水稻的耐淹性状及其Sub1基因

近4年来, 水稻(Oryza sativa L.)耐淹性状及其分子机理的研究取得了长足的进展。水稻植株的耐淹性状主要由Submergence-1 (Sub1) 基因控制。Sub1通过调节乙烯和赤霉素介导的反应, 抑制淹水期间的伸长生长和减缓碳水化合物消耗来控制耐淹性状。文章介绍了水稻应对淹涝胁迫的两种策略, 影响耐淹性状的主要生理因素, Sub1基因定位以及它控制耐淹性状的机理; 阐述了Sub1基因在我国杂交水稻生产方面的应用前景。     

Evolutionary analysis of the Sub1 gene cluster that confers submergence tolerance to domesticated rice

BACKGROUND AND AIMS: Tolerance of complete submergence is recognized in a small number of accessions of domesticated Asian rice (Oryza sativa) and can be conferred by the Sub1A-1 gene of the polygenic Submergence-1 (Sub1) locus. In all O. sativa varieties, the Sub1 locus encodes the ethylene-responsive factor (ERF) genes Sub1B and Sub1C. A third paralogous ERF gene, Sub1A, is limited to a subset of indica accessions. It is thought that O. sativa was domesticated from the gene pools of the wild perennial species O. rufipogon Griff. and/or the annual species O. nivara Sharma et Shastry. The aim of this study was to evaluate the orthologues of the Sub1 locus in the closest relatives of O. sativa to provide insight into the origin of the gene and allelic variation of the Sub1 locus. METHODS: Orthologues of the Sub1 genes were isolated from O. rufipogon and O. nivara by use of oligonucleotide primers corresponding to the most highly conserved regions of the Sub1 genes of domesticated rice. The phylogenetic relatedness of Sub1 genes of O. sativa and its wild relatives was evaluated. KEY RESULTS AND CONCLUSIONS: Both O. rufipogon and O. nivara possess two Sub1 gene orthologues with strong sequence identity to the Sub1B and Sub1C alleles of cultivated rice. The phylogeny of the Sub1 genes of the domesticated and wild rice suggests that Sub1A arose from duplication of Sub1B. Variation in Sub1B alleles is correlated with the absence or presence of Sub1A. Together, the results indicate that genetic variation at the Sub1 locus is due to gene duplication and divergence that have occurred both prior to and after rice domestication.     

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F₃ families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F₂ plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region. Received: 15 December 1999 / Accepted: 18 February 2000     

Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of α-amylase production in flooded rice plants

Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of α-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A.     

A marker-assisted backcross approach for developing submergence-tolerant rice cultivars

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC₂F₂ generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC₃F₂ double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3–3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Introgression of dual abiotic stress tolerance QTLs (Saltol QTL and Sub1 gene) into Rice (Oryza sativa L.) variety Aiswarya through marker assisted backcross breeding

Salinity and submergence are two very prominent abiotic stress conditions affecting rice yield adversely in the coastal agro ecosystem. Marker Assisted Backcross Breeding (MABB) is an efficient and fast track molecular tool to incorporate a desired stress tolerant QTL/gene into an improved cultivar. The present study was carried out for the introgression of Saltol QTL responsible for salinity tolerance and Sub1 gene responsible for submergence tolerance into the high yielding rice variety Aiswarya independently through MABB. Final objective of the study is to develop dual stress tolerant (tolerance to salinity and submergence) Aiswarya rice variety by pyramiding the both target QTLs introgressed BC₂F₂ progenies having maximum background homozygosity. The donors of Saltol QTL and Sub1 gene used in the present study were FL478 and Swarna Sub1, respectively. Based on the background genome analysis of the introgressed plants, the plants with > 85–90% background similarity were selected for pyramiding of Saltol QTL and Sub1 gene into the elite background of rice variety Aiswarya. Those selected introgressed lines with Saltol QTL and Sub1 gene will be again crossed to pyramid both Saltol QTL and Sub1 gene into the rice variety Aiswarya. Such a mega rice variety pyramided with dual stress tolerant QTLs is the expected outcome of this study and can be recommended for cultivation in the flood prone saline coastal agroecosystem.     

Genes/QTLs affecting flood tolerance in rice

The adaptation of deepwater rice to flooding is attributed to two mechanisms, submergence tolerance and plant elongation. Using a QTL mapping study with replicated phenotyping under two contrasting (water qualities) submergence treatments and AFLP markers, we were able to identify several genes/QTLs that control plant elongation and submergence tolerance in a recombinant inbred rice population. Our results indicate that segregation of rice plants in their responses to different flooding stress conditions is largely due to the differential expression of a few key elongation and submergence tolerance genes. The most important gene was QIne1 mapped near sd-1 on chromosome 1. The Jalmagna (the deepwater parent) allele at this locus had a very large effect on internal elongation and contributed significantly to submergence tolerance under flooding. The second locus was a major gene, sub1(t), mapped to chromosome 9, which contributed to submergence tolerance only. The third one was a QTL, QIne4, mapped to chromosome 4. The IR74 (non-elongating parent) allele at this locus had a large effect for internal elongation. An additional locus that interacted strongly with both QIne1 and QIne4 appeared near RG403 on chromosome 5, suggesting a complex epistatic relationship among the three loci. Several QTLs with relatively small effects on plant elongation and submergence tolerance were also identified. The genetic aspects of these flooding tolerance QTLs with respect to patterns of differential expression of elongation and submergence tolerance genes under flooding are discussed. Received: 13 December 1999 / Accepted: 14 March 2000<@head-com-p1a.lf>Communicated by G. Wenzel     

Mapping QTLs for submergence tolerance in rice using a population fixed for <Emphasis Type="Italic">SUB1A</Emphasis> tolerant allele

Submergence is a widespread problem of rice production, especially in low-lying areas in South and Southeast Asia. Despite the success of Sub1 mega varieties, repeated instances of prolonged and severe flooding in stress-prone areas suggests that the SUB1 gene is no longer sufficient in those regions and requires improved varieties with increased tolerance. A study was conducted to identify quantitative trait loci (QTLs) associated with submergence tolerance using 115 F₇ recombinant inbred lines (RILs) derived from the cross of Ciherang-Sub1, a popular Indonesian cultivar carrying the SUB1 gene that has relatively higher tolerance to submergence compared to the performance of most other Sub1 lines and the submergence and stagnant flooding tolerant IR10F365. As the tolerant allele at SUB1A on chromosome 9 was fixed in this mapping population, additional QTLs responsible for submergence tolerance were expected to be revealed. Genotyping with an Infinium 6K SNP chip resulted in 469 polymorphic markers that were then used for QTL mapping. Phenotyping was performed under complete submergence with two replicates. A major QTL for submergence derived from Ciherang-Sub1, named qSUB8.1, was detected on chromosome 8 with a LOD score of 10.3 and phenotypic variance of 27.5%. Additionally, a smaller QTL, also derived from Ciherang-Sub1, was detected on chromosome 2 with a LOD score of 3.5 and phenotypic variance of 12.7%. There was no digenic interaction detected between these QTLs suggesting their independent action. The QTLs detected in this study can be used in marker-assisted selection to further improve the tolerance of other Sub1 varieties.     

Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond

Background and Aims

Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F₁ hybrids heterozygous for the Sub1A-1-tolerant allele.

Methods

Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data.

Key Results

All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent.

Conclusions

Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids.Key words: Oryza sativa, Sub1, marker-assisted backcrossing, mega varieties, submergence tolerance, recombinant, hybrid, abiotic stress     

A major locus for submergence tolerance mapped on rice chromosome 9

Submergence stress is a widespread problem in rice-growing environments where drainage is impeded. A few cultivars can tolerate more than 10 days of submergence, but the genes conferring this tolerance have not been identified. We used randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to map submergence tolerance in 169 F₂ plants and the resulting F₃ families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851. IR40931-26 inherited strong submergence tolerance from the unimproved cultivar FR13A. Eight-day old F₃ seedlings were submerged for 14–16 days in 55-cm deep tanks, and tolerance was scored after 7 days recovery on a scale of 1 (tolerant) to 9 (susceptible). The tolerant and susceptible parents scored 1.5 and 8.4, respectively, and the F₃ means ranged from 1.6 to 8.9. Two bulks were formed with DNA from F₂ plants corresponding to the nine most tolerant and the nine most susceptible F₃ families. Of 624 RAPD primers used to screen the bulks, five produced bands associated with either tolerance or susceptibility. These markers were mapped to a region of chromosome 9 by linkage to RFLP markers. A submergence tolerance quantitative trait locus (QTL), here designatedSub1, was located ca. 4 cM from the RFLP marker C1232 and accounted for 69% of the phenotypic variance for the trait.     

The key regulator of submergence tolerance,SUB1A,promotes photosynthetic and metabolic recovery from submergence damage in rice leaves

The submergence‐tolerance regulator, SUBMERGENCE1A (SUB1A), of rice (Oryza sativa L.) modulates gene regulation, metabolism and elongation growth during submergence. Its benefits continue during desubmergence through protection from reactive oxygen species and dehydration, but there is limited understanding of SUB1A's role in physiological recovery from the stress. Here, we investigated the contribution of SUB1A to desubmergence recovery using the two near‐isogenic lines, submergence‐sensitive M202 and tolerant M202(Sub1). No visible damage was detected in the two genotypes after 3 d of submergence, but the sublethal stress differentially altered photosynthetic parameters and accumulation of energy reserves. Submergence inhibited photosystem II photochemistry and stimulated breakdown of protein and accumulation of several amino acids in both genotypes at similar levels. Upon desubmergence, however, more rapid return to homeostasis of these factors was observed in M202(Sub1). Submergence considerably restrained non‐photochemical quenching (NPQ) in M202, whereas the value was unaltered in M202(Sub1) during the stress. Upon reaeration, submerged plants encounter sudden exposure to higher light. A greater capability for NPQ‐mediated photoprotection can benefit the rapid recovery of photosynthetic performance and energy reserve metabolism in M202(Sub1). Our findings illuminate the significant role of SUB1A in active physiological recovery upon desubmergence, a component of enhanced tolerance to submergence.     

Rice with SUB1 QTL possesses greater initial leaf gas film thickness leading to delayed perception of submergence stress

Background and AimsSubmergence tolerance in rice is primarily attributed to the action of the SUB1 gene, but other associated traits such as leaf gas film (LGF) thickness, leaf hydrophobicity, porosity and leaf density have been known to aid submergence tolerance in rice. However, association of these traits with SUB1 quantitative trait locus (QTL) has not been demonstrated. In this study, we aim to investigate (1) whether the presence of the SUB1 QTL in the genetic background has any influence on the thickness of the LGF and (ii) whether its removal has any impact on stress perception and submergence tolerance in Sub1 and non-Sub1 rice.MethodsWe examined 12 genotypes (including both Sub1 and non-Sub1 types) for different leaf traits such as initial LGF thickness, leaf hydrophobicity, tissue porosity and leaf density in order to work out the relatioship of these traits to the SUB1 QTL in rice. Furthermore, we investigated the changes in the gene expression profile and different metabolic processes in selected genotypes in the presence and absence of their LGF to study its impact on stress perception and adaptation.Key ResultsThe initial thickness of the LGF and hydrophobicity seemed to have a highly positive correlation with the presence of the SUB1 QTL in the genetic background of rice; however, other leaf traits such as porosity and density seemed to be independent of it. Artificial removal of the LGF resulted in partial loss of tolerance, showing increased ethylene production and early induction of anoxia-related genes (SUB1A-1, ACS5, Ramy3D and ADH1) which manifested symptoms such as increased stem elongation, faster chlorophyll and starch breakdown, and partial loss of quiescence in SUB1-containing rice genotypes. Stripping of the LGF resulted in early and enhanced induction of SUB1A-1, indicating a quicker perception of stress.ConclusionsThe presence of SUB1 in the genetic background positively influences surface hydrophobicity and the concomitant LGF thickness of rice. Furthermore, LGF helps in terms of providing better ethylene dissipation and reduced in planta accumulation, owing to the slowing down of ethylene-induced leaf senescence under submergence stress.     

Molecular Genetics of Submergence Tolerance in Rice: QTL Analysis of Key Traits

Flash flooding of young rice plants is a common problem forrice farmers in south and south-east Asia. It severely reducesgrain yield and increases the unpredictability of cropping.The inheritance and expression of traits associated with submergencestress tolerance at the seedling stage are physiologically andgenetically complex. We exploited naturally occurring differencesbetween certain rice lines in their tolerance to submergenceand used quantitative trait loci (QTL) mapping to improve understandingof the genetic and physiological basis of submergence tolerance.Three rice populations, each derived from a single cross betweentwo cultivars differing in their response to submergence, wereused to identify QTL associated with plant survival and variouslinked traits. These included total shoot elongation under water,the extent of stimulation of shoot elongation caused by submergence,a visual submergence tolerance score, and leaf senescence underdifferent field conditions, locations and years. Several majorQTL determining plant survival, plant height, stimulation ofshoot elongation, visual tolerance score and leaf senescenceeach mapped to the same locus on chromosome 9. These QTL weredetected consistently in experiments across all years and inthe genetic backgrounds of all three mapping populations. SecondaryQTL influencing tolerance were also identified and located onchromosomes 1, 2, 5, 7, 10 and 11. These QTL were specific toparticular traits, environments, or genetic backgrounds. Allidentified QTL contributed to increased submergence tolerancethrough their effects on decreased underwater shoot elongationor increased maintenance of chlorophyll levels, or on both.These findings establish the foundations of a marker-assistedscheme for introducing submergence tolerance into agriculturallydesirable cultivars of rice.     

Physiological and Molecular Basis of Susceptibility and Tolerance of Rice Plants to Complete Submergence

Rice plants are much damaged by several days of total submergence.The effect can be a serious problem for rice farmers in therainfed lowlands of Asia, and runs contrary to a widespreadbelief amongst plant biologists that rice is highly tolerantof submergence. This article assesses the characteristics ofthe underwater environment that may damage rice plants, examinesvarious physiological mechanisms of injury, and reviews recentprogress achieved using linkage mapping to locate quantitativetraits loci (QTL) for tolerance inherited from a submergence-tolerantcultivar FR13A. Progress towards identifying the gene(s) involvedthrough physical mapping of a dominant tolerance locus on chromosome9 is also summarized. Available physiological evidence pointsaway from responses to oxygen shortage as being inextricablyinvolved in submergence injury. An imbalance between productionand consumption of assimilates is seen as being especially harmful,and is exacerbated by strongly accelerated leaf extension andleaf senescence that are ethylene-mediated and largely absentfrom FR13A and related cultivars. DNA markers for a major QTLfor tolerance are shown to be potentially useful in breedingprogrammes designed to improve submergence tolerance.     

Differential metabolic regulation governed by the rice SUB1A gene during submergence stress and identification of alanylglycine by 1H NMR spectroscopy

Although the genetic mechanism of submergence survival for rice varieties containing the SUB1A gene has been elucidated, the downstream metabolic effects have not yet been evaluated. In this study, the metabolomes of Oryza sativa ssp. japonica cv. M202 and cv. M202(Sub1) were profiled using (1)H NMR spectroscopy to compare the metabolic effect of submergence stress and recovery on rice in the presence or absence of SUB1A. Significant changes were observed in the NMR resonances of compounds in pathways important for carbohydrate metabolism. The presence of SUB1A in M202(Sub1) was correlated with suppression of carbohydrate metabolism in shoot tissue, consistent with the role of SUB1A in limiting starch catabolism to fuel elongation growth. The absence of SUB1A in M202 was correlated with greater consumption of sucrose stores and accumulation of amino acids that are synthesized from glycolysis intermediates and pyruvate. Under submergence conditions, alanine, a product of pyruvate metabolism, showed the largest difference between the two varieties, but elevated levels of glutamine, glutamate, leucine, isoleucine, threonine, and valine were also higher in M202 compared with the M202(Sub1) variety. The identification and characterization of alanylglycine (AlaGly) in rice is also reported. After 3 days of submergence stress, AlaGly levels decreased significantly in both genotypes but did not recover within 1 day of desubmergence with the other metabolites evaluated. The influence of SUB1A on dynamic changes in the metabolome during complete submergence provides new insights into the functional roles of a single gene in invoking a quiescence strategy that helps stabilize crop production in submergence-prone fields.