Application of molecular markers to find out classificatory determinants of rice ( Oryza sativa L . )

The present communication is aimed to find out determinants of molecular marker based classification of rice (Oryza sativa L) germplasm using the available data from an experiment conducted for development of molecular fingerprints of diverse varieties of Basmati and non Basmati rice adapted to irrigated and aerobic agro-eco systems. The distance-matrix based dendrogram was developed following UPGMA method that grouped all the 44 varieties agro-eco systems wise. We examined the clustering of Basmati and nonBasmati varieties in detail. The results suggest that the easily distinguishable simple characters viz. plant height, photosensitivity, leaf color, awn’s color, awns’ number etc., governed by major genes did not contribute significantly towards classification of rice genotypes used. The complex characters such as adaptation to agro-eco systems and rice quality may play vital role in determination of molecular marker based classification of rice genotypes.


INTRODUCTION
The traditional varieties of Basmati rice are tall, long duration, photosensitive and contained minimum genetic diversity.Therefore, this crop could not be genetically benefitted even during green revolution era.However, low yielding, disease susceptible traditional varieties of Basmati rice still remained in cultivation due to their high quality that fetch high premium price in the local as well as international market.
Lodging and disease appearance in such basmati rice varieties results into poor productivity and inferior quality (DelaCruz and Khush, 2002) which in turn results into less remuneration to the farmers in high input agriculture (Singh and Gupta, 1985).Furthermore, short duration and input efficient rice varieties of inferior quality like Sarbati resembling with basmati in appearance became a challenge for Basmati trade.Due to high price difference such varieties are used for blending in export consignments that loses national *Corresponding author.E-mail: devisingh11@gmail.com.Tel: +919358918146.Fax: +91 121 2888505.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License credibility in the global market.Triggering efforts, to promote production and maintaining purity of Basmati rice, Government of India has declared this traditional basmati growing area as Agri Export Zone for Basmati rice (Singh et al., 2011;Singh and Sirohi, 2005).Traditionally used morphological and physico-chemical parameter does not provide more precise techniques for maintenance of purity of the commodity (Nagaraju et al., 2002).Therefore, foolproof instant safeguards are required to be developed to detect adulteration of any type of rice blended in the export consignments In parallel, reliability and precision of such discriminatory functions will apparently depend upon adequacy of molecular markers used and characters contributing towards estimates of genetic diversity.Once adequate molecular markers and efficient determinants are identified, further studies will be intensified to get information at finer level which could be potent to precisely detect admixture in commercial varieties (Botstein et al., 1980;Vos et al., 1995).Alternatively, it is needed to find out characteristics/determinants contributing to genetic diversity (Singh, 2010) which is the basis of classification/clustering of genotypes.
So, the present study was undertaken with the objective to fingerprint/characterize different varieties of Basmati and non-Basmati rice adapted to different agroecological systems by using SSR and its alternative approach inter-SSR assays (Zietkiewicz et al., 1994, Singh et al., 2000a, 2000b) and to examine the results in order to work out efficient determinants for molecular marker based classification.

Plant materials and molecular markers assay
Forty four varieties of Basmati and non-Basmati rice along with their variety wise place of development/ origin and visibly distinguishable characteristics are listed in Table 1.The genomic DNA of 44 rice varieties was isolated using CTAB method (Moller et al., 1992).10 ISSR and 28 SSR molecular markers (Table 4) were used for genetic analysis.The ISSR-PCR technique (Zietkiewicz et al., 1994) was used to enhance the speed of sensitivity of detection of molecular markers.The 5', 3' anchored ISSR, and SSR primers synthesized by Bangalore Genei were used.Each reaction mixture (20 ul) that used ISSR and SSR primers for amplification consisted of 10x assay (10 mM Tris HCl, pH 8.0, 50 mM KCl with 3.0 mM MgCl2): 1 U of Red Taq DNA polymerase, 1.0 mM each of dATP, dTTP, dCTP and dGTP, 10 μm of primer (Bangalore Genei) and approximately 25 ng of genomic DNA for Inter-SSR and SSR, respectively.
The PCR amplification conditions for ISSR analysis were as follows: initial extended step of denaturation at 94°C for 4 min, followed by 45 cycles of denaturation at 94°C for 1 min, primer annealing at 50°C for 1 min, elongation at 72°C for 2 min, followed by extension step at 72°C for 7 min.For SSR analysis, the number of cycles was 35 and annealing temperature was 55°C, respectively.PCR products were mixed with 5 μl of gel loading dye (1x buffer, Bromophenol blue, 0.1%; xylene cyanol 0.1%; and glycerol in water, 50%).The amplification products were electrophoresed on 1.5 to 3% SFR agrose gel at 3 to 5 V/cm in 1x Singh et al. 43 TAE buffer.Genomic DNA was quantified by UV absorbance at 260 nm and 280 nm, using UV Bio-Rad spectrophotometer.

Data analysis and detection of genetic diversity
Forty-four rice varieties were used to estimate genetic diversity.Polymorphic products from ISSR-PCR and SSR-PCR assays were calculated qualitatively for presence (1) or absence (0).The proportion of bands that have been shared between any of the two varieties averaged over loci ISSR and SSR primers were used as the measure of similarity.Genetic diversity was calculated using the following formula (Botstein et al., 1980): Pij is the frequency of the jth allele for marker i and the summation extends over n alleles.The calculation was based on the number of bands in ISSR and the number of bands per primer for SSR.
Cluster pattern was based on distance matrices by using the Unweighted pair group method analysis (UPGMA) program in WINBOOT software (Yap and Nelson, 1996).The diversity or similarities between varieties were given in the form of dendrogram (Figure 1).For the sake of brevity, dendrogram for combined ISSR-SSR assays is given in Figure 1.

Evaluation of grain and quality
For determination of the length breadth (L/B) ratio, kernel length, breadth of milled and cooked rice, aroma and alkali spreading value (ASV) (Dela Cruz and Khush, 2000), 20 fully developed wholesome milled rice kernels were measured.The kernel elongation ratio (ER) was estimated as ratio of length of the cooked kernels to that of uncooked kernel.Determination of aroma was based on panel reports.Traditional Basmati varieties Taroari Basmati and evolved basmati variety Pusa Basmati-1 were used as standards.The strength of the aroma was scored sample wise (Sood and Siddiq, 1978) as Strongly Scented (SS) and Mildly Scented (MS).ASV was estimated based on visual rating of starchy endosperm (Little et al., 1978).Twenty rice kernels were incubated in 1.7% potassium hydroxide solution in a Petri dish for 23 h at 30°C.
In addition, evaluation of quality was also made by a panel of experts for overall acceptability ratings on account of its acceptability for appearance, cohesiveness, tenderness on touching and chewing and, aroma, taste, elongation and flaky texture on cooking (Anonymous, 2007).

RESULTS AND DISCUSSION
The molecular marker based available techniques are used to estimate genetic diversity that may correspond to contribution of different characteristics in the categoryzation of germplasm.Development of molecular markers was cost effective by using RFLPs, AFLP, microsatellites, inter-SSR and RAPDs.Primary applications of molecular markers are: molecular characterization, classification of germplasm and mapping of the genes.Therefore, these markers are available in public domain.A variety of SSR markers, the most robust molecular markers is potent enough to tag the genes with small differences of 2 to 3 nucleotides for development of genetic maps and Table 1.Rice varieties (Oryza sativa) consisted of Basmati, scented non-Basmati, non-scented fine and coarse rice groups adapted to Irrigated and aerobic agro-eco systems across Asian countries.The present data used for characterization of rice genotypes was generated involving ten ISSR, 20 eight SSR molecular markers and 44 Basmati and non-Basmati rice varieties.The 22 varieties of Basmati and non-Basmati rice adapted to Irrigated agro ecosystem considered presently were developed in Indo-Gangetic plains of Northern India (Singh et al., 2000a) where rice is cultivated mainly in Irrigated puddled conditions (puddle that is, anaerobic conditions, the traditional method of rice cultivation in Northern India).The rest 22 rice genotypes collected from four different sources (1) IRRI, the Philippines (2) Sri Lanka (3) Central India (4) Indo-Gangetic plains of Northern India, were adapted to aerobic agro-ecosystem that is, direct seeding rice (unpuddled).Out of 22 varieties adapted to aerobic conditions, two varieties Sathi white and Sathi black were Farmers' varieties collected from Indo-Gangetic plains of Northern India.Both the Sathi strains/ farmers' varieties mature in 75 days (seed to seed) with minimal water requirement and remained in cultivation in aerobic conditions over decades in the area of their adaptation before deployment of irrigation facilities.Also, Vallabh Basmati-21, the variety recently released for Irrigated agro-eco system (Singh et al., 2011) has expressed excellent harvests in aerobic conditions (Singh, 2008).
In ISSR-PCR assay, all the 44 rice varieties were classified agro-ecosystem wise into two major distinct clusters.Twenty two ( 22) varieties of irrigated agro ecosystem were further grouped into 5 clusters based on variations in quality.Out of these, 11 varieties of super fine quality were distributed over 3 clusters depending upon minor variations in quality.Four traditional Basmati varieties Ranbir Basmati, Type 3, Basmati 370 and Taroari Basmati along with four evolved basmati varieties Pusa-1121, HB-1, CSR-30 and PB-1 were clustered together as also reported earlier by Nagaraju et al. (2002).
None of the variety was clustered with Vallabh Basmati 21 and therefore, it was put in a separate cluster.The improved dwarf genotype MAUB 57 recently identified as an elite genotype of Basmati rice for UP and Haryana by AICRP (Anonymous, 2007) and Super Basmati popular in Punjab were clustered together in a separate cluster consistently in all the three assays.The rest 11 varieties of non-basmati rice adapted to irrigated agro-eco system were grouped into two different clusters.
Furthermore, all the 22 varieties adapted to aerobic conditions were further grouped into two sub clusters.Three varieties BG 352, BG 358 and Sathi Black were grouped into one sub cluster.The rest 19 genotypes were distributed over five sub clusters.Within agro-eco systems, the distribution of varieties in the main as well as in sub clusters reflected independence of their geographical origin.The varieties of aerobic rice despite having their origin in the Philippines, Sri Lanka, Northern India (Sathi strains) and Central India were grouped in only one major cluster indicating presence of common base sequence repeats in the genomic DNA corresponding to ISSR markers used.Such DNA sequences could perhaps have evolved, due to different kinds of selection pressures required to acquire fitness well for survival, in due course of time.The explanation could also be forwarded that all the genotypes of common gene pool would have exchanged over the locations collected there from not long before.In other words, the time passed after shifting of such germplasm was too short to attain adaptation as a consequence of a new genetic mechanism which could have evolved in due course of time.Such sequences were detected by the molecular markers used herein.Therefore, it might be concluded that the base sequences which determined ISSR markers were corresponding to the gene complexes responsible for quality and adaptation reactions.
Examination of SSR-PCR assay reflected, minor variations in clustering pattern.However, clustering of the 8 varieties of basmati rice involving traditional and evolved groups remained the same.Likewise, both the evolved varieties of basmati rice MAUB-57 and Super Basmati were consistently grouped in a separate cluster.PS-2511 and Vallabh Basmati-21 adapted to irrigated agro ecosystem were clustered with varieties of aerobic rice, that is, Sathi Black, BG-375-2, and BG-358.Vallabh Basmati-21 being adapted to aerobic conditions might have genes common to those adapted to aerobic conditions.
However, it could not be detected by ISSR markers.It suggested that PS-2511 requires further evaluation in aerobic agro ecosystem for adaptation reactions.Additionally, it might be concluded that SSR markers were, in general, more robust and therefore, could detect even the minor variations more efficiently in the genomic DNA.Such variations are taken as a basis of molecular characterization of the genotypes (Wu and Tanksley, 1993;Yang et al., 1994).
Furthermore, the clustering pattern of joint assay was more similar to that of ISSR clustering pattern.However, it varied slightly when joint assay was taken into consideration for DNA profiling of these varieties (Figure 1).PS-2511, a super fine non Basmati variety was also included into the cluster containing traditional as well as evolved varieties of basmati rice.PS-2511 expressed quality characteristics of Basmati of inferior quality (Table 2) reflecting its limited closeness to Basmati group.This is why; despite continues efforts, PS-2511 could not be popularized as Basmati among consumers.It also holds true in the present context as reflected by its inconsistent clustering with Basmati group.However, its molecular lineage with Basmati was detected only by ISSR+SSR joint assay (Figure 1).However, impact of adaptation and quality on classification of varieties remained distinct.
In parallel, all the genotypes suitable for aerobic conditions were clustered in a distinct major cluster almost consistently in all the three assays.More variation was noticed in clustering pattern, in general, among the varieties adapted to irrigated agro-eco system over all the three analyses.The varied minor genetic changes which have occurred spontaneously during course of evolution to acquire fitness in a particular agro-eco system seem to be the only cause.It could be an evolutionary adaptation that serves to discriminate between genotypes on the basis of their quality.Examination of dendrogram (Figure 1) and the results discussed above suggested that clustering of 44 rice genotypes was mainly based on two factors that is, adaptation and quality (Table 1 and 2).
Both adaptation and quality of rice are complex traits.Adaptation is governed by several gene complexes.Adaptation is a function of the genes possessed by the variety for the regulation of biochemical and physiological processes during growth and development and how well these are matched with the available environmental resources and possible hazards.The adaptation results from a genetic mechanism which influences the matching of their growth and development processes within the environment (Fisher and Scott, 1993).Thus, process of adaptation is viewed as changes in the genetic constitution of genotypes as they accumulate genes or a change in gene frequencies within population which better match growth and development within the environment (Byth, 1981;Clements et al., 1983;Cooper and Hammer, 1996).Adaptation is more complicated but its elegant genetic studies are lacking.Quality of rice is the resultant effect of a number of component traits.
Contrarily, quality of Basmati rice is a complex character consisting of 13 physicochemical component characteristics (Table 2).In addition, finally quality is judged/ examined by a panel of experts.Each component character is governed by different genetic mechanisms.Comprehensive information about genetic mechanism of quality components is available in the literature and some of such studies are listed in the Table 3. Though, aroma is an essential component of Basmati.However, among non-Basmati rice genotypes such as PS-2511 also carried aroma.
The study reveals that visibly distinguishable qualitative characters like plant height, photosensitivity, disease resistance, colour, size and number of awns, and sensory characters such as aroma etc. could not significantly influence clustering of genotypes.Therefore, the varieties differ for such characters could not be identified by using such molecular tools.For example, tall and dwarf collections of farmers' variety Sarbati could not be identified by the markers presently used and, therefore, were clustered together in a single cluster in all the three cases.Likewise, morphologically distinct genotypes MAUB-57 and Super Basmati were also clustered together.Such results indicated limited utility of molecular markers to the breeders and traders.It appears selfexplanatory that small size of DNA sequences of genes responsible for such characters does not have significant bearing on the clustering of these varieties.DNA sequences of small size representing genes responsible for such characters could not be detected by the markers used.However, trait specific markers could be used to detect such characters.In such cases it seems easier to classify the varieties on the basis of morpho-physicochemical characteristics and application of molecular markers seems of limited value.In the study, impact of any individual component character of quality on classification could not be noticed.None of the variety was classified on the basis of aroma alone.DNA

Table3.
Studies describing genetic mechanisms of quality components of rice.

Table 2 .
The component characteristics of rice quality (approved by Government of India and acceptable in the global trade) were scored at quality laboratories, DRR, Hyderabad (AP) and CRRI Cuttack (Orissa).For the sake of brevity, results only of some of basmati varieties Vallabh Basmati-21, MAUB-57, Taroari Basmati and Pusa Basmati-1 obtained at DRR, Hyderabad are given below.

parameter Quality standards approved by DAC, GOI and expressed by Basmati varieties Approved by DAC, GOI and also acceptable in global trade Quality parameters expressed by commercially popular and recently released/identified varieties of Basmati rice
AICRP-All India Coordinated Research Project, DRR-Directorate of Rice Research, Hyderabad, India.CRRI-Central Rice Research Institute, Cuttack (Orissa), India.DAC-Department of Agriculture and Co-operation, GOI-Government of India.

Table 4 .
Contd.QTLs responsible for several component characters of adaption as well as quality were spread over the whole genome and were detected by the corresponding molecular markers used.Therefore, respective genes responsible for some crucial components of quality and adaptation which greatly influenced clustering of varieties of both the agro-eco systems seem to be largely represented by ISSR and SSR markers' DNA sequences.Clustering of the genotypes used herein greatly depended upon adap-tation to IRRI gated and aerobic agro-eco systems within limits of regulations of the genetic mechanism evolved for adaptation, represented by ISSR and SSR sequences.Likewise, within irrigated agro-eco system quality determined classification of varieties.