting and by measuring leaf relative water content (RWC). The leaf samples were collected from both control (well-watered) and stressed plants (at 60–65% of RWC), and the standard test for CMS was carried out in the laboratory. There was no significant difference (P>0.05) in RWC between the two parental lines as well as among the 104 lines, indicating that all the plants were sampled at a uniform stress level. However, a significant difference (P<0.05) in CMS was observed between the two parental lines and among the population. No significant correlation was found between CMS and RWC, indicating that the variation in CMS was genotypic in nature. The continuous distribution of CMS and its broad-sense heritability (34%) indicates that CMS should be polygenic in nature. A linkage map of this population comprising of 145 RFLPs, 153 AFLPs and 17 microsatellite markers was used for QTL analysis. Composite interval mapping identified nine putative QTLs for CMS located on chromosomes 1, 3, 7, 8, 9, 11 and 12. The amount of phenotypic variation that was explained by individual QTLs ranged from 13.4% to 42.1%. Four significant (P<0.05) pairs of digenic interactions between the detected QTLs for CMS were observed. The identification of QTLs for this important trait will be useful in breeding for the improvement of drought tolerance in rice. This is the first report of mapping QTLs associated with CMS under a natural water stress condition in any crop plants. Received: 8 September 1999 / Accepted: 13 October 1999     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Δ¹³C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE. Electronic Supplementary Material Supplementary material is available for this article at     

Multiple genome alignment for identifying the core structure among moderately related microbial genomes

Background

LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown.

Results

We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded.

Conclusion

The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate the functional role of these enigmatic proteins.     

QTLs linked to leaf epicuticular wax,physio-morphological and plant production traits under drought stress in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Drought stress is the major constraint to rice (Oryza sativa L.) production and yield stability in rainfed ecosystems. Identifying genomic regions contributing to drought resistance will help to develop rice cultivars suitable for rainfed regions through marker-assisted breeding. Quantitative trait loci (QTLs) linked to leaf epicuticular wax, physio-morphological and plant production traits under water stress and irrigated conditions were mapped in a doubled haploid (DH) line population from the cross CT9993-5-10-1-M/IR62266-42-6-2. The DH lines were subjected to water stress during anthesis. The DH lines showed significant variation for epicuticular wax (EW), physio-morphological and plant production traits under stress and irrigated conditions. A total of 19 QTLs were identified for the various traits under drought stress and irrigated conditions in the field, which individually explained 9.6%–65.6% of the phenotypic variation. A region EM15_10-ME8_4-R1394A-G2132 on chromosome 8 was identified for leaf EW and rate of water loss i.e., time taken to reach 70% RWC from excised leaves in rice lines subjected to drought stress. A large effect QTL (65.6%) was detected on chromosome 2 for harvest index under stress. QTLs identified for EW, rate of water loss from excised leaves and harvest index under stress in this study co-located with QTLs linked to shoot and root-related drought resistance traits in these rice lines and might be useful for rainfed rice improvement.     

Candidate genes for drought tolerance and improved productivity in rice (Oryza sativa L.)

Candidate genes are sequenced genes of known biological action involved in the development or physiology of a trait. Twenty-one putative candidate genes were designed after an exhaustive search in the public databases along with an elaborate literature survey for candidate gene products and/or regulatory sequences associated with enhanced drought resistance. The downloaded sequences were then used to design primers considering the flanking sequences as well. Polymerase chain reaction (PCR) performed on 10 diverse cultivars that involvedJaponica, Indica and local accessions, revealed 12 polymorphic candidate genes. Seven polymorphic candidate genes were then utilized to genotype 148 individuals of CT9993 × IR62266 doubled haploid (DH) mapping population. The segregation data were tested for deviation from the expected Mendelian ratio (1:1) using a Chi-square test (<1%). Based on this, four candidate genes were assessed to be significant and the remaining three, as non-significant. All the significant candidate genes were biased towards CT9993, the female parent in the DH mapping population. Single-marker analysis strongly associated (<1%) them to different traits under both well-watered and low-moisture stress conditions. Two candidate genes,EXP15 andEXP13, were found to be associated with root number and silicon content in the stem respectively, under both well-watered and low-moisture stress conditions     

The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling,drought and salt stress response

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1‐silenced peppers and CaLEA1‐overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus‐induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl‐treated leaves. CaLEA1‐OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild‐type plants because of enhanced stomatal closure and increased expression of stress‐responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA‐mediated cell signaling.     

Mapping QTLs associated with drought avoidance in upland rice

The identification of molecular markers linked to genes controlling drought resistance factors in rice is a necessary step to improve breeding efficiency for this complex trait. QTLs controlling drought avoidance mechanisms were analyzed in a doubled-haploid population of rice. Three trials with different drought stress intensities were carried out in two sites. Leaf rolling, leaf drying, relative water content of leaves and relative growth rate under water stress were measured on 105 doubled haploid lines in two trials and on a sub-sample of 85 lines in the third one. Using composite interval mapping with a LOD threshold of 2.5, the total number of QTLs detected in all trials combined was 11 for leaf rolling, 10 for leaf drying, 11 for relative water content and 10 for relative growth rate under stress. Some of these QTLs were common across traits. Among the eleven possible QTLs for leaf rolling, three QTLs (on chromosomes 1, 5 and 9) were common across the three trials and four additional QTLs (on chromosomes 3, 4 and 9) were common across two trials. One QTL on chromosome 4 for leaf drying and one QTL on chromosome 1 for relative water content were common across two trials while no common QTL was identified for relative growth rate under stress. Some of the QTLs detected for leaf rolling, leaf drying and relative water content mapped in the same places as QTLs controlling root morphology, which were identified in a previous study involving the same population. Some QTL identified here were also located similarly with other QTLs for leaf rolling as reported from other populations. This study may help to chose the best segments for introgression into rice varieties and improvement of their drought resistance.     

QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice

Field resistance is defined as the resistance that allows effective control of a parasite under natural field conditions and is durable when exposed to new races of that parasite. To identify the genes for field resistance to rice blast, quantitative trait loci (QTLs) conferring field resistance to rice blast in Japanese upland rice were detected and mapped using RFLP and SSR markers. QTL analysis was carried out in F₄ progeny lines from the cross between Nipponbare (moderately susceptible, lowland) and Owarihatamochi (resistant, upland). Two QTLs were detected on chromosome 4 and one QTL was detected on each of chromosomes 9 and 12. The phenotypic variation explained by each QTL ranged from 7.9 to 45.7% and the four QTLs explained 66.3% of the total phenotypic variation. Backcrossed progeny lines were developed to transfer the QTL with largest effect using the susceptible cultivar Aichiasahi as a recurrent parent. Among 82 F₃ lines derived from the backcross, resistance segregated in the expected ratio of resistant 1 : heterozygous 2 : susceptible 1. The average score for blast resistance measured in the field was 4.2 ± 0.67, 7.5 ± 0.51and 8.2 ± 0.66, for resistant, heterozygous and susceptible groups, respectively. The resistance gene, designated pi21, was mapped on chromosome 4 as a single recessive gene between RFLP marker loci G271 and G317 at a distance of 5.0 cM and 8.5 cM, respectively. The relationship to previously reported major genes and QTLs conferring resistance to blasts, and the significance of marker-assisted selection to improve field resistance, are discussed. Received: 8 June 2000 / Accepted: 24 November 2000