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MOLECULAR CHARACTERIZATION OF SUBMERGENCE TOLERANCE GENES AND LOCUS IN THE DEEP-WATER RICE CULTIVARS

Most of the rice cultivars exhibit suspension of growth when submerged to overcome the reduced availability of oxygen. When the situation continues, majority of the cultivars unable to recover after the flood recedes. However, there are fortunately some rice genotypes that can withstand such submerged condition for up to two weeks by adapting two totally opposite mechanisms. One type of cultivars elongates enormously at a very short span of time and the leaves come above the water level. In the second type, they remain under water without any growth. Cultivars of both types tolerate the submergence but the first category easily lodges when flood water recede. In those lines, yields are reduced drastically. In this study, we focus on characterize the genetic variation at the Sub1 locus and to associate its relevance, if any, to submergence tolerance among the deep water landraces. As a first step, seeds of some rice cultivars collected from North-east Indian regions were initially selected for the characterization of genetic variation. The PCR based analysis involving several genes known to be associated with submergence tolerance did not reveal much difference. However, Southern hybridization revealed certain differences between submergence tolerant and susceptible cultivars. Although we did not notice major difference with regard to Sub1 genes when tried with EcoRI and BamHI, differences were noticed with adh1 and RAmy3C genes. Representative, Southern analysis showed the genetic variation among the deep-water cultivars as compared to Swarna and Sub1-Swarna. It is possible that deep-water rice cultivars may not differ in their genome at Sub1 locus but they respond through SNORKEL genes under submergence.


INTRODUCTION
Rice is a semi-aquatic plant. It can live, adopt and grow under water logging. Rice plants survive longer than other cereals under water. However, complete submergence is serious for the farmer as severe flooding results in reduced grain productivity and quality. Unfortunately, sudden and uncontrollable flash floods up to half a meter deep are common and last for several days. Resulted from overflowing rivers, low height earth barriers made to stop erosion, or accumulation from higher ground, and higher tides. Flooding in these areas, along with drought and salinity are main constraints affecting the yield.
Typically, deep-water rice responds by increasing in cell division and/or elongation of cells leading to overall increase of internodes underwater stems. When plants are submerged under water, the levels of ethylene goes up triggering several mechanisms that include increased production of gibberellic acids (GA) and degradation of growth promoting abscisic acid (ABA) (Kende et al., 1998;Hattori et al., 2009). An interesting feature of GA mediated response is rapid elongation of stem that can reach up to 25 cm/day. Later some studies (Hattori et al., 2009) have shown that such growth is regulated at least three different quantitative trait loci (QTLs). One of the most studied loci, the SNORKEL is located on chromosome 12 which encodes two ethylene responsive factor (ERF) proteins [SNORKEL1 (SK1) and SNORKEL2 (SK2)] containing DNA binding motif, one of the typical motif for any transcription factor. It is also now well established that SK1 and SK2 are not present in the cultivars that do not elongate like deep-water rice and survive under submerged conditions for a longer duration (Hattori et al., 2009). Such elongation growth response of these deep-water rice ensures that sufficient aerial tissue like leaves is in contact with the air for exchange of oxygen and to continue photosynthesis to meet the required energy for the submerged tissues (Bailey-Serres, Voesenek, 2010).
Despite of huge efforts in screening and selection of submergence tolerant plants, not much is known until the Sub1 locus was identified in rice. Systematic studies lead to molecular mapping of a major QTL, designated as SUBMERGENCE 1 (sub1) onto rice chromosome 9. The Sub1 locus has been estimated to contribute for about 70% of submergence tolerance in rice (Xu and Mackill, 1996). Based on elaborate crosses made between tolerant and susceptible lines and segregation pattern of the F1 progeny, rice breeders postulated that the submergence trait is a quantitative trait. Xu et al., (2000) further narrowed it to less than a cM region on chromosome 9 using a large number of F2 population. Besides the Sub1 QTL, there are three more QTLs identified in deep-water-rice on chromosome 1, 3 and 12 (Nemoto et al., 2004). Oxygen, a major electron acceptor drops below the optimized level so that the plant energy consumption is reduced by shutting off the metabolism involving sugars, proteins and amino acids and lipid to conserve energy (Geigenberger, 2003). This conserved energy is redirected to the translation and transcription of stress proteins that are needed for the plant survival. These stress proteins are called anaerobic polypeptides (ANP) (Drew, 1997). Major ANPs are sucrose synthetase (SUS), glucose phosphate isomerase, alcohol dehydrogenase (ADH), pyruvate decarbonxylase (PDC), and lactase dehydrogenase. They are all part of the fermentation and the glycosis pathway.

Plant materials
Total six rice cultivars expressed well-known submergence tolerance mechanisms were collected for this study. Rice cultivars namely IR64, Swarna, Sub1-Swarna were collected from IARI (Indian Agricultural Research Institute), New Delhi, India. These varieties response to submergence condition by non-elongation mechanism. Other cultivars named Khongan, Taothabi and Murshi response to submergence by putative elongation mechanism, which were selected from Manipur, North-east Indian region.

Experimental conditions to assess submergence tolerance
Experiments involving submergence tolerance test have been conducted using two week old seedlings by submerging completely in the water in a clear glass tanks. Duration of the submergence was 1-2 weeks depending on the type of experiement. They were then kept 1-2 weeks outside the tanks for recovery.

Genomic DNA isolation and molecular characterization
Genomic DNA isolation was carried out following the protocol of Murray and Thompson (1980). All the primers used in this study were designed using oligoanalyzer program. For primer designing, genome sequence (Phytozome database or EST sequence of rice, extracted through "blastx" were used. (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). While designing primers, GC content was maintained between 40 -60% and range of Tm was chosen between 55 -65 o C wherever possible. Primers and conditions applied for PCR were shown on table 1. Following PCR amplification program was set up: Initial denaturation at 94 o C for 3 min followed by thermal cycling at 94 o C for 30s; annealing at 50 o C to 60 o C for 30 -45s and extension at 72 o C for 1 min 30s for 35 cycles. Final extension was given at 72 o C for 5 min. The obtained amplicons were analyzed by running electrophoresise on 0.8 -1.0% agarose gel.

Radioactive labelled probe synthesis for Southern hybridization
The PCR -amplified fragment of the target genes were labeled by α-P 32 dCTP using a nick translation system (Invitrogen, Life Technologies, India) according to the manufacturer's instruction. The probes were synthesized one day before and stored at -20°C.

Submergence tolerance mechanisms in deepwater -rice cultivars
One set of experiments involving submergence have been conducted using two week old seedlings by submerging them in the water for two weeks and kept one week for recovery. Interestingly, the Taothabi and Khongan both elongated above the water levels all the times while Sub1-Swarna did not elongate and normal Swarna elongated slightly. The experiment clearly shows the two opposite phenotypical responses during submergence. First type is elongation mechanism to escape from flooded conditions regarding GA3 pathway and another type expressed no elongation, conserved energy and used it for new-growth (Figure 1). Two varieties namely Murshi, Niphu Thokpi remained their plant height in complete submergence, familiar to Sub1-Swarna's response, then were dismissed next experiements (or their results, if have, do not need to be mentioned).
In order to characterize the genetic variation in the North-east Indian rice, Southern hybridization and PCR based approaches were used. Sub1-Swarna that has been developed through traditional plant breeding methods through introgression of Sub1 QTL was included in this study for a better comparison and was used as a positive Sub1 gene control. Swarna variety was used as a negative Sub1 control. Main study focused on the effects of other unknown and novel genes, if any, on the submergence tolerance. The IR64, a popular variety susceptible to submergence conditions, is also included.

PCR based analysis to detect genetic variation among the rice cultivars
Also, PCR based approach was followed for the amplification of important genes involved in submergence tolerance and few housekeeping genes were utilized in the PCR experiments. PCR primers were designed for Sub1A1, Sub1B and Sub1C genes along with alcohol dehydrogenase 1 and 2 (ADH1, ADH2), pyruvate decarboxylase (PDC1, PDC2, PDC4), rice amylase (RAmy3C), and sucrose synthase (SUS) genes. All the primers were tested in these cultivars for the presence of the above mentioned genes. No significant no differences were noticed in the PCR based amplification, excepted Sub1A ( Figure 2). So far Sub1 genes (Sub1B and Sub1C) have been amplified in all the varieties tested and cloned while Sub1A has been amplified in Sub1-Swarna, Khongan and Taothabi but not in Swarna. PCR based amplification of three genes (Sub1A, Sub1B and Sub1C) showed the conserved nature of the present in Sub1 loci. However, the data also showed certain variations among the genotypes studied. It is likely that Sub1A gene plays a key role in submergence tolerance while Sub1B and Sub1C do not play an important role. Indeed, a trait of non-elongation in submergence tolerance mechanism is regulated by Sub1A gene (Aaron, Schmitz et al., 2013;. However, this gene also presents in the escape mechanism (Khongan and Taothabi vars.) which responded with submergence by fast elongation (stem or leaves). So that the interesting question is why Sub1A gene does not play its function in these deep-water rice varieties (stop stem elongation in submergence condition). Is a flash flooded responded mechanism independent or dependent with escape (stem elongation) mechanism? If it is independent, is there any third mechanism in submergence tolerance?

Southern hybridization to detect genetic variation among rice cultivars
For this purpose, genomic DNAs isolated from six cultivars (Murshi, Taothabi, Khongan, Swarna, Sub1-Swarna and IR64) were digested with EcoRI or BamHI restriction enzymes and hybridized with the coding regions of a number of genes associated with submergence tolerance in rice.
Representative, Southern hybridization blots showing the genetic variation among the North-east cultivars as compared to Swarna and Sub1-Swarna are shown in Figure 3. Arrows indicate the differences in the size of the bands. Although we did not notice major difference with regard to Sub1A and Sub1B when tried with EcoRI and BamHI. Differences were noticed with adh1 and RAmy3C genes.
Note the difference in Khongan as compared to other cultivars when genomic DNA was digested with EcoRI and probed with adh1. Similarly, no difference was observed in IR64 as compared to other cultivars when genomic DNA was digested with BamHI and probed with RAmy3C. Several genotypes having Sub1 QTL have been studied to understand and identify the genetic diversity (Sarkar et al., 2011). These studies revealed that greater genotypic variability in terms of various parameters such as plant height, survival rate, etc., associated with submergence tolerance. More recently, Goswami et al., (2015) also showed genetic diversity at Sub1 loci among the genotypes that are known to exhibit submergence tolerance in India. On the whole, these studies have indicated the potential genetic resources in the deep-water rice that can be deployed in lowland-flood prone areas through marker assisted breeding or through genetic transformation approaches to increase yield.