Identification of QTLs for stagnant flooding tolerance in rice employing genotyping by sequencing of a RIL population derived from Swarna × Rashpanjor.

In lowland rice ecosystems stagnant flooding or partial submergence has a significant negative impact on important yield attributing traits resulting in substantial grain yield reduction. Genetics of this stress is not yet studied intensively. Rashpanjor (IC 575321), a landrace from India, was identified and used as the tolerant donor for stagnant flooding and was crossed with high yielding variety Swarna to develop the RIL population for the present investigation. Yield and yield attributing traits of 180 F2:8 lines in rainfed non-stressed and stressed (stagnant flooding with 45 ± 5 cm standing water) conditions were recorded in the wet season of 2018 and stress susceptibility and tolerance indices of yield component traits were deduced. Homo-polymorphic high-quality SNPs between two parents derived from genotyping by sequencing were employed and 17 putative QTLs for plant height, shoot elongation, panicle number, grain weight, panicle length in control and stagnant flooding conditions were identified. Tolerance and susceptibility indexes for these traits were detected in chromosomes 1, 3, 4, 5, 6, 10, 11, and 12 with PVE ranging from 6.53 to 57.89%. Two major QTLs clusters were found for stress susceptibility index of grain and panicle weight on chromosome 1 and plant height in non-stress condition and stress tolerance index of elongation ability on chromosome 3. Putative functional genes present either in associated non-synonymous SNPs or inside the QTL regions were also predicted. Some of them were directly associated with ethylene biosynthesis and encoding auxin responsive factors for better adaptation under stagnant flooding and also coded for different transcription factors viz. NAC domain-binding protein, WRKY gene family, and MYB class known for ROS scavenging and production of metabolites to enhance tolerance to stagnant flooding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01107-x.

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice.

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m-1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K+ concentration and low Na+- K+ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.

Natural leaf senescence: probed by chlorophyll fluorescence, CO2 photosynthetic rate and antioxidant enzyme activities during grain filling in different rice cultivars.

Natural leaf senescence was investigated in four rainfed lowland rice cultivars, FR 13A (tolerant to submergence), Sabita and Sarala (adapted to medium depth, 0-50 cm stagnant flooding) and Dengi (conventional farmers' cultivar). Changes in the levels of pigment content, CO2 photosynthetic rate, photosystem II photochemistry and anti-oxidant enzyme activities of flag leaves during grain-filling stage were investigated. Chlorophyll content, photochemical efficiency of photosystem II and CO2 photosynthetic rate decreased significantly with the progress of grain-filling. Likely, the activities of antioxidant enzymes namely, superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase decreased with progress of grain-filling. A substantial difference was observed among the four cultivars for the sustainability index (SI) of different photosynthetic parameters and antioxidant enzyme activities; SIs of those parameters, in general, were lower in low yielding cultivar FR 13A compared to the other three cultivars. Among the four cultivars Sabita gave maximum grain yield. Yet, SI of Pn was greater in Sarala and Dengi compared to the Sabita. SIs of electron transport (ETo/CS), maximal photochemical efficiency (Fv/Fm), area above Fo and Fm, catalase and ascorbate peroxidase were also greater in Sarala and Dengi. The data showed that among the different Chl a fluorescence parameters, PI could be used with greater accuracy to distinguish slow and fast senescence rice cultivars during grain-filling period. It was concluded that maintaining the vitality of rice plants during grain-filling gave guarantee to synthesize carbohydrate, however greater yield could be realized provided superior yield attributing parameters are present.

Introgression of SUB1 aggravates the susceptibility of the popular rice cultivars Swarna and Savitri to stagnant flooding.

Identification of the Sub1 gene for tolerance to flash flooding and its introgression into high-yielding rice cultivars are major targets in rice breeding for flood-prone rice agro-ecosystems for ensuring yield stability. However, knowledge is scant on the response of the modified genotypes under stagnant flooding (SF) to meet the challenge of finding a superior allele that may confer greater resilience to the plant under a stress-prone environment. In pursuance, we have tested the response of Sub1-introgression in two popular rice varieties, Swarna and Savitri to SF by comparing the biochemical factors in the control of flag leaf senescence and its primary production mechanisms of the parental lines' versus Sub1-introgressed lines. The activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GR), and ascorbate peroxidase (APX) increased while various parameters of primary production like total chlorophyll content, stomatal conductance (gs), normalized difference vegetation index (NDVI) and photosynthetic activity (Pn) decreased progressively with passage of time in the flag leaf of the cultivars during the post-anthesis period and SF-treatment increased the enzyme activity while depressing primary production further. Introgression of Sub1 had no influence on these activities under control conditions but widened the margin of effects under SF. It was concluded that the functional ability of flag leaf in mega rice cultivars like Swarna and Savitri decreased significantly by SF because of an ethylene-mediated promotion of senescence of the flag leaf. The enhancement of antioxidant enzyme activity by SF could not sustain the stability of primary production in the flag leaf. The introgression of the Sub1 gene made the cultivars more vulnerable to SF because the gene induced overexpression of ethylene.

Introgression of Sub1 (SUB1) QTL in mega rice cultivars increases ethylene production to the detriment of grain- filling under stagnant flooding.

In the recent time, Submergence1 (Sub1)QTL, responsible for imparting tolerance to flash flooding, has been introduced in many rice cultivars, but resilience of the QTL to stagnant flooding (SF) is not known. The response of Sub1-introgression has been tested on physiology, molecular biology and yield of two popular rice cultivars (Swarna and Savitri) by comparison of the parental and Sub1-introgression lines (SwarnaSub1 and SavitriSub1) under SF. Compared to control condition SF reduced grain yield and tiller number and increased plant height and Sub1- introgression mostly matched these effects. SF increased ethylene production by over-expression of ACC-synthase and ACC-oxidase enzyme genes of panicle before anthesis in the parental lines. Expression of the genes changed with Sub1-introgression, where some enzyme isoform genes over-expressed after anthesis under SF. Activities of endosperm starch synthesizing enzymes SUS and AGPase declined concomitantly with rise ethylene production in the Sub1-introgressed lines resulting in low starch synthesis and accumulation of soluble carbohydrates in the developing spikelets. In conclusion, Sub1-introgression into the cultivars increased susceptibility to SF. Subjected to SF, the QTL promoted genesis of ethylene in the panicle at anthesis to the detriment of grain yield, while compromising with morphological features like tiller production and stem elongation.

Distinction and characterisation of rice genotypes tolerant to combined stresses of salinity and partial submergence, proved by a high-resolution chlorophyll fluorescence imaging system.

Chlorophyll a fluorescence (ChlF) parameters measured with fluorescence imaging techniques were used to investigate the combined effect of salt and partial submergence stress to understand photosynthetic performance in rice (Oryza sativa L.). ChlF parameters such as maximal fluorescence (Fm), variable fluorescence (Fv=Fm -F0), the maximal photochemical efficiency of PSII (Fv/Fm) and the quantum yield of nonregulated energy dissipation of PSII (Y(NO)) were able to distinguish genotypes precisely based on their sensitivity to stress. Upon analysis, we found the images of F0 were indistinguishable among the genotypes, irrespective of their tolerance to salt and partial submergence stress. On the contrary, the images of Fm and Fv/Fm showed marked differences between the tolerant and susceptible genotypes in terms of tissue greenness and the appearance of dark spots as stress symptoms. The images of effective PSII quantum yield, the coefficient of nonphotochemical quenching (qN) and the coefficient of photochemical quenching (qP) captured under different PAR were able to distinguish the tolerant and susceptible genotypes, and were also quite effective for differentiating the tolerant and moderately tolerant ones. Similarly, the values of electron transport rate, qN, qP and Y(NO) were also able to distinguish the genotypes based on their sensitivity to stress. Overall, this investigation indicates the suitability of chlorophyll fluorescence imaging technique for precise phenotyping of rice based on their sensitivity to the combined effect of salt and partial submergence.

Distinction and characterisation of salinity tolerant and sensitive rice cultivars as probed by the chlorophyll fluorescence characteristics and growth parameters.

Soil salinity is a major abiotic stress that limits rice productivity worldwide. The problem is intense - particularly in areas with extremely dry and hot climatic conditions. Designing an effective phenotyping strategy requires thorough understanding of plant survival under stress. The investigation was conducted using 12 rice cultivars differing in salinity tolerance. Among these cultivars, seedling survival on day 10 of salt treatment (12dSm-1) was above 85% during wet season and 75% during dry season in FL478, AC39416, Pokkali and Kamini. Highly salt-tolerant cultivars maintained greater proportion of green leaf and chlorophyll content under salt stress. Unlike sensitive cultivars, tolerant cultivars taken up less Na+ and more K+, resulting in lower Na+:K+ ratio in leaf and sheath. Normalised chlorophyll a fluorescence data revealed that the Fv/Fm and PIABS values decreased on days 3 and 7, respectively, of salt stress in susceptible rice cultivar. Salinity factor index (SFI) calculated by giving different weights to relative PIABS values after variable days of salinity stress clearly distinguished the level of tolerance among rice cultivars. The SFI can be used for grouping of moderately to highly salt-tolerant cultivars based on their tolerance level. We conclude that maintenance of greater proportion of green leaf, and restricted transport of Na+ to sheath and leaf helps the plant to counteract adverse effects of salinity on rice growth.

Distinction and characterisation of submergence tolerant and sensitive rice cultivars, probed by the fluorescence OJIP rise kinetics.

Rice (Oryza sativa L.) plants experience multiple abiotic stresses when they are submerged. In addition to the effects of submergence on gas exchange, water also creates shading of submerged plants. It is believed that responses to submergence are actually responses to low light stress, although during complete submergence in addition to low light other environmental factors like reduce movement of gases affect the plant growth, and therefore, the consequences of submergence are not always alike to shade. We monitored the extent to which shade and submergence change the plant height, chlorophyll a fluorescence characteristics and CO2 photosynthetic rate in three Indica rice cultivars, namely Sarala, Kalaputia and Khoda, which differed in submergence tolerance. There were both similarities and dissimilarities between the consequence of shade and submergence on rice plants. Under shade conditions, elongation growth was greater in submergence tolerant cultivars than the sensitive cultivar, whereas elongation growth was greater under submergence in sensitive cultivar. The reduction in chlorophyll content, damage to PSII, and decrease in CO2 photosynthetic rate was more notable under submergence than the shade conditions. Our results show that several JIP-test parameters clearly distinguish between submergence tolerant and sensitive cultivars, and responses to submergence among different rice cultivars differ depending on their sensitivity to submergence. There were different interactions between cultivar and shade (~low light) and cultivar and submergence.