Morpho-Genetic Screening of the Promising Rice Genotypes under Salinity Stress

Selection of new rice (Oryza sativa L.) variety tolerant to soil salinity is one of the most important issues to avoid salt stress in rice production. In this study, the morphological characterization was performed on 5 rice genotypes under two salt stress treatments EC8 and 12 dS/m at seedling, EC 12 dS/m at vegetative and EC8 dS/m at reproductive stages. Morphological characters and Na/K uptake ratio were surveyed to evaluate salt stress effects. At vegetative stage, all the genotypes have survived. However, at reproductive stage all the genotypes were affected by salt stress except for the Binadhan-10 that survived the whole life cycle. A total of 160 SSR markers were used which revealed 209 alleles among the 5 rice genotypes. Interestingly, 4 SSR markers RM105, RM125, RM178 and RM549 with highest PIC (0.67) value have scored the highest level of genetic diversity value (0.72). These markers could be used to pyramiding the major and minor salt stress related genes via marker assisted selection in rice. The present investigation on variant salt stress responsiveness of 5 promising rice genotypes will be of great value in rice breeding.


Introduction
Rice (Oryza sativa L.) is one of the most important cereals in the world.There are about 380 million hectares of saline soils on the earth's surface and these are widely distributed in arid and semi-arid as well as seasonally dry coastal areas (Mansuri et al., 2012).Soil salinity is considered as one of the major factors that reduce plant growth in many regions in the world.Salt stress is one of the most important abiotic stresses that adversely affect natural productivity and causes significant crop loss worldwide.The salinity has affected crop production in Pakistan, India and other regions including the US.Worldwide 20% of all irrigated land is salt-affected (45 million out of 230 million ha).Eventually, soils in the arid and semiarid regions have excessive concentrations of soluble salts, which adversely affect plant growth (Aref & Rad, 2012).In Bangladesh, 2.8 million ha of rice lands are currently affected by salinity (Islam et al., 2012) and further degradation will have detrimental consequences on food security due to limited land resources.In China, salt affected extensively soils from tropical area to temperate zones, from the coast to the inland and from the semi-arid to desert regions.Clearing the permanent deep-rooted vegetation is a major cause of salinization in non-irrigated areas; this practice causes the water table to rise and salts to accumulate in the low-lying areas.Salinity induces osmotic stress and reduces water uptake into plant roots (as less water is taken up, cell expansion growth slows down and the stomata close, ultimately affecting photosynthesis).Plants regulate water transport under salinity stress because a sufficient amount of water is indispensable for the cells to maintain their growth and vital cellular functions such as photosynthesis and metabolisms (Horie et al., 2012).Attempts to improve the salt tolerance of crops through conventional breeding program have met very limited success; due to the complexity of the salt tolerance traits, which implied morphological and physiological factors.Salinity is mainly due to the osmotic (Na + /K + ) imbalance in response to excessive accumulation of Na + (Gregorio et al., 1997).Evaluation of genotypes in farmers field not reliable due to heterogeneous conditions.Landraces are currently being exploited as preferred potential donors of salt tolerance traits because of their local adaptation.Salinity effects varied along with different growth stages in rice, thus depressed yield grain production (Mansuri et al., 2012;Shankar et al., 2011).Morpho-genetic screening of rice genotypes during all the plant developmental stages could lead to identify the salt tolerant genes.Molecular markers could now be used to evaluate their effects towards the phenotypes and thereby selecting for favorable alleles at those loci using Marker Assisted Selection scheme that aims to accelerate genetic advancement in rice (Gregorio, 1997).Due to genetic similarities between cultivated rice species, the transfer of useful genes from one to another is possible.Thus, the evaluation of genetic diversity of rice genotypes could provide valuable information for genetic improvement of salt tolerant rice.Therefore, in the present study, 5 genotypes comprised of salt sensitive, moderately tolerant and tolerant genotypes were subjected to a diversity study utilizing SSR markers.The objective of this study was to characterize genetically 5 rice genotypes and found molecular markers linked to the salt tolerance which could be used for markers assisted selection in rice breeding program.

Experiment Design
Screening of genotypes was done under controlled environment conditions at IRRI phytotron, following the method described by Gregorio et al. (1997).A randomized complete block design with three replications was used for each entry at seedling, vegetative, and reproductive stages respectively; each plot contains 20 plants for the seedling stage and 4 plants for the vegetative and reproductive stages.The middle 5 plants of each row were sampled for evaluating the morphological data.Root length was evaluated as an average value of the 5 representative plants randomly selected from each replicate (Appendix 1).Modified standard evaluation system (SES) of visual salt injury at seedling stage (IRRI, 1996) was used to group the genotypes in different salt injury categories (Table 1).

Screening of Rice Genotypes for Salinity Tolerance at Seedling Stage
Salinization was imposed three weeks starting from 7 th day of seedling growth following the protocol of Gregorio (1997): two pre-germinated seeds were sown per hole on a nylon-mesh covered by styrofoam seedling float.The seedling floats were put on black trays filled with tap water and then placed inside IRRI phytotron.Three days after seeding (when seedlings are well established), water in the trays was replaced with hydroponic solution (consisting of  water soluble fertilizer solution, use 1 g/l tap water with 300 mg of Fe 2 SO 4 •7H 2 O added per liter of solution).This solution can be used for 30 days.On the 7 th day, salinity treatment of EC=12 dS/m was done by adding sodium chloride (NaCl) with the pH level maintained at 5.0.EC and the pH level were measured thereafter for each day.Due to evapotranspiration there is loss of solution in the tray.Every day, the tray was filled up with tap water and pH adjusted to 5.0.The pH of the solution must be maintained at pH 5.0 to make available nutrients to the plants.Modified standard evaluation score was used to rating the visual symptoms of salt toxicity.This scoring discriminated the sensitive genotype from the tolerant and the moderately tolerant ones.Test entries were rated at fourteen and twenty-one days after initial salinization or when the sensitive genotype is dead.
Nine different treatments were designed based on the rice growing stage and salinization condition: 1.

Screening of Rice Genotypes at Reproductive Stage
Salinity of EC 8 dS/m was done from PI to maturity.The culture solutions were changed to normal or salinized after each treatment stage.

Biomass Measurement
Fresh and dry weights of both the shoots and the roots were evaluated for analyzing biomass.The fresh weight of each genotype in each treatment (three plants per variety per replication) was measured and then plants were placed inside labeled paper bags.Drying was done inside a convection oven (65 o C) for three days and measured for the dry weight.
Percentage of biomass for each of the sample was obtained using the formula: Percent biomass = Percent biomass growth reduction was also obtained using the formula: Percent growth reduction = Percent growth reduction = N is the normal nutrient solution and S is the nutrient solution with salinity.

Measurement of Yield Parameters
Each of the panicles per plant was threshed and measured panicle dry weight.Percent reductions in number of filled grains, unfilled grains and 100 grain weight were obtained using the formula: Percent growth reduction =

Molecular Analysis
DNA extraction: A modified mini-prep CetylTrimethyl Ammonium Bromide (CTAB) DNA extraction method developed at IRRI was followed in this experiment (Zheng et al., 1995).Leaf samples from all varieties were collected at 21 days after seeding.The simplified mini-scale procedure for DNA isolation was done at the Molecular Marker Applications Laboratory (MMAL), IRRI.

Physiological Analysis
Na + /K + analysis: Leaf samples from representative genotypes were collected and grinded.Extraction was done with 10 mg fresh tissue and 0.1 N acetic acid in water bath at 90 °C for 2 hrs.The samples were then cooled overnight at room temperature.Samples were filtered and diluted with water.Na + /K + were determined through the use of a flame photometer.Contents in parts per million (ppm) were converted to (milimol per gram) mmol/g by using the following formula of IRRI (Thomson et al., 2010): Where, x represents sodium content (ppm).

Morphological Characterization
The five rice genotypes were evaluated for the following morphological parameters such as percentage reduction in fresh weight and dry weight of shoot and root biomass, differences in length for both root and shoot under normal and salinized treatments.The variation was observed between the genotype under salinized condition and their counterparts under normal condition for plant height, shoot length, root length, biomass, and percent reduction of growth.

Salinity Screening at Seedling Stage
With EC = 12 dS/m of salinity stress, the 5 genotypes have different phenotypic response at seedling stage, which allowed to grouped them into three categories: moderately tolerant, sensitive and highly sensitive, with scores of 5, 7, and 9, respectively (Table 2) based on the modified standard evaluation score (SES) of visual salt injury at seedling stage (IRRI, 1996).On other hand, the results of salinity stress at EC = 8 dS/m also showed varied genotypic responses.The genotypes were grouped into three categories: moderately tolerant, sensitive and highly sensitive, with scores of 3, 5, and 7 respectively, based on SES scores (Table 3).The screening analysis showed that NSIC Rc222 had a high percent reduction in biomass for root and shoot mmol g ( ) 100 / 22.9 x mmol g ( ) 100 / 39.09 y fresh weight content (52.03% and 54.43%) whereas the lowest percent reduction was obtained from Binadhan-10 (root fresh weight-17.85%and shoot fresh weight-18.02%).On other hand, NSIC Rc222 had a high percentage reduction for root and shoot dry weight content (53.25% and 52.98%).However, the lowest percent reduction obtained was 12.88% by NSIC Rc182 for root dry weight and 11.25% by Binadhan-8 for shoot dry weight, respectively.All genotypes were observed to have relatively longer roots (ranging from 17.00 cm to 22.54 cm) and shoot length (ranging from 46.00 cm to 62.94 cm) as compared to the salinized treatments for root (ranging from 14.71 cm to 17.93 cm) and shoot (ranging from 31.91 cm to 47.07 cm) length (Figure 1A).

Salinity Screening at Vegetative Stage
In vegetative stage screening, Binadhan-10 had a high percent reduction for root (67.34% and 74.18%) and shoots biomass (79.08% and 77.68%) based on fresh weight and dry weight, respectively while the lowest percent reduction in root biomass was obtained from NSIC Rc182 (21.32% and 28.83%).And the lowest percent reduction in shoot biomass content was explained by Binadhan-8 (9.97% and 18.15%).Comprehensively, the root length of the salinized genotype ranged from 19.00 cm to 30.50 cm while that of normal treatments from 26.50 cm to 31.33 cm.Meanwhile, the shoot length of the salinized genotypes ranged from 80.33 cm to 98.50 cm while that of the normal treatments from 104.00 cm to 111.00 cm.The results of the seedling and vegetative stages seemed to have the same tendency as illustrated in (Figure 1B).

Salinity Screening at Reproductive Stage
At reproductive stage, NSIC Rc222 has expressed highest percent reduction for root biomass for T 4 (66.84%),T 5 (62.58%), and T 7 (79.49%)based on dry weight content while the lowest percentage reduction was obtained in IR64 for T 4 (3.35%), in NSIC Rc182 for T 5 (1.61%), and in Binadhan-8 for T 7 (0.76%).Furthermore, NSIC Rc222 had highest percent reduction in biomass for shoot fresh weight for T 4 (36.00%) and T 7 (79.79%)as compared to the moderately tolerant IR64 and tolerant Binadhan-8.The root length under normal condition ranged from 25.27 cm to 30.33 cm while that of the salinized condition ranged from 17.23 cm to 29.77 cm.At the same time, the shoot length in normal condition ranged from 93.90 cm to 108.77 cm and that of the salinized condition ranged from 78.87 cm to 105.80 cm (Figures 1C, 1D and 1E).The variable measured for assessing the effects of salinization set-up on the yield components of 5 rice genotypes were: days to flowering, panicle length, and percent reduction in filled grains number, unfilled grains number, and 1000-grain weight.Salinity causes delayed in flowering and panicle initiation.For T 4 treatment, Binadhan-8 (74%) showed highest filled grains along with NSIC Rc222 (68%) whereas NSIC Rc182 showed the smallest value (32%).In addition, 62% of unfilled grains number reduction was observed in NSIC Rc222 and Binadhan-10 while the lowest was for IR64 (11%).For 1000-grain weight, highest reduction was observed for NSIC Rc182 (26.27%) and the lowest for NSIC Rc222 (1.19%) (Figure 1F).In case of T 5 , the reduction in filled and unfilled grains were 73% and 47% for NSIC Rc222, and 15% and 18% for NSIC Rc182, respectively as the two extreme values compared to others genotypes.For 1000-grain weight, highest reduction was for IR64 (9.84%) and lowest reduction was for NSIC Rc182 (1.75%) (Figure 1G).In T 7 treatment all the varieties showed differences for all measured variables.NSIC Rc222 showed highest mean for percent reduction in both filled and unfilled grains number (90% and 69%, respectively), but Binadhan-8 had the smallest mean (4%).In case of 1000-grain weight NSIC Rc182 (4.94%) had the smallest value (Figure 1H).NSIC Rc222 had a high percent reduction in biomass for shoot fresh weight such as 54.43% at seedling stage (T 1 and T 2 ), 65.09% at vegetative stage (T 3 ), and 52.08%reproductive stage (T 4 ) screening, as compared to the tolerant NSIC Rc182 with 35.30%, 17.57% and 48.15%, Binadhan-8 with 18.02%, 9.97% and 50.46%, moderately tolerant Binadhan-10 with 23.68%, 79.08% and 16.00%, and IR64 with 43.65%, 48.83% and 24.09%, respectively (Figures 1A, 1B and 1C).There was a relationship between the genotypes SES scores (Tables 1 and  2) and percent reduction in root-shoot biomass for fresh weight and dry weight content (Figure 1).Root and shoot length at seedling, vegetative and reproductive stages screening in all treatments were reduced in the different salinized treatments (Figures 1A,1B,1C,1D and 1E).The percent reduction in dry weight and fresh weight of the root biomass was higher than that of the shoot biomass for all the genotypes.
This indicated that reduction in filled grains, unfilled grains and 1000-grain weight among the different genotypes was caused by salinity stress.

Physiological Characterization
The five rice genotypes were evaluated for the following physiological parameters: Shoot Na + /K + ratios for both normal and salinized treatments.

Seedling Stage
In the seedling stage, the ratio of Na + /K + in the genotypes under normal treatment were lower compared to that under salinized treatment.Under salinized treatment, the Na + /K+ ratio was significantly different among the genotypes.The Na + /K + ratio of the tolerant genotypes (NSIC Rc182, Binadhan-8, Binadhan-10) and moderately tolerant genotype (IR64) was lower than that of the sensitive NSIC Rc222 (Figure 2A).The difference in the ratio of Na + /K + under normal condition for all genotype was not significant.
Figure 2A.Mean comparison of the each genotype of rice across Na + /K + absorption (impose salinity EC = 8 dS/m at seedling stage only)

Vegetative Stage
At vegetative stage the ratio of Na + /K + among the genotypes under normal and salinized treatments was different.
In the salinized condition, the average value of Na + /K + for NSIC Rc222 (0.12) and NSIC Rc182 (0.09) were slightly higher than that of the control (IR64) while Binadhan-8 (0.08) has shown similar value compared to the control(0.08).Further, the Na + /K + ratio of Binadhan-10 (0.06) was relatively lower than that of the control.In normal condition, 3 genotypes such as NSIC Rc222 (0.06), Binadhan-8 (0.06) and Binadhan-10 (0.07) have explained a relatively low value of Na + /K + while that of the NSIC Rc182 (0.13) was high as compared to the control (0.08), respectively.No difference was found in Na + /K + ratio for the control (IR64) under both the saline and normal condition (Figure 2B).

Reproductive Stage
At the reproductive stage, genotypes have lower values of Na + /K + ratio under normal treatment than under salinized treatment, which varied for flag leaf and the third leaf from the top.Comparatively, the Na + /K+ absorption rate in the third leaf is higher than in the flag leaf.However, the average Na + /K+ values between the third leaf and flag leaf under normal treatment were different for all the genotypes.In reproductive stage screening, this Na + /K + ratio the tolerant genotypes seemed to be lower than that of sensitive genotypes.In case of flag leaf, Na + /K + ratio of tolerant genotypes was averagely higher compared with the normal treatment.Tolerant and moderately tolerant genotype NSIC Rc182, Binadhan-8, Binadhan-10 and IR64 had lower Na + /K + ratio than sensitive NSIC Rc222 in third leaf under salinized condition.In normal condition Na + /K + ratio also varied for all the genotypes for third leaf and for flag leaf (Figures 2C and 2D).

Molecular Characterization
Among the 161 SSR markers used to amplify PCR products in 5 rice genotypes, 98 which showed clear bands that could be scored and used for genetic analysis.A total of 209 alleles were detected, which ranged from 1 to 4 alleles with an average of 2.13.Four markers out of the 101 amplified 4 alleles and 25 showed highest frequency of major allele.On other hand, the PIC values calculated ranged from 0.27 to 0.67 with an average of 0.41.It is observed that the polymorphism information content (PIC) was inversely proportional to the frequency of the major allele.According to Ram et al. (2007), a PIC value higher than 0.50 indicates high degree of polymorphism.In this study, 19 SSR markers were found to score PIC value more than 0.50, thus indicated that these markers could be used for performing genetic diversity analysis for the traits.This implied that these markers were effective for screening salinity tolerance of the 5 genotypes.Accordingly, 4 out of the 19 SSR markers have showed 0.72 as the highest level of genetic diversity value with 0.48 as mean (Table 4).The UPGMA was constructed in this study based on genetic similarity values of the SSR markers alleles from all the rice genotypes.The Result showed two major groups, group I and II.The cluster analysis showed significant genetic variation among the rice genotypes studied.The first group clustered NSIC Rc222 as salt sensitive genotype while the second group included the genotypes Binadhan-8, NSIC Rc182, Binadhan-10 and IR64 (Figures 3A and 3B) with relatively tolerant ability against salt stress.

Figure 1A .
Figure 1A.Mean comparison of the each genotype of rice across all physiological traits for seedling stage (EC = 8 dS/m) Note.*S = Salinized; N = Normal; G-R = Growth Reduction; FW = Fresh Weight; DW = Dry Weight.

Figure 1B .
Figure 1B.Mean comparison of the each genotype of rice across all physiological traits for vegetative stage (impose salinity EC = 12 dS/m at vegetative stage only) Note.*S = Salinized; N = Normal; G-R = Growth Reduction; FW = Fresh Weight; DW = Dry Weight.

Figure 1C .
Figure 1C.Mean comparison of the each genotype of rice across all physiological traits for reproductive stage (impose salinity EC = 8 dS/m at reproductive stage only) Note.*S = Salinized; N = Normal; G-R = Growth Reduction; FW = Fresh Weight; DW = Dry Weight.

Figure 1D .
Figure 1D.Mean comparison of the each genotype of rice across all physiological traits for reproductive stage (impose salinity EC = 8 dS/m at seedling and vegetative stages only) Note.*S = Salinized; N = Normal; G-R = Growth Reduction; FW = Fresh Weight; DW = Dry Weight.

Figure 1E .
Figure 1E.Mean comparison of the each genotype of rice across all physiological traits for reproductive stage (impose salinity EC = 8 dS/m at vegetative and reproductive stages only) Note.*S = Salinized; N = Normal; G-R = Growth Reduction; FW = Fresh Weight; DW = Dry Weight.

Figure 1F .
Figure 1F.Mean comparison of the each genotype of rice across all yield traits (impose salinity EC = 8 dS/m at reproductive stage only) Note.*PL = Panicle length; S = Salinized; N = Normal; FG = Filled grain; UFG = Unfilled grain; TGW = Total grain weight; MC = Moisture content.

Figure 1G .
Figure 1G.Mean comparison of the each genotype of rice across all yield traits (impose salinity EC = 8 dS/m at seedling and vegetative stages only) Note.*PL = Panicle length; S = Salinized; N = Normal; FG = Filled grain; UFG = Unfilled grain; TGW = Total grain weight; MC = Moisture content.

Figure 1H .
Figure 1H.Mean comparison of the each genotype of rice across all yield traits (impose salinity EC = 8 dS/m at vegetative and reproductive stages) Note.*PL = Panicle length; S = Salinized; N = Normal; FG = Filled grain; UFG = Unfilled grain; TGW = Total grain weight; MC = Moisture content.

Figure 2B .
Figure 2B.Mean comparison of the each genotype of rice across Na + /K + absorption (impose salinity EC = 12 dS/m at vegetative stage only)

Figure 2C .
Figure 2C.Mean comparison of the each genotype of rice across Na + /K + for 3 rd and flag leaf (impose salinity EC = 8 dS/m at reproductive stage only)

Figure 3A .
Figure 3A.Dendrogram of 5 rice genotypes showing the genetic similarity based on SSR marker using the UPGMA cluster analysis

Table 1 .
Modified standard evaluation system (SES) of visual salt injury at seedling stage(IRRI, 1996)

Table 2 .
Average salinity stress reactions at EC = 12 dS/m of five rice genotypes at the seedling stage (T 1 )

Table 3 .
Average salinity stress reactions at EC = 8 dS/m of five rice genotypes at the seedling stage (T 2 )

Table 4 .
Statistics Allelic variation of 98 SSR loci in the 5 rice genotypes surveyed