Biochemical Analysis of Rice

Rice (Oryza sativa (2n = 24) is a monocot plant and belongs to the Poaceae family and Oryzoidea subfamily. It occupies almost one-fifth of the total land area under world cereals. It covers about 148 million hectares annually that is roughly 11 percent of the world-cultivated land. It is life for more than half of humanity and in past, it shaped the cultures, diets, and economies of billions of people in the world (Farooq et al., 2009). More than 90 percent of the world’s rice is grown and consumed in Asia where 60 percent of the world population lives. The world major rice consuming countries are China, India, Egypt, Indonesia, Malaysia, Bangladesh, Vietnam, Thailand, Myanmar, Philippines, Japan, Brazil, South Korea and USA that consume 135, 85, 39, 37, 26, 18, 10, 10, 9.7, 8.7, 8.1, 5.0 and 3.9 million metric ton, respectively (Meng et al., 2005; USDA, 2003-04).

• Biochemical and nutritional aspects of rice

Rice is a major source of macro and micronutrients for human being. It feeds more than two billion people worldwide and is the number one staple food in Asia. It provides over 21 percent of the calorific needs of the world’s population and up to 76 percent of the calorific intake of the population of South East (SE) Asia (Fitzgerald et al., 2009). It is mostly consumed as a polished grain, which usually lacks its nutritional components such as minerals and vitamins 41 P. Lucca et al., Genetic engineering approaches to enrich rice with iron and vitamin A, Physiol. Plant. 126 (2006), pp. 291-303. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (7)( Lucca et al., 2006). Since the advent of molecular techniques, recently genetically modified rice verities have been developed, which contains more nutritional aspects like minerals and vitamins in endosperm (Vasconcelos et al., 2003; Paine et al., 2005; Fitzgerald et al., 2009). The major value-added nutritional protein constituents of the rice.

• Rice Position in Pakistan

In Pakistan, besides its importance as a food crop, rice is the second important component of daily diet of bulk of the population after wheat. About 23% of the total foreign exchange earnings is shared by rice and thus called as ‘Golden Grain of Pakistan’ (Shah et al., 1999). Around one third of total production is annually exported and two third is locally consumed to meet food needs. Rice is also used in dishes for special occasions (Sagar et al., 1988). Pakistan is the third largest rice exporting country. In Pakistan, rice occupies about 10% of the total cultivated area, accounts for 6.1% of value added in agriculture and 1.3% in gross domestic product. Production of rice during 2007-08 was estimated at 5,540 thousand tones, 10.4% higher than last year with 6.1% increase in yield per hectare (Anonymous, 2006).

Area, production and yield of rice for the last 5 years are shown in Fig. 1. Varieties of basmati rice, sub-species of indica, are economically important due to the high quality of the grain and constitute an important source of revenue for two major rice-growing countries in Asia (Pakistan and India). The international market for basmati rice has always been higher than that of the moderate varieties. Pakistan’s annual rice export stands at about 2.5 million tons, which earn a total of 513.0 million dollars for the country (Anonymous, 1998). During the year 2005-2006 rice export was about one billion US$ (Bashir et al., 2007).

Rice growing areas of Pakistan

Depending upon the irrigation water availability, rice can be grown in any part of the country from sea level up to 2500m height. Pakistan has a climate and a potential in soil that permits the expectations of a most bright future for the productions of rice. Considering temperature difference, optimum sowing seasons and the varietals performance, rice growing areas can be divided in four ecological zones (Salim et al., 2003; Table-1.2).

Rice is grown in all four provinces of Pakistan. However, the acreage under rice varies greatly from one province to another. The Punjab and Sindh are the major rice growing provinces with about 59% and 33%, respectively of the total rice in the country. The remaining 5% of the area is planted in Baulochistan and 3% in NWFP (Bhatti and Anwar, 1994). Despite the fact that it’s cultivated area is far smaller than wheat (more than 7.24 million), it has a great impact on national economy due to two reasons. Firstly, rice is the only crop which can be grown successfully in vast chunks of salt-ridden and water-logged areas where it facilitates not only the reclamation of land for the cultivation of other crops but also provide food.

Secondly, superior quality basmati has a consistently increasing demand in the foreign countries. Consequently, there is a great scope for augmenting the foreign exchange earning by exporting it in bigger quantity. In view of these facts, it is highly desirable to increase the production and improve the quality of rice the quality is particularly more important from the “trade view point, as it is instrument entail in increasing and then sustaining the demand in the foreign market in competition with other rising exporting countries. There in no denying the fact that purity is the very sole of quality. The impurities not only restrict the export trade, but also inflict losses to the growers, millers and the consumers alike. Therefore, these should possibly be minimized (Saleem et al., 2003).

• Major rice varieties in Pakistan

More than 20 rice varieties have been released for general cultivation in Pakistan (Bashir et al., 2007). A general description of agronomical and physiochemical characteristics of these varieties.

• Importance of Basmati Rice in Pakistan

There are thousands of rice varieties and landraces, which differ with respect to plant and grain characteristics. Of these, aromatic (Basmati) rice constitutes a small but special group that is regarded as best in grain quality, superior aroma and usually used for special dish preparation (Khush and dela Cruz, 2001). Quality of rice may be considered from the view point of size, shape and appearance of grain, milling quality and cooking properties (Dela Cruz and Khush, 2000). Pakistan is famous for the production and export of Basmati rice. The origin of the word “Basmati” can be trade to the word “Basmati” meaning earth recognized by its fragrance. The Hindi word “Bas” was derived from the Pakrit word “BAS” and has a Sanskrit root” Vassy” (Aroma), while “Mati” originated from “Mayup” (ingrained from the origin). In common usage Vas is pronounced as “Bas” and while combining “Bas and Mayup”, the later changed to “Mati’ thus the word Basmati (Ahuja et al., 1995; Gupta, 1995).

The fragrance of basmati rice is most closely associated with the presence of 2-acetyl-1-pyrroline (Buttery et al., 1983; Lorieux et al., 1996; Widjaja et al., 1996; Yoshihashi et al., 2002). Although many other compounds are also found in the headspace of fragrant rice varieties (Widjaja et al., 1996) possibly due to secondary effects related to the genetic background of the rice variety, 2-acetyl-1-pyrroline is widely known to be the main cause of the distinctive basmati and jasmine fragrance. The desirability of fragrance has resulted in strong human preference and selection for this trait. Non-fragrant rice varieties contain very low levels of 2-acetyl-1-pyrroline, while the levels in fragrant genotypes are much higher (Widjaja et al., 1996).

Basmati rice occupies a prime position in the Indian subcontinent and is becoming increasingly popular in Middle East, Europe, USA and even in non-traditional rice growing countries such as Australia (Bhasin, 2000). High-quality, traditional Basmati rice varieties command premium prices, more than three times that of non-Bamati rices in the world market due to its exquisite aroma, superfine grain characteristics and excellent cooking (extra elongation, soft and flaky texture) qualities (Bhasin, 2000; Singh et al., 2000a; Khush and dela Cruz, 2002). Basmati rice traditionally grown in the Himalayan foothills regions of Pakistan and India, and the name is traditionally associated with this region. Basmati rice is the result of centuries of selection and cultivation by farmers (Khush, 2000).

Cultivation of basmati rice in mainly confined to the Kallar tract (Gujranwala, Sheikhupura and Sialkot districts) of Punjab province. Basmati rice always fetch a higher price in the domestic as well as in the international market due to their peculiar quality features such as pleasant aroma, fine grain, extreme grain elongation (7.6mm long) and soft texture on cooking. In spite of hard competition from India, Thailand and the United States, Pakistan enjoys a good position in the global trade of aromatic rice and every year earns a lot of foreign exchange (Akram and Sagar, 1997).

Genetic Diversity in Rice

Diversity among organisms is a result of variations in DNA sequences and of environmental effects. The diversity in crop varieties is essential for agricultural development for increasing food production, poverty alleviation and promoting economic growth. The available diversity in the germplasm also serves as an insurance against unknown future needs and conditions, thereby contributing to the stability of farming systems at local, national and global levels (Singh et al, 2000). In crop improvement program, genetic variability for agronomic traits as well as quality traits in almost all the crops is important, since this component is transmitted to the next generation (Singh, 1996). Study of genetic divergence among the plant materials is a vital tool to the plant breeders for an efficient choice of parents for plant improvement. Genetically diverse parents are likely to contribute desirable segregants and/or to produce high heterotic crosses. Parents identified on the basis of divergence for any breeding program would be more promising (Arunachalam, 1981). In early 1970’s, public authorities felt the need that genetic resources should be collected, maintained and conserved, especial focus was on important food crops e.g wheat, rice, barley etc (Hawkes 1983; Bellon et al., 1998; Barry et al., 2007). This was the first official attempt to preserve genetic diversity. Currently different genetic diversity assessment methods including morphological, biochemical and molecular markers are available.

• Morphological Markers used to study genetic diversity

Morphological evaluation is the oldest and considered as the first hand tool for detection of genetic variation in germplasm (Smith and Smith, 1989). It is cheap and convenient. It requires not any in depth knowledge at genomic or proteomic level. However, morphological markers are relatively less effective for genetic diversity analysis due to sensitivity to environmental influences and developmental stage of the plant (Werlemark et al., 1999). It takes long time, requires seasonal changes and quite laborious. The genetic variability for some of the traits needed for high yield performance and stress tolerance is limited in cultivated germplasm. This is because a small core of adapted progenitors has been used repeatedly in rice breeding programs such that the genetic base of rice has become narrow (Moncada et al. 2001; Hargrove et al. 1980; Dilday 1990). Introgression of genes from other rice species can provide genetic variation to improve rice and meet the challenges affecting rice production. Morphological traits including both qualitative and quantitative ones are used to evaluate genetic relationship among genotypes (Goodman 1972; Bajracharya et al., 2006). Fida et al. (1995) reported the evaluation of elite rice genotypes for agronomic traits during 1992 at NARC, Islamabad. All the genotypes possessed similar grain quality. Agronomic evaluation was used for screening of lines with desired performance by Akram et al. (1995), in field leading to the identification of varieties possessing longer and fine grains as donors for utilization in breeding programmes aimed for the improvement of grain length in Basmati rice. Iqbal et al. (2001) morphologically evaluated selected landraces for paddy yield and other important agronomic traits as a propose to select parents for hybridization program. All the landraces possessed some desirable agronomic traits so these proved effective in rice breeding programmes. Koutroubas et al. (2004) described variation in grain quality traits among some European rice lines. They concluded that these lines could be used as parents for introgression of desired traits into different rice cultivars grown in Europe. They also suggested that the interrelations among grain quality traits found in these lines could be useful to study the relationship among their grain quality components and for improving selection criteria. Nabeela et al. (2004) evaluated fifteen agronomical important traits in landrace genotypes of rice collected from various parts of Pakistan. A significant amount of genetic variation was displayed for most of the traits examined. The coefficient of variation was more than 10% for all the characters with exception of grain length. Seven accessions with best performance for individual character were identified, by exploiting their genetic potential. These genotypes can have a beneficial use in the breeding programs. Nepali rice landrace diversity was evaluated by Bajracharya et al. (2005) by using morphological traits as one of the parameter for selection. The genotypes varied only for few quantitative traits controlled by major genes; husk color, seed coat and panicle traits. Agronomic characterization also helped to decide which traits need to be improved for further crop improvements. Zaman et al. (2005) studied fifteen different rice varieties which showed that the different morphological characteristics such as the yield, tiller number per hill and filled grains per panicle did not contribute towards the total divergence. This suggested that the breeding improvement of these morphological characteristics have the little possibility. Little phenotypic variation at farm level was observed in Vietnamese rice by Fukuoka et al. 2006, which was considered to be the result of genetic drift and selection by the farmers, on farm conservation of the landraces of rice is considered to be under a force to decrease phenotypic diversity. Different phenotypic profiles contribute to the conservation of regional genetic diversity of the landraces of rice. Veasey and colleagues (2008) investigated the genetic variability among different rice species from South in a greenhouse experiment. They showed a significant difference (p<0.001) among different investigated species. The highest phenotypic variability was observed in two different rice species by multivariate analyses.

Keeping in view these benefits, morphological variation is a selection criterion for plant scientists among landrace genotypes. Though the environmental factors also play an important role in morphological variation but the knowledge of agro-morphological diversity and the distribution pattern of variation among crop species could be an invaluable aid in germplasm management and crop improvement strategies. Zeng et al. (2003) studied ecogeographic and genetic diversity based on morphological characters of rice landraces (Oryza sativa L.) in Yunnan, China. A great difference in ecological diversity index of rice resources between prefectures or counties in Yunnan province exists. Kayode et al. (2008) studied the relationship in geographical pattern and morphological variation of 880 rice landrace in Côte d’Ivoire for 13 agro-morphological characters. Result of the phenotypic frequency showed differential distribution of landraces with height, heading and maturity period which reflected the distribution pattern of different Oryza sativa landraces in Côte d’Ivoire that proved useful in germplasm management and breeding programs. The altitudinal distributions of grain length, grain width, grain length to width ratio and grain weight were evaluated by Siddiqui and coworkers in 2007. It was noticed that grain length decreased with the increase in altitude, while the grain width increased with the increase in altitude, resulting into a decrease in length to width ratio with the increase in altitude. Considering the change in altitude as a difference in habitat and environment, it can be assumed that Pakistan rice cultivars show a wide variation between and within locations. It may be concluded that the Pakistan rice genetic resources comprise of great diversity for grain morphological characteristics. The prevailing diversity for grain type (shape and size) and pericarp color has distinct correlation to its geographical distribution in terms of altitude. Morpho-physiological traits are an important tool in hands of plant breeders for identification and purity testing of rice varieties. Sharief et al. (2005) investigated the genetic purity of four different rice varieties on the basis of morphological characteristics at their different growth stages. All of the varieties were identified by different morphological characteristics in terms of flag leaf area, grain color, seed width, number of tillers, time of heading, absent awing, slemma, palea pubescence, plant height, and culm diameter.

• Biochemical markers for analysis of diversity

Seed proteins are very helpful in genetic diversity evaluation in cereal crops because the seeds of these crops have nutritional value. Glutelin, globulin and prolamin are important seed proteins in rice. Variation in these proteins at subunit level changes the quality of rice. Various tools were used to assess variability at peptide level. Biochemical markers have some disadvantages being tissue specific and affected by environmental and developmental changes. These disadvantages could be eliminated by the use of seed storage protein as they are conservative in nature and least effected by environmental changes. (Thanh et al., 2006) Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is useful method not only for revealing variations but also for identification of a variety in seed storage proteins. Four protein fractions (albumin, globulin, gliadin and glutenin) separated by SDS-PAGE as biochemical marker for evaluating polymorphism in three spelt wheat varieties. Very significant difference was observed at protein profile level in old cultivars and new breeding lines (Dvoracek and Curn 2003). Sengupta and Chattopadhyay (2000) identified twelve rice varieties on the basis of banding pattern obtained by SDS-PAGE. Aung et al. (2003) investigated 350 local rice cultivars from different regions of Myanmar. These were analyzed by using SDS-PAGE and IEF. Various cultivars differed in their SDS-PAGE profiles. Padmavathi et al. (2002) evaluated seven aromatic and five non-aromatic rice cultivars using SDS-PAGE. Two bands of 60.3 and 51.3KDa were polymorphic for their presence in both aromatic and non-aromatic genotypes and suggested that these polymorphic bands can be used as markers for verification of hybridity in crossing programme. Rehana et al. (2004) investigated twenty accessions of Pakistani rice germplasm for total seed protein by using SDS-PAGE, to determine the magnitude of genetic variation with respect to geographical distribution. Variation in protein banding pattern with respect to various geographical regions was evaluated and it was suggested that the inter-specific variations were more pronounced as compared to intra-specific variations. Variation in banding profile of globulin and glutelin was used as identification tool for differentiating coarse, fine and super fine rice cultivars by Thind and Sogi (2005). Jahan et al. (2005) studied protein diversity in 576 rice cultivars from Bangladesh and SDS-PAGE was used for separation. Thanh et al., 2006 used seed storage protein profiles of different varieties including rice for evaluation of genetic purity and variability.

• Molecular markers for diversity analysis

Variation in a DNA sequence is known as DNA polymorphism. This quality of DNA can be used as a marker to assess diversity in the genome of any organism. An ideal DNA marker must have any of the following qualities: Highly polymorphic in nature, co-dominant inheritance, frequent occurrence in genome, selective neutral behaviour, easy access/availability, easy and fast assay, high reproducibility and easy exchange of data between laboratories (Joshi et al., 1999). DNA-based molecular markers/DNA fingerprinting can increase screening efficiency in breeding programs in a number of other ways. For example, they provide: the ability to screen in the seedling stage for traits that are expressed late in the life of a plant (i.e. grain or fruit quality, male sterility, photoperiod sensitivity), the ability to screen for traits that are extremely difficult, expensive, or time consuming to score phenotypically (i.e. root morphology, resistance to quarantined pests or to specific races or biotypes of diseases or insects, tolerance for certain abiotic stresses such as drought, salt, or mineral deficiencies or toxicities), the ability to distinguish the homozygous versus heterozygous condition of many loci in a single generation without the need for progeny testing (since molecular markers are co-dominant), and the ability to perform simultaneous marker-aided selection for several characters at one time.

• Random Amplified Polymorphic DNAs (RAPDs)

Randomly-amplified polymorphic DNA markers (RAPD) are arbitrary sequence markers developed by Welsh and McClelland in 1991. This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. On an average, each primer directs amplification of several discrete loci in the genome, making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals. However, due to the stoichastic nature of DNA amplification with random sequence primers, it is important to optimize and maintain consistent reaction conditions for reproducible DNA amplification. They are dominant markers and hence have limitations in their use as markers for mapping, which can be overcome to some extent by selecting those markers that are linked in coupling. RAPD assay has been used by several groups as efficient tools for identification of markers linked to agronomically important traits, which are introgressed during the development of near isogenic lines. though it is less popular due to problems such as poor reproducibility faint or fuzzy products, and difficulty in scoring bands, which lead to inappropriate inferences but it is still applied as markers in variability analysis and individual-specific genotyping has largely been carried out,. Raghunathachari et al. (2000) differentiated a set of 18 accessions from Indian scented rice by random amplified polymorphic DNA (RAPD) analysis. The RAPD analysis offered a rapid and reliable method for the estimation of variability between different accessions, which could be utilized by the breeders for further improvement of the scented rice genotypes. Porreca et al. (2001) reported confirmation of genetic diversity among 28 rice cultivars, different for biometric traits, biological cycle and suitability to water limitation, using RAPD markers. High level of polymorphism was found between japonica and indica subspecies, whereas japonica cultivars with long grains (tropical) resulted to be genetically different from the short grains genotypes (temperate). Genetic relationships among indica and japonica cultivars and between tropical and temperate japonica was estimated. Variability among the varieties could lead to good heterotic combinations between japonica genotypes. Neeraja et al. (2002) determined genetic diversity in a set of landraces in comparison to a representative sample of improved rice varieties, using random amplified polymorphic DNA (RAPD). Analysis of 36 accessions using 10 arbitrary decamer random primers, revealed 97.16% polymorphism. Similarity values among the landraces ranged from 0.58 to 0.89 indicating wide diversity. The landraces and improved varieties formed separate clusters at 0.65 similarities suggesting that genetically distant landraces could be potentially valuable sources for enlarging and enriching the gene pool of improved varieties. Kwon et al. (2002) evaluated genetic divergence among 13 Tongil type rice cultivars and the relationship between genetic distance and hybrid performance in all possible nonreciprocal crosses between them assessed. These results indicate that GDs based on the microsatellite and random amplified polymorphic DNA (RAPD) markers may not be useful for predicting heterotic combinations in Tongil type rice and support the idea that the level of correlation between hybrid performance and genetic divergence is dependent on the germplasm used. Rabbani et al. (2008) evaluated the genetic polymorphism and identities of several Asian rice cultivars by using random amplified polymorphic DNA technique. On the basis of analysis performed on similarity matrix by using UPGMA, they grouped 40 cultivars into three main clusters correspondent to aromatic, non-aromatic and japonica group, and a few independent cultivars. The cluster analysis placed most of the aromatic cultivars close to each other showing a high level of genetic relatedness. But the clusters produced by the aromatic cultivars were distinct from those of non-aromatic and japonica types. In this study, several improved and obsolete cultivars originating from diverse sources did not produce well defined distinct groups and indicated no association between the RAPD patterns and the geographic origin of the cultivars used. Amita et al. (2005) performed molecular and hybridization studies to investigate variation patterns in O. meridionalis by producing 119 polymorphic RAPD markers from 12, 10-mer operon primers. In addition, they detected 67 alleles by using 11 SSR primers. They showed speciation in O. meridionalis a with respect to its geographic distribution in northern Australia and Irian Jaya. Santhy et al. (2003) tested application of RAPD markers for the identification of three rice (Oryza sativa L.) hybrids and their parental lines i.e. CMS female parent (A line), maintainer (B line) and pollen parent (R line), using 17 random oligonucleotides. It was possible to distinguish each of these genotypes, following a combination of selected primers. The results are discussed in view of its application for the purpose of Plant Variety Protection and for testing the genetic purity of A line and hybrid seed lots.

• Simple Sequence Repeats Analysis

Microsatellites or simple sequence repeats (SSRs) are simple tandemly repeated di- to penta-nucleotide sequence motifs. Microsatellite data are also commonly used to assess genetic relationships between populations and individuals through the estimation of genetic distances (e.g. Beja-Pereira et al., 2003; Ibeagha-Awemu et al., 2004; Joshi et al., 2004; Sodhi et al., 2005; Tapio et al., 2005). The most commonly used measure of genetic distances is Nei’s standard genetic distance (DS) (Nei, 1972). Because of microsatellite abundance and even distribution in nuclear genomes of eukaryotes and some prokaryotic genomes, they offer valuable good source of polymorphism, which make them a promising class of genetic markers. The high levels of polymorphism performed by these markers; they are mostly referred as SSLP (simple sequence length polymorphism). Li et al. (2004) examined genetic diversity within and differentiation between the indica and japonica subspecies, including 22 accessions of indica and 35 of japonica rice by using five microsatellite loci from each chromosome having total 60 loci. Evaluating on chromosome-based comparisons it is concluded that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. By applying chromosome-based comparisons they suggested that the extent of the indica-japonica differentiation varied substantially, ranging from 7.62% in chromosome 3 to 28.72% in chromosome 1. At 15 of the SSR loci, traditional and crossbred Basmati rice varieties amplified different alleles than those in the indica and/or japonica rice varieties. During this study the identified SSR markers, which can be used to differentiate among the traditional Basmati varieties and between traditional Basmati and other crossbred Basmati or long grain, non-Basmati rice varieties. Genetic relationships among rice genotypes as determined by UPGMA cluster analysis and three-dimensional scaling based on principal component analysis showed that the three traditional Basmati rice varieties are closely related and have varying degree of similarity with other crossbred Basmati rice varieties Priyanka et al. (2004). Amanda et al. (2004) classified 234 accessions of rice into five distinct groups corresponding to indica, aus, aromatic, temperate japonica, and tropical japonica rices using 169 microsatellite markers. Yunbi et al. (2004) evaluated diversity in 236 rice accessions by applying 113 restriction fragment length polymorphism (RFLP) and 60 simple sequence repeat (SSR) loci at DNA level. Higher value of polymorphism information contents (0.66) was recorded for SSR markers as compared to RFLP (0.36). A diverse subset of 31 rice cultivars was identified that embodied 95% of RFLP and 74% of SSR alleles. This subset was useful in developing core collections and an efficient source of genetic diversity for future crop improvement. Zhang et al. (2005) evaluated the potential of discriminate analysis (DA) to identify candidate markers linked with agronomic traits among inbred lines of rice (Oryza sativa L.). A sum of 218 lines originating from the US and Asia were planted in field plots of Texas. Data were collected for 12 economically important traits, and DNA profiles of each inbred line were produced using 60 SSR and 114 RFLP markers. Model-based methods revealed population structure among the lines. Associated marker alleles pointed to the same and different regions on the rice genetic map when compared to previous QTL mapping experiments. Results of the study suggested that candidate markers associated with agronomic traits can be readily detected among inbred lines of rice. Bajracharya et al. (2005) estimated genetic diversity of rice landraces collected from different locations of Nepal based on agro-morphological variability and microsatellite marker polymorphism. They 39 microsatellite (simple sequence repeats, SSR) markers among these collected accessions by using 10 different names. After studying all these qualitative and quantitative traits they concluded that these accessions showed low morphological diversity having an average Shannon Weaver diversity index of 0.23. Among the studied traits only 16 morphological traits showed significant variation among the accessions. Discriminant function analysis showed that only 36% of accessions could be clustered according to name by morphological traits. Only one SSR locus was polymorphic, distinguishing only one accession. Genetic differences among new rice lines (NERICA), developed by cross breeding of African rice (Oryza glaberrima) with high yielding Asian rice (Oryza sativa subsp. japonica), were explored by using simple sequence repeat markers (Semagn et al. 2006). Michael et al. (2006) characterized 330 rice accessions, including 246 Indonesian landraces and 63 Indonesian improved cultivars, by studying 30 fluorescently-labeled microsatellite markers. By using genetic diversity analysis they characterized the Indonesian landraces as 68% indica and 32% tropical japonica, having an indica gene diversity of 0.53 and a tropical japonica gene diversity of 0.56