EVALUATION OF RICE GERMPLASM UNDER SALT STRESS AT THE SEEDLING STAGE THROUGH SSR MARKERS

Twenty eight rice germplasms were used for identification of salt tolerant rice genotypes at the seedling stage at the experimental farm and Biotechnology laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh during February 2009 to October 2009. Phenotyping for salinity screening of the rice genotypes was done using salinized (EC level 12 dS m-1) nutrient solution in hydroponic system. Genotypes were evaluated for salinity tolerance on 1-9 scale based on seedling growth parameters following modified Standard Evaluation Scoring (SES) of IRRI. Phenotypically, on the basis of SES and % total dry matter (TDM) reduction of the genotypes viz. PBSAL-614, PBSAL-613, PBSAL-730, Horkuch, S-478/3 Pokkali and PBSAL (STL)-15 were found to be salt tolerant; on the other hand Iratom-24, S-653/32, S-612/32, S-604/32, S-633/32, Charnock (DA6), BINA Dhan-6 and S-608/32 were identified as salt susceptible. For genotyping, ten SSR markers were used for polymorphism, where 3 primers (RM127, RM443 and RM140) were selected for evaluation of salt tolerance. In respect of Primer RM127, 7 lines were found salt tolerant and 11 lines were moderately tolerant and 10 lines were susceptible. Nine tolerant, 9 moderately tolerant and 10 susceptible lines were found when the primer RM140 was used and primer RM443 identified 8 lines as tolerant, 9 lines as moderately tolerant and 11 lines as susceptible. Thus, the salt tolerant lines can be used in further evaluation for salinity tolerance and the SSR markers used in this study are proving valuable for identifying salt tolerant genes in marker assisted breeding.


Introduction
Rice (Oryza sativa L.) belongs to the family Gramineae.It is the staple food crop in Bangladesh and one of the most important cereal crops throughout the world.This staple food ranked first position by production (28931 thousand metric tons) during the year 2007-08 among all cereals in Bangladesh (BBS, 2009).Total rice cultivated area 10575 thousand hectares.One of such efforts is to cultivate rice with elevated level of salt tolerance on salt affected marginal lands.Over 30 percent of the net cultivable area of Bangladesh lies in the coastal zone.Out of 2.85 million hectares of coastal and offshore land, about 1.5 million hectares are affected by varying degrees of salinity.The coastal saline soils are distributed unevenly in 64 Upazila of 13 District, covering portions of eight agro-ecological zones (AEZ) of the country (Seraj and Salam, 2000).Salinity affects plants at all stages of development, but sensitivity sometimes varies from growth stage to the next.Several studies indicated that rice is tolerant during germination, becomes very sensitive during early seedling stage (2-3 leaf stage), gains tolerance during vegetative growth stage becomes sensitive during pollination and fertilization and then become increasingly more tolerant at maturity (Pearson et al., 1966;IRRI, 1967).However, some studies reported that at fertilization, rice is not sensitive to salinity (Kaddah et al., 1975).Hence, early seedling stages were used to know the response of the rice plant to salinity.To screen the salt tolerant variety, we need reliable technique.IRRI standard protocol for salinity screening is such type of technique (Gregorio, 1997).Conventional breeding is time consuming and depends on environmental conditions.Molecular marker technology offers a possibility by adopting a wide range of novel approaches to improve the selection strategies in rice breeding.SSR or microsatellite markers are proved to be ideal for making genetic maps (Islam, 2004;Niones, 2004), assisting selection (Bhuiyan, 2005) and studying genetic diversity in germplasm.
Microsatellite marker analysis is promising to identify major gene locus for salt tolerance that can be helpful for plant breeders to develop new cultivars.From these points of view the present study were undertaken for determine the phenotypic performance of rice germplasm under salinized conditions at the seedling stage and to identify salt tolerant rice lines from twenty eight rice germplasm using SSR markers.

Plant materials
The experiment was carried out at the experimental farm and Biotechnology Laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh from February 2009 to October 2009.In this study twenty eight rice germplasm were used for salinity screening at the seedling stage following International Rice Research Institute (IRRI) standard protocol (Gregorio et al., 1997)

Phenotypic study of salinity tolerance at seedling stage
The screening for salt tolerance were done using modified standard evaluation score (SES) of visual salt injury of the seedlings (Table 1).
Hydroponic system with IRRI standard protocol (Gregorio et al., 1997) was used at the glasshouse to evaluate salt tolerance of rice germplasm.Nutrient solution (Yoshida et al., 1976) was used in hydroponic system for screening salinity tolerance at the seedling stage.The modified standard evaluation score (SES) of IRRI was used to assess the visual symptoms of salt toxicity (Table 1).This scoring discriminates the tolerant, moderately tolerant and susceptible rice lines.

CTAB mini preparation DNA extraction
The modified Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method was used to extract DNA from the plant leaves.The following steps were followed in PCR-based DNA marker analysis.Healthy leaf samples were collected from 25-day old seedlings of each germplasm for isolation of genomic DNA.Leaf sample was washed in distilled water and ethanol.The collected leaf samples were then put into polythene bags with tags, placed on ice and stored in a -80oC freezer.The leaf samples were ground with pestle and mortar to collect DNA.Strict hygiene was maintained during the DNA extraction by autoclaving all glassware, micropipette tips, PCR tubes, distilled water, reagents and buffer solutions.
Isolated genomic DNA contains a large amount of RNA and pigments, which cause over estimation of DNA concentration during spectrophotometer reading.Therefore, the DNA samples were evaluated qualitatively using agarose gel electrophoresis.
For checking of DNA concentration, two methods can be applied: 1)  DNA and 2) Spectrophotometry.For this study only  DNA was used.After electrophoresis, the gel was taken out carefully from the gel chamber and transferred in a prepared ethidium bromide solution for staining.Staining was done for 20 minutes and then placed on the UV transilluminator in the dark chamber of the Image Documentation System.

Amplification of microsatellite markers and evaluation of genotypes
Ten primers of random sequence were selected for amplification of the DNA sequences.Primers were evaluated based on intensity of bands, consistency within individual, presence of smearing and potential for population discrimination.In this experiment, OSR14, OSR17 RM152, RM443, RM127, RM140, RM9, RM169, RM18 and RM21 primers were used for polymorphism.Out of ten primers, three polymorphic SSR markers viz., RM127, RM443 and RM140 were selected to evaluate 28 rice germplasm for salt tolerance.The PCR cocktail had total volume of 15.0 µl reaction mixture including 2 µl DNA based on salinity protocol, was placed in the PCR tubes and run in the DNA thermal cycler.The PCR tubes were set on the wells of the thermocycler plate and the machine was run according to the programme.
The germplasms having similar banding pattern to Pokkali were considered as tolerant, banding pattern having similar to Iratom-24 were considered as salt susceptible and having banding pattern at the medium position between Pokkali and Iratom-24 were considered as medium tolerant In each marker, allelic bands were designated as T for tolerant, S for susceptible and MT for medium tolerant.

Phenotypic variation of rice genotypes under salinized and non-salinized conditions at the seedling stage
Twenty eight genotypes of rice seedlings were used for screening salinity tolerance.After two or three days of salinization, salt stress symptoms were started.Several salt injuries observed in the salinized conditions which were leaves became yellowish, drying of leaves, shoot growth reduction, root growth reduction, stem became thin and week, stunted growth of seedlings and seedling drying occurred.These symptoms were also observed by several researchers (Bhuiyan, 2005;Islam, 2004;Niones, 2004;Bonilla et al., 2002).The seedlings in the non-salinized condition showed normal growth over the salinized condition.Salt tolerant seedlings were distinguished from the sensitive seedlings when grown in salinized condition.
The salinity tolerant lines showed no symptom of salt injury.In the salinized setup, twenty eight germplasms showed wide variation in phenotypes under salt stress.Among the 28 germplasms, seven were found salt tolerant viz.Pokkali, Horkuch, PBSAL (STL)-15, PBSAL-613, PBSAL-730, PBSAL-656 and S-478/32 (Table .3),ten were moderately tolerant and eleven were susceptible.According to the SES of IRRI (Gregorio et al., 1997) a score from 1-9 was used for grading the genotypes based on visual salt injury where, 1= highly tolerant and 9= highly susceptible.This scoring differentiated the susceptible genotypes from the tolerant and moderately tolerant genotypes.Islam et al. (2007) also observed wide variation in phenotypes from tolerant (score 3) to highly susceptible (score 9) using modified SES of IRRI standard protocol.Such types of results were also found by several researchers (Javed et al., 2006;Maiti et al., 2006).They showed that the increase in salinity level reduced the seedling height.Munns and Tester (2008) also reported that salinity might directly or indirectly inhibit cell division, consequences leaves and stems of the affected plants become stunted.
From the above results, it can be said that TMD reduction increased under salt stress.Javed et al. (2006) reported that increase in salinity level reduced the fresh biomass and dry biomass.Roy et al. (2002) reported that reduction of dry biomass (% TDM) increased with the increased of salinity level.

Genotyping of salinity tolerance
Identification of molecular markers tightly linked to salt tolerant genes can serve as landmarks for the physical localization of such genes facilitating marker assisted selection (MAS).

Screening twenty eight rice germplasmss for salt tolerance using SSR markers
In this experiment RM152, RM443, RM127, RM140, RM9, RM169, RM18 and RM21 primers were used for polymorphism survey of twenty eight rice germplasms.Out of these primers, three polymorphic SSR markers viz., RM127, RM443 and RM140 were shown highly polymorphic and these three markers were selected to evaluate 28 rice germplasms for salt tolerance.According to the phenotypic performance, Pokkali was considered as tolerant and Iratom-24 was considered as susceptible.The lines having similar banding pattern to Pokkali were considered as tolerant and similar to Iratom-24 were considered as salt susceptible and having both banding pattern (Pokkali and Iratom-24) were considered as moderately tolerant (Table 5).
In respect of primer RM127, 7 lines were found tolerant at salt stress.In the same reaction, 11 lines were moderately tolerant and 10 lines were susceptible with compared to the tolerant variety Pokkali and susceptible variety Iratom-24 (Fig. 1).
Nine lines were detected as tolerant, 10 susceptible and 9 moderately tolerant lines were found with compared to the tolerant variety Pokkali and susceptible variety Iratom-24 with the reaction with RM140 (Fig. 2).
With RM443, 8 tolerant lines were identified.In the same reaction, 11 saline susceptible lines and 9 moderately tolerant lines were also found with compared to the tolerant variety Pokkali and susceptible variety Iratom-24 (Fig. 3).
These three primers i.e.RM127, RM140 and RM443 showed polymorphisms in the studied twenty eight rice germplasms because they showed different banding patterns and discriminate tolerant lines from moderately tolerant and susceptible with relation to the tolerant variety Pokkali and susceptible variety Iratom-24.The SSR markers were also reported as highly polymorphic in IR29 × Pokkali for tagging salt tolerant genes (Islam, 2004;Niones, 2004).El-Refaee et al. (2006) also reported that 80% of the tested SSR primers showed polymorphic pattern in rice while they studied 272 SSR primers on nine rice genotypes.Sultana et al. (2007) found RD-2586, TBDB-100, PNR-519 and Dhol Kochuri as tolerant; BRRIdhan 40 and Y-1281 as moderately tolerant regarding the marker RM115 and concluded that genotypic screening for salt tolerance using molecular marker is more trustworthy and effective.Islam et al. (2007) also identified the genotypes viz.
From the phenotypic data of twenty eight rice genotypes, based on SES and percentage TDM reduction PBSAL-614, PBSAL-613, PBSAL-730, Horkuch, S-478/3 and Pokkali were identified as salt tolerant rice genotypes whereas Iratom-24, S-653/32, S-611/32, S-604/32, S-633/32, Binadhan-6 and S-608/32 were identified as salt susceptible rice genotypes.Keshrail, S-611/32, S-546/32, S-530/32, S-603/32, S-445/32, S-375/32, PBSAL-614, STM-1 were identified as moderately salt tolerant rice genotypes.Finally, salt tolerant rice lines were identified both phenotypically (salinity screening at the seedling stage) and genotypically using twenty eight rice germplasms.The tested markers RM127, RM140 and RM443 could be efficiently used to identify salt tolerant lines in rice and can also be used in marker-assisted selection (MAS) for breeding, quantitative trait loci (QTL) mapping, studying genetic diversity in germplasms and gene pyramiding in rice salinity breeding.Microsatellite marker analysis is promising to identify major gene locus for plant breeders to develop new cultivars.The selected salt tolerant rice lines could be further tested in saline areas to observe yield potentiality for developing high yielding salt tolerant varieties suitable for saline areas.

Table 2 .
The sequence and size of the microsatellite markers used for the study

Table 4 .
Performance of plant height, root length and total dry matter at the seedling stage (30DAS) under salinized and non-salinized conditions

Table 5 .
Genotypic performance of twenty eight rice germplasm using SSR markers