Screening of Some Rice ( Oryza sativa L . ) Genotypesfor Salinity Tolerance Using Morphological and Molecular Markers

Salinity is one of the major abiotic stresses, which adversely affects the crop productivity. Thirty rice genotypes of diverse origin including three salt tolerant check varieties, Binadhan-8, Binadhan-10 and Pokkali, were used to evaluate salt tolerance at seedling stage and to determine the genetic diversity using microsatellite markers. Salinity screening was done at the seedling stage using hydroponic system following IRRI standard protocol. Three salinity levels as 6dSm-1, 8dSm-1, and 10dSm-1 were used along with control. Data were recorded on root length, shoot length and dry weight and the genotypes were scoredbased on modified standard evaluation score (SES) for visual injury. Sixteen SSR markers were used to study the genetic variation within 30 rice genotypes. A total of 65 alleles with an average of 4.06 allele per locus were detected among 30 rice genotypes. The polymorphism information content (PIC) value ranged from 0.24to 0.86 with an average of 0.51. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram revealed four clusters. Among them cluster I identified 5 salt tolerant genotypes and cluster IV separated one tolerant and one moderately tolerant genotype. Based on SES evaluation and molecular analysis genotypes Balam, THDB, Q-31, Ab.Hai, BR-5, FR13A ware salt tolerant; Moulota, Superhybrid, Y-1281, Binadhan-16 weremoderate salt tolerant. This information could be useful for selection of suitable genotypes for developing salt tolerant rice variety through molecular breeding.

A large part of the world's population is having rice as a staple food, especially in the East, South, Southeast Asia, tropical Latin America and West Indies.Rice (Oryza sativa) belonging to the family Graminae and subfamily Oryzoidea comprises of two main types: indica and japonica in Asia.The indica type is from tropical and japonica is from temperate and subtropical Asia.
Oryza glaberrima originates from inland delta of the Niger River.Nowadays, the Asian species (O.sativa) is cultivated more than the African species (O.glaberrima), mainly for its higher yield potential (Wopereis et al.2013).In Bangladesh the dominant food crop is rice, accounting for about 75% of agricultural land use.Agriculture sector contributes about 17% to the country's Gross Domestic Product (GDP) (BBS, 2016).Rice is grown in three seasons (Aus, Aman and Boro) of the year in Bangladesh.Salt stress is one of the major abiotic stresses, which adversely affect the crop productivity (Yasseen et al. 2010; Joseph and Jini 2010).It causes reduction of crop yield and alteration in plant metabolism, including a reduced water potential, ion imbalances and toxicity and sometimes severe salt stress may even threaten survival (Joseph and Jini 2011).So the need of the time is to develop plants with resistance to salinity.Rice is a highly polymorphic crop species with wide geographic distribution (Nemati et al.,2011).Bangladesh is endowed with a great diversity of rice landraces in its vast traditional land area.After green revolution the traditional rice landraces were eliminated majorly by high yielding varieties.In recent scenario abiotic stress tolerance study has much significance due to global warming.Systemic study and characterization of such landraces is important for utilization of appropriate attribute based donors.Landraces of rice played a very important role in the local food security and sustainable development of agriculture, in addition to their significance as genetic resource for rice genetic improvement (Tang et al.,2002).
Simple Sequence Repeats (SSR) can presently be short motif nucleotides (Dhar et al., 2012).DNA fingerprinting using SSR markers is playing an important role to identify gene for salt tolerance.They have become a popular type of codominant molecular marker in genetic analysis and plant breeding application (Choi etal.,2011) and also been useful in integrating genetics, physical and sequence based maps of rice that provides breeders and geneticists with efficient tool to link phenotypic and genotypic variations.SSR or microsatellite markers are proved to be ideal for making genetic maps (McCouch et al., 2002), marker assisting selection (Afiukwa et al, 2016), DNA fingerprinting analysis (Chakravarti et al., 2016)and studying genetic diversity (Roy et al.2015;Islam et al. 2018) in genotype.These experiments explore and evaluate the pattern and extent of genetic variability and relatedness among 30 rice genotypes at the molecular levels using SSR markers and to help in the identification and differentiation of landraces with different genetic make-up.The generated information will enable maximized selection of diverse parents and selecting appropriate parental genotypes in breeding programme for improving salt tolerance of elite cultivars based on genetic similarity and clustering data together with variations of tolerance to salt.

Plant culture
The experiment was conducted at the Glass House and Biotechnology Laboratory, Bangladesh Institute of Nuclear Agriculture (BINA).Rice genotypes were collected from BINA.A list at Table 1 is given below to mention the name origin and source of 30 genotypes.

Screening of varieties for tolerance to salinity at seedling stage
All genotypes were screened for salt tolerance at seedling stage in hydroponic system using in hydroponic, system using International Rice Research Institute (IRRI) standard protocol (Gregorio et al., 1997).Youshida et al.(1976) solution was used as nutrient solution that was renewed every 8 days and was maintained at pH 5.1.The nutrient solution was salinized by adding crude salt to obtain desired EC (6dSm -1 ,8dSm -1 &10dSm -1 ).The standard evaluation system (SES) of IRRI was followed to assess the visual symptoms of salt toxicity (Gregorio et al., 1997).Initial and final scoring was done at 14 th d and 21 st d respectively after salinization.Besides root length, shoot length and root / shoot ratio were recorded at three different levels of treatment along with the control.The experiment was laid in completely randomized design with three replications.

Genotyping of salinity tolerant rice genotypes
Modified CTAB mini prep was used for DNA extraction for 21-day-old seedling (Stein et al., 2001).A total of 46 SSR primer were used covering all 12 chromosomes.Among these, sixteen primers were showed polymorphic.Each PCR carried with 10.0µl reactions containing 2.0 µl 5X Green buffer, 1.2 MgCl 2 (10mM), 0.5µl dNTPs (10mM), 0.5µl primer forward, 0.5µl primer reverse, 0.2 µl taq polymerase, 4.1 µl RNase free water and 1.0 µl of each template DNA samples.PCR was maintained as initial denaturation at 94 0 C for 5 min, followed by 35 cycles of denaturation at 94 0 C for 30 sec, annealing at55 0 C for 30 sec extension at 72 0 C for 1.0 min; and final extension by 5.0 min at 72 0 C. Then amplified fragments were separated on 8.0% (w/v) native polyacrylamide gels, those were performed at 70-80V for 1.5-2.5h in 1× TBE [Tris-borate-ethylenediaminetetraacetic acid (EDTA)] buffer, and the gels were stained with ethidium bromide for 25-30 min, kept in dark, and then visualized using an UVPRO (Uvipro platinum, EU) gel documentation unit linked to a PC.

SSR data analysis
The size of amplified fragments was measured by comparing the migration of amplified fragments with that of a known size fragments of molecular weight marker, 100 base pair DNAladder, using Alpha-Ease FC 5.0 software (Alpha Innotech,USA).Genetic diversity of cultivers by SSRs was evaluated with the number of alleles and the polymorphism information content (PIC) value, which is an estimate of the discriminatory power of a SSR marker locus.Statistics shows including the number of alleles per locus, major allele frequency, gene diversity and PIC values were calculated using Power Marker version 3. 25.The band profiles for each SSR primer were scored for distinct and reproducible bands as present (1) or absent (0).Jaccard's similarity

Screening of rice genotypes for salinity tolerance at seedling stage
Thirty genotypes of rice seedlings were used for screening salinity tolerance.Salt stress are applied at 7 th days old seedling.After two or three days of salinization, salt stress symptoms were observed.Several symptoms of salt injury like yellowing of leaves, drying of leaves, and reduction in root growth, reduction of shoot growth and stem thickness and in many cases dying of seedlings were detected within 2-weeks continuous salt stress of 6,8,10 dSm -1.Some other symptoms like rolling, tip whitening were also noticed.Above all of these, reduction in leaf area was the first symptom.Salinity suppresses the growth of leaves in the plants and eventually completes cessation of growth and premature senescence of leaves.Overall, the seedlings growth was suppressed under salinity stress.On the other hand, the seedlings in the non-salinized (control) 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 minor symptoms of salt injury.Among these 30 genotypes, according to the SES of IRRI, at 6 dSm -1 , some genotypes showed highly tolerant (HT) e.g.Binadhan-8, THDB, Pokkali, Binadhan-10, FR13A, Super hybrid, Binadhan-16, BR-5, Moulota, Ab.Hai, Balam and Q-31.The genotypes BRRI dhan29, BRRI dhan46, BINA-E-02, R3027, BRRI dhan39, BINA-MV-10, Y-1281 was tolerant (T) and rest of the genotypes were moderately tolerant (MT).
Whereas, at 8 dSm -1 ,12 of them found as tolerant (T), 7 were moderately tolerant (MT) and rest of the genotypes were susceptible (S).At 10 dSm -1 , 9 of them found as tolerant (T), 4 were moderately tolerant (MT), 6 were susceptible (S) and rest of them were highly susceptible (HS).The performance of these genotype under at different salinize conditions is given at Figure 1.

Molecular characterization of rice using SSR markers
Analysis of genetic diversity is important for rice improvement that can be obtained through DNA fingerprinting techniques, which is capable of exhibiting large number of loci for extensive variability.Genotypes collected from different location and origin wareanalysed using a highly repeatable PCR based fingerprinting assay known as Simple Sequence Repeat (SSR) or microsatellites markers.

Allelic and loci variation within the genotypes
SSR markers are widely used for fingerprinting and diversity studies on rice cultivars and wild relatives due to its high polymorphic rates, which can be identified even at individual rates.
The microsatellite loci were also polymorphic.Total 65 polymorphic alleles were generated by 16 SSR primers in the studied 30 rice genotypes.
The number of polymorphic allele varied from 2 (RM508) to 9 (RM336) with an average of 4.06 allele (Table 5).The bands obtained from other genotypes were compared to the band obtained from salt tolerant variety like Binadhan-8, Binadhan-10 and Pokkali which were used as salt tolerant check verities in this study because these are widely known as salt tolerant.The detailed result which was obtained after analysis of fingerprinting data are briefly presented (Figure 2-5) and discussed below.

Genetic diversity and major allele
The highest genetic diversity (0.91) was observed in loci RM336 and the lowest genetic diversity (0.28) was observed in loci RM508 with a mean diversity of 0.56 (Table 5).It was observed that marker detecting the lower number of alleles showed lower genetic diversity than those which detected higher number of alleles which revealed higher genetic diversity (Herrera et al. 2008;Rana et al. 2018).Major allele is defined as the allele with the highest frequency and also known as most common allele at each locus.The size of the different major alleles at different loci ranges from 100bp (RM277) to 312 bp (RM594) (Table 5).On an average, 60 % of the 30 rice lines shared a common major allele ranging from 30% (RM336) to 80% (RM508, RM517, RM7175) at each locus.

PIC value
Polymorphism information content (PIC) value is a reflection of allele diversity and frequency among the varieties.PIC value of each marker can be evaluated on the basis of its alleles.PIC varied significantly for all the studied SSR loci.
In the present study, the level of polymorphism among the 30 rice genotypes were evaluated by calculating PIC values for each of the 16 SSR loci.The PIC values ranged from 0.28 (RM508) to 0.86(RM336) with an average of 0.51 per locus (Table 5).

Table 1 .
List of genotypes with their origin and Source of collection

Table 4 .
Dry Weight and its reduction (%) in 30 rice genotypes upon salinity stress compared with control condition at seedling stage Ploense et al. 2002; Hakim et al. 2010; Chunthaburee et al. 2016.

Table 5 .
Data of sample size, major alleles, PIC value, genetic diversity and heterozygosity found among 30 rice genotypes for 16 microsatellites (SSR) markers The lowest PIC value observed 0.28 for RM 508.PIC value observed in our study was consistent with the pervious works ofLu et al. 2005; Hossain et al. 2012; Sajib et al. 2012.Considering the PIC value and genetic diversity data RM 336 would be best for screening the studied 30 rice