Discovery of a Novel Submergence Tolerance Gene in Rice for Better Flood Adaptation

In Bangladesh, several million hectares of land are flooded during monsoon - located in rainfed lowland to stagnant conditions, deepwater, and tidal wet-lands. In some year, Aman seedlings may completely submerge for about 7-12 days. Large economic loss occurs, if crops are damaged. In deepwater areas, there are shallow deepwater (1.0 meter depth) and very deep water (more than 1.0 meter depth). Due to the climate change, threat of extreme flooding is too much in duration and depth.

Different types of rice varieties selected by farmers in flood prone areas.  In rainfed areas- semi-dwarf Aman varieties with submergence tolerance could survive for about 7-12 days under flood water. For stagnant condition (30-50 cm depth) - tall varieties were selected. Slow stem-elongating deepwater rice could survive in 0.5-1.0 meter shallow flooded areas; whereas, fast-elongating floating rice could survive in 3 meter deep flooded areas. Tidal wet-lands having tidal influence (60-90 cm) - traditional tall varieties are grown.

Long time submergence may kill Aman crops. Submergence tolerance is ability to survive 7-12 days of complete submergence, having lower underwater leaf chlorosis- new leaf emergence, after water subsides. Submergence tolerance was investigated intensively by Dr. Mackill at IRRI, as well by Dr. Gomasta at BRRI. The variety, FR13A from Orissa, India found as tolerant. Flood adaptation is attributed to two mechanisms: (a) submergence tolerance FR13A can survive for 7-12 days under submergence (b) Elongation by which tall deep-water varieties avoid submergence.

Escape from submergence, achieved by speeding-up of shoot elongation of deepwater rice. Flood escaping of highly submergence susceptible deepwater rice is conferred by stems elongation to keep pace with rising water. Under water elongation is hormonally driven, by accumulated ethylene that sensitizes gibberellins (GA)-dependent elongation, cell division and starch mobilization. About 0.7 million ha of deep water rice (Dudlaki, Fulkori etc) having elongation ability are still grown. Breeders transferred tallness into high-yielding varieties and developed BRRI dhan91 for shallow flooded areas.

In contrast, mechanisms associated with highly submergence tolerance are reduced elongation and low synthesis of ethylene. Reduced elongation under submergence is vital for survival, because elongating plants would collapse after water recedes. The symptoms of injury are not much pronounced in tolerant varieties and they show new leaf emergence after few days of recovery. After submergence, decline in photosynthetic ability, stem carbohydrate and chlorophyll content of leaves progresses slowly in tolerance varieties than in those are susceptible.

Xu and Mackill identified two ethylene response factor (ERF) like genes: SUB1A-1 and SUB1C-1 in submergence-tolerant variety FR13A. A single submergence locus, SUB1 on chromosome 9, controlled most of variation for submergence tolerance. Two markers RM219 and RM464A tightly linked to Sub1 locus were identified and transferred into semi-dwarf popular varieties, using Marker Assisted Backcrossing (MAB). New submergence tolerance varieties BRRI dhan51 and 52 with SUBA gene exhibited limited elongation- are grown in Bangladesh, might resist floods that destroy vast tracts of paddy.

In tolerant semi-dwarf varieties (IR40931), the highest expressed gene is SUB1-A1; expression of SUB1C-1 is lower. Under submergence, plant produces Gibberellic Acid (GA) that stimulates stem elongation. In tolerant varieties, mechanisms associated with higher submergence tolerance are over expression of SUB1A-1 that suppresses excess production of GA and suppresses elongation- helps plants to survive under flood water. However, semi-dwarf tolerant variety (SUB1A-1) having defective GA mutant sd-1 allele also induces reduced plant height/less GA - that have influence on suppression of harmful elongation.

Under flood water, submergence intolerant tall rice variety elongates strongly- become tall and dies within 4-5 days. In a susceptible tall variety, the highest-expressed gene is SUB1C and the SUB1A expression is lower - that showed higher susceptibility. Tolerance level is lower in high elongating plants through activating GA and leaf chlorosis. Dominant Sd1 allele in tall deepwater rice encodes excess GA to promote elongation. In intolerant deepwater variety, lower expression of SUB1A-2 gene and high expression of SUB1C-2 provides more stem elongation and induces high susceptibility.

Tall Deepwater rice expresses lower submergence tolerance in presence of GA3 under submergence. Thus, elongation ability and submergence tolerance has opposite direction for flood adaptation. Submergence tolerant varieties experience less depletion in photosynthetic ability, dry-matter, carbohydrate and chlorophyll than susceptible varieties under submergence. Under flash floods, limited elongation was associated with submergence tolerance.  Present research findings demonstrated that elongation and submergence tolerance could not be incorporated in same genotype- as those two traits show antagonistic expression.

Combining SUB1 gene into tall varieties has enormous opportunities under flooded conditions. Efforts are needed to develop improved flood adaptable rice varieties having both submergence tolerance and stem elongation for climate change adaptation. Although FR13A and local deep water cultivars have been used as source of submergence tolerance and elongation respectively - additional sources are needed. It is realized that rice diversity is crucial to find additional sources for submergence tolerance and tallness.

Most useful area of biotechnology is to identify new sources of genes. A linked chain of 4 bases constitute DNA molecule; called a gene - stores genetic information. Knowledge on gene located on different chromosomes is important. From rice genetic map, it is known that position of markers/SUB-1 gene for submergence tolerance is on chromosome-9 and Sd-1 on chromosome-1. Markers linked with Sub1 QTLs and Sd1 marker linked with tallness/GA3 synthesis could measure intra-specific variation in diverse                                                                                                                                                                                                                                                                        garmplasm.

Basic research on gene isolation, markers and cDNA synthesis has become important for understanding a gene. Single nucleotide polymorphism (SNP) emerged with discovery of restriction enzymes (REs) that specifically cut double-stranded DNA molecule. Molecular restriction digestion-based haplotype diversity at SUB1A and SUB1C loci was analyzed among 160 flood prone rice cultivars by Masuduzzaman et al. (2011). Steps for identifying new sources of gene are: Isolating DNA and PCR amplification, restriction digestion, comparing molecular and phenotypic data, c-DNA synthesis, gene expression analysis and selection of new sources.

Donor plants could provide source DNA. General protocols for DNA isolation are available.  To obtain DNA comprising a gene- DNA is first extracted from cells. Two CAPS markers, GnS2 and ERF173 closely linked to SubA and Sub1C alleles respectively were used as diagnostic markers. PCR (Polymerase Chain Reaction) is a technique for amplifying DNA (or RNA), using taq polymerase and two specific flanking primers. Gel electrophoresis is used to separate PCR products and DNA of interest could be isolated.

PCR products were used for restriction digestion. Restriction digestion was assayed with enzyme Cac8 I and Alu I to generate polymorphisms at Sub1C and Sub1A loci, respectively. Restriction analysis was performed in 20 µl volume, using 7µl PCR products with 13µl of reaction mixture with 0.35 µl of respective restriction enzyme.  The mixtures were incubated at 370C for digestion. Digested PCR products were separated in polyacrylamide gel and visualized. Molecular weight for cut and un-cut bands was measured.

A linear arrangement of alleles at different SNPs on a chromosome is called haplotype. Information provided by SNPs is most useful, when gene based haplotypes are examined. SNP based association with phenotype was analyzed to determine allelic variants (presence or absence of SNP) and to differentiate tolerant and intolerant specific Sub1A and Sub1C alleles. Haplotype-based associations with elongation class were determined. Tolerant accessions having possible sources of novel tolerance genes were identified comparison with known source.

Genetic variation was assayed by haplotype analysis, based on single nucleotide polymorphism at SUB1 locus in occurrence of monomorhpic bands in different individuals. Restriction enzyme produced a clear polymorphism between tolerant and intolerant genotypes. Haplotype diversify analysis at SUB1 locus has advantage to find new source of tolerant gene. Tall varieties Madabaru and Tillakkachari were found as submergence tolerance without SUB1A1 allele. These varieties are different from FR13A and suspected to carry different novel submergence tolerant genes and tallness.                                                                                                                                                                                                                               
Semi-quantitative RT-PCR (Reverse Transcriptase-PCR) was performed for proofing novelty of genes through synthesis of stable double stand c-DNA from single stand m-RNA. For c-DNA synthesis- RNA was extracted, RNA integrity was assessed and 100 ng of high-quality total RNA was used. RT-PCR was performed, using RT-PCR kit in a 25 µl reaction mixture of 22 µl of RT-PCR reagent and 3µl of total RNA. For one reaction, RT-PCR reagent mixture contained: RNase-free water 6.75 µl, 12.5 µl of 1X one-step RT-PCR, 1 µl gene specific primers, and 0.5 µl RT-PCR Taq. Then, tubes were transferred to a thermal cycler for RT-PCR amplification.

A gene/DNA transcribed to mRNA that encodes specific protein to produce a specific trait/phenotype. Any small change (single nucleotide polymorphisms) in a gene sequence could alter the phenotype- that change could be detected through gene expression. To quantify expression of SUB1A and SUB1C- gel electrophoresis was carried out using c-DNA template. Intensity of each band was measured- expression values of SUB1A and SUB1C genes were used for grouping varieties.

In Tillakkachari- higher expression of both SUB1A2 and SUB1C2 alleles that confer higher tolerance- distinct from previous tolerant and intolerant varieties. A novel mechanism favored to express elongation and submergence tolerance in a same direction for better flood adaptation. In general, elongating type expresses high susceptibility, but Tillakkachari is moderate elongating - expressing higher submergence tolerance. The products of SD-T2 trigger elongation via gibberellins without adverse effects on submergence tolerance.

These results suggested that synergistic interactions of two alleles involve: beneficial SD-T2 allele for elongation and novel SUB1A-2 allele for submergence tolerance. This research finding is an innovation for incorporating both tallness and submergence tolerance in same genotype - published in peer reviewed journals. We pyramided SD-T2 and SUB1A2 alleles into background of a robust tall and lodging tolerance modern deepwater (BR10260-5-15-21-6B) genotype. This tall entry (165 cm tall, & 5.8 t ha-1) survived in 1.0-1.5 meter of flood water having submergence tolerance.

Tall and submergence tolerance improved deepwater rice BR10260-5-15-21-6B (proposed as BRRI dhan109) also provide genetic gains in robustness and lodging tolerance, more chlorophyll content, more stem carbohydrate, vigorous root systems, more biomass and yield. Introgression of SD-T2 and SUB1A2 into tall rice will widen the adaption from flash floods to deepwater areas. Further, SD-T2 and SUB1A2 alleles could be transferred in to tall varieties using Marker Assisted Backcrossing.

Research on flood adaptable rice could be progressed based on higher knowledge and innovation with support from policy makers for greater food security.


A S M Masuduzzaman, PhD is
Chief Scientific Officer, Bangladesh
Rice Research Institute, Gazipur.