Physio-Biochemical and Microsatellite Based Profiling of Lowland Rice ( Oryza sativa L . ) Landraces for Osmotic Stress Tolerance

Global climate changes accelerate a wide range of abiotic stresses leading to a series of physiological, biochemical and molecular changes that adversely affect the growth and productivity of rice (Oryza sativa L.). In this background, there is limited knowledge and profiling reports of lowland, less popular rice landraces in relation to their osmotic stress tolerance. Laboratory mediated physiological and biochemical screening for a selected set of 20 lowland rice landraces was done in induced drought and salinity stress conditions. Varietal genetic diversity and inter-relatedness were assessed by using osmotic stress tolerance linked forty-five rice microsatellite markers. For representing the allelic diversity of the studied microsatellite loci across the selected genotypes, a microsatellite panel was constructed and PIC values of all used microsatellite markers were calculated. The obtained database can be used for varietal identification, characterization and genetic information in relation to osmotic stress tolerance.


Introduction
Rice (Oryza sativa L.) is the principal staple food as over 50% of the world's population depends on it for about 80% of their food requirements [1].Rice is grown under diverse ecological conditions (irrigated, lowland, upland, coastal, drought and flood prone areas) in tropical Asian countries such as India.Global climate change accelerates a series of abiotic stresses, especially drought and salinity (osmotic stress), which impose serious threats for rice production worldwide [2].By the end of the 1980 (post Green-revolution time), conventional breeding mediated rice yield's potential was increased in different experimental trials [3].During this agricultural modernization era, a large number of rice landraces have been lost due to the invasion of high-yielding rice varieties [3].Although less productive, these landraces have been reported to be highly tolerant to a number of biotic and abiotic stresses [4,5] and harbour a good number of other quality traits [6,7].Unlike high-yielding varieties (whose variability is limited due to homozygosis), the rice landraces are endowed with tremendous genetic variability, as they are not subjected to subtle selection over a long period of time [8,9].For proper utilization and incorporation of these indigenous rice lines from the natural crop resources, identification of the allelic variant that results in plant phenotype is the utmost important.Conservation of these genetic resources is, therefore, much more useful since such lines have diverse gene pool providing the potentiality to withstand in varied environmental fluctuations.Their wide phenotypic and genotypic responses can also be considered for their varietal characterization and proper identification [10].In this context, molecular evaluation in form of genetic diversity for available landraces has become important in modern-day crop improvement [11,12].Such rice genetic resources were being used for incorporating genetic variability into rice breeding programs to generate new cultivars with broadened genetic bases [3] and allow new useful allelic combinations [13,14].Genetic profiling among the diverse rice lines presently grown in West Bengal (a state of In-Physio-Biochemical and Microsatellite Based Profiling of Lowland Rice (Oryza sativa L.) Landraces for Osmotic Stress Tolerance 53 dia) has been assessed by a number of workers [15][16][17][18][19] using various molecular marker systems.Microsatellites, also known as simple sequence repeats (SSRs), a recent and popular marker system, are abundant and well distributed throughout the rice genome [20][21][22], being used in rice for genomic diversity analysis [23][24][25].Several traits linked rice SSR markers have been established for drought [26,27], salinity [28,29] and submergence [30,31] tolerance.Besides the molecular characterization at DNA level, physiological performances under induced osmotic stresses and quantity of bioactive compounds (proline, chlorophyll, total protein, salicylic acid, catalase, peroxidase etc.), having a significant role in adaptation to altered environmental stress conditions, are also necessary for the proper characterization of rice lines in relation to stress tolerance.
In spite of erosion of a number of rice landraces, West Bengal is still today a rich source of indigenous rice lines having the hidden genetic potential that needs to be explored [16].There were no previous reports on the stud-ied rice landraces for their osmotic stress tolerance properties.Keeping this as an objective of the present research, a total number of 20 lowland indigenous less popular lines' suffixes with "Sail" were genotyped for the first time with 45 osmotic stress tolerance linked reported SSRs in addition to physiological and biochemical screening to make a systematic and multi-informative profile of selected varieties in relation to osmotic stress tolerance.

Plant Materials
A total of 20 indigenous, lowland, less popular West Bengal rice landraces suffix with "Sail" were selected for this investigation.The detailed description of these landraces is given in Table 1.

Physiological Screening
For physiological screening, drought stress was induced by varying concentrations (−0.5, −1.0 and −1.5 MPa) of Polyethylene Glycol 6000 (PEG) [32] and salinity was induced by three different concentrations (100, 200 and 300 mM) of sodium chloride (NaCl) solution [33].Stresses were imposed during seed germination and seedling growth for the studied landraces after the mercuric chloride mediated surface sterilization.Germination percentage (G%) and seedling growth (in form of shoot and root length) were monitored and recorded on 15 th day after seed sowing.Grains were considered germinated when both the plumule and radical had extended to more than 2 mm [34].

Biochemical Profiling
Estimation of some selected bioactive compounds commonly varying with different degree of osmotic stresses was quantified.Surface sterilized rice grains were allowed to germination and growth in a plastic pot contained artificial soil and set for 20 days at 28˚C ± 2˚C with 9 h photoperiod.Then the plantlets were transferred to culture tubes for treatment with 20 ml solutions of different PEG and NaCl concentrations (as used in physiological screening) and set for 24 h.Then, proline, chlorophyll and total protein contents were quantified from the fresh leaf tissues using standard protocols [35][36][37].

Statistical Analysis
For different physio-biochemical parameters the estimated values were analyzed statistically.Mean values of studied traits (germination percentage, root and shoot length); proline, total chlorophyll and total protein contents were subjected to analysis of variance (ANOVA) using Microsoft Excel version 10.0 as per the method of Panse and Sukhatme [38] to test the significance at 5% level of probability (P < 0.05) for each character which employed to obtain, assemble, classify and to interpret voluminous quantitative data.

Genomic DNA Isolation, PCR Amplification and SSR Genotyping
Leaf and other aerial plant tissues from five days old germinating seedlings were used for isolation of rice genomic DNA following a prestandarized protocol [39] of our laboratory.The isolated genomic DNA samples were adjusted to 25 ng/μl concentration.Based on available published reports [18,[40][41][42], forty five osmotic stress tolerance linked SSR markers were selected.The detailed information of the selected SSR markers was obtained from the Gramene database (www.gramene.org).PCR amplification, polymorphism screening and analysis of the amplified products were done [18].Individual alleles (in the form of different mol.wt. of the amplified product from a single loci) of all SSR markers were scored to prepare a binary matrix based on the presence (1) or absence (0) of a particular allele.A pair-wise genetic similarity coefficient matrix between all possible pairs of the studied genotypes were calculated from such 1/0 matrix using SPSS (version 10.0) and a dendrogram was constructed based on Ward's method using complete linkage between groups and Euclidean distance using STATIS-TICA software.Polymorphism information content (PIC value) of each SSR was also determined following a simplified formula: where i = 1 to n, P ij represents the frequency of j th allele for the i th band scored for a particular marker [43].

Physiological Screening
Germination percentage: Seed G% of the studied rice landraces on 15th day was significantly low at the highest concentrations of both PEG (−1.5 MPa) and NaCl (300 mM).With increasing doses of stressors (PEG and NaCl), G% gradually decreased when compared with the control set (Figures 1(a) and (b)).The significant exception found for three genotypes (Lati Sail, Punjab Sail and Rup Sail) showing higher G% than the control for lower doses.Although, both PEG and NaCl inhibit seed germination, PEG showed more inhibitory effects than that of NaCl.Malik Sail, Neizer Sail and Rani Sail showed highest germination (100%) in all osmotic stress conditions.Chapri Sail, Punjab Sail, Rup Sail and Sankar Sail showed 100% germination in all NaCl doses, but not in PEG.Days to seed germination was also delayed with the increasing concentration of stressors.
Growth pattern: The phenotypic responses in form of shoot and root length in induced osmotic stresses (by PEG and NaCl treatment) were varied greatly.Shoot length in control was maximum (5.7 cm) in Malik Sail, whereas the minimum (3.2 cm) was obtained for Mallik Sail.Overall shoot growth in three different doses of PEG were more than the values found in three different NaCl concentrations as presented in Figures 2(a

Biochemical Profiling for Proline, Chlorophyll and Protein Content
Chlorophyll (mg), proline (µg) and total protein (mg) content per g of mature normal leaf tissue, in control and all stress conditions, showed a considerable variation for studied twenty rice landraces (Table 2).Among the all rice cultivars, Sankar Sail showed the highest and Nekrai Sail showed the lowest quantity of chlorophyll in all the concentrations of PEG and NaCl.Protein content in the lower osmotic stresses, i.e. −0.5 MPa PEG and 100 mM NaCl, reveal that Laxmi Sail has gave the highest values (95.2 and 109, respectively) and Chapri Sail has gave the lowest values (56.7 and 58.2, respectively).

Analysis of Variance (ANOVA)
The analysis of variance revealed highly significant (p < 0.05) differences among the physio-biochemical responses (Table 3) in different osmotic stress conditions (in both PEG and NaCl).

Discussion
Analysis of variance (ANOVA) indicates the presence of considerable amount of variability amongst the different studied cultivars for all studied physio-biochemical parameters.Reduction in seed G% was higher in PEG compared to NaCl indicates that at equivalent osmotic potential, seed germination was better in NaCl than the PEG as reported by Gulzar and Khan [44].The explanation of the higher inhibitory effects of PEG than NaCl lies in ion or solute entry into the seed [45].It was clear from the above results that increased concentration of both PEG and NaCl reduce seedling growth.Such type of stress tolerance is characterized by an extensive root growth and small reduction of shoot growth in stressed conditions [46].Pradhan et al. [47] reported that root length and rootlet number were increased due to severe moisture stress.
Castillo et al. [48] and Cha-um et al. [49] have been reported that the quantity of different biochemical components like protein, chlorophyll, proline, etc. in mature rice tissues were changed under drought and/or salinity stress conditions.Chlorophyll pigment also tends to decline rapidly under osmotic stresses [50].Presence of high proline content is a good index for osmotic stress tolerance and under stressed condition, the cellular protein degrades and consequently, the proline content increases [51].
Range of the SSR genotyped amplified alleles (2 -5 per locus) was much similar to the earlier studies on another set of rice genotypes [17,18].The overall poly-   morphic frequency of biallelic markers was higher than the tri-, tetra-and pentaallelic SSRs [11].The number of SSR amplified alleles and their PIC values also depend upon the repeat motif and the repeat sequence of the microsatellites [52].Temnykh et al. [53] showed that (CTT) and (AT) rich repeat motifs amplified with higher efficiency and revealed greater overall polymorphism.

Conclusion
The physiological screening and fingerprint profile, with the aid of quantified biochemical compounds in different stress tissue of the selected traditional rice lines are the first attempt to make their overall multi-informative profiling in relation to osmotic stress tolerance.Osmotic stresses, mainly drought and salinity, adversely affected the germination percentage as well as shoot and root length of germinated seedlings which are necessary to have optimum plant growth for crop production.Chlorophyll and protein contents were declined in increasing stress doses compared to the control; whereas proline was accumulated at the higher concentration in the mature leaves with an elevated stress dose.The 45 sets of SSR markers provide a positive assessment by their ability to produce unique DNA fingerprinting profile of traditional rice genotypes leading to their genetic relationship and diversity.Microsatellite profiling combined with physiological and biochemical screening revealed that Malik Sail and Rani Sail showed full germination at both osmotic stress (due to PEG and NaCl) conditions and were recommended as tolerant genotypes; whereas Laxmi Sail, Mihinagra Sail, Ratan Sail and Raghu Sail were proved to be susceptible to drought and salinity stress for all aspects.The obtained data can be used for varietal identification and the construction of a database of such vulnerable rice landraces of West Bengal.This investigation would be more significant and useful if it can be used for more numbers of trait linked SSR markers spanning all twelve rice chromosomes.

Fingerprinting results reveal that 38 Figure 4 .
Figure 4. Microsatellite panel of studied rice landraces against used 45 SSRs.andRM 219) to 0.765 (for RM 3412 and RM 328) with an average PIC value of 0.524.As overall observation, the SSR loci RM 3412 followed by RM 328, RM 206 and RM 493, were found to be superior for analysis of allelic diversity in this study.Similarity matrix revealed that the similarity coefficient value ranged from 0.0 (between Balaram Sail-Nekrai Sail and Rup Sail-Sindur Sail) to 0.98 (between Lati Sail and Nagra Sail).The dendrogram (Figure5) showed four major clusters: Cluster I, II, III and IV.Within the first cluster, Chapri Sail and Kabirraj Sail are very much close to each other; whereas in second cluster, Rup Sail and Sindur Sail are closely related.In Cluster-III and IV, the pairs of Rani Sail-Ratan Sail and Neizer Sail-Punjab Sail are closely linked, respectively.
SOV = source of variation, df = degree of freedom, MSS = mean sum of square, * indicates significant at 5% level of probability (p < 0.05).

Figure 5 .
Figure 5. Clustering of twenty rice landraces based on 45 SSR fingerprint.

Table 4 . Amplicon size range, total scorable bands, total alleles, number of unique alleles, polymorphic alleles and percentage of polymorphism for each SSR marker.
[54]his context, it was observed that SSRs (RM 493, RM 206 and RM 3412) with (CT)-rich di or tri-nucleotide repeats were effective for producing the highest number Biochemical and Microsatellite Based Profiling of Lowland Rice (Oryza sativa L.) Landraces for Osmotic Stress Tolerance 61 of alleles as well as highest values of PIC than other SSR markers.While variation in the size of SSR alleles results from a change in the number of repeats is the consequence of sequence changes at the primer binding site(s)[54].The genetic relationship among the studied rice genotypes after SSR based molecular profiling has also partially confirmed by the physio-biochemical screening in relation to osmotic stress tolerance.Malik Sail and Rani Sail showed 100% germination in all stressor (PEG and NaCl) concentrations and were placed in a single cluster and thus initially proved as stress tolerant genotypes.Rup Sail and Sankar Sail showed 100% germination in only NaCl conc., but not in PEG and were placed in an another cluster.For selection of the parental rice genotypes for the development of a mapping and/or breeding population, Laxmi Sail, Mihinagra Sail, Ratan Sail and Raghu Sail were proved as stress susceptible cultivars and were placed in one cluster.On the other hand, Rup Sail-Sindur Sail and Punjab Sail-Neizer Sail pairs were found to be closely linked together in other two clusters, respectively.For future rice breeding program, the probable parental combinations derived from the partially completed analyses will be further studied for selection of the proper parental genotypes.