Biochemical Analysis of Rice

06 Feb 2018

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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, and a Fst of 0.38 between the two groups. All of the improved varieties sampled were indica, and had an average gene diversity of 0.46. Genetic diversity of rice (Oryza sativa L.) cultivars that are historically significance to rice breeding and production in Argentina were evaluated at the DNA level (Giarrocco et al., 2007). Sixty-nine accessions were surveyed with 26 simple sequence repeat (SSR) markers revealing the genomic relationship among cultivars. O. sativa accessions into two major O. sativa groups, indica and japonica, and the japonica group into the subgroups, tropical and temperate. These clusters agree with the pedigree information available on the accessions and almost all Argentina-released cultivars grouped within the japonica cluster. Application of DNA polymorphism analysis revealed genomic relationships in Argentine rice germplasm, generating a database useful for cultivar identification, local germplasm conservation, and breeding programs. Barry et al. (2007) Rice genetic diversity partitioning between farms, varieties and, within-variety diversity, were analysed in two villages of Maritime Guinea with contrasted agro ecological conditions. One thousand and two hundred individual plants belonging to 45 accessions collected in eight farms were genotyped using 10 SSR markers. The molecular variance was evenly shared between and within accessions, while the farm effect was almost nil. Local varieties had a multi-line genetic structure. The number of multilocus genotypes was proportional to the utilisation rate of the variety in the village. This varietal structure could mainly be explained by the migration phenomenon and the high varietal turnover. Compared to allelic diversity, multilocus genotypic diversity seemed to be the most suitable indicator of the quantitative distribution of diversity at different management scales (accession, farm and village). The within- and between-farm FST values were in the same order of magnitude. The within-farm diversity was not farm-specific but quantitatively high, i.e. up to 50% of the total genotypic diversity of a given village. Given the relative importance of the within-variety diversity, the in situ approach stands out as the most effective solution. As farms do not host specific diversity the in situ approach could be implemented by working with a small number of farms. Thomson et al. (2007) classified and evaluate genetic diversity of traditional and improved Indonesian rice germplasm using microsatellite markers. A set of high quality Indonesian varieties, including Rojolele, formed a separate cluster within the tropical japonicas separating japonica, indica and tropical japonica groups of rice. This germplasm presents a valuable source of diversity for future breeding and association mapping. Jayamani et al. (2007) used simple sequence repeat (SSR) markers to detect a significantly high degree of polymorphism in rice (Oryza sativa L.) which are particularly suitable for evaluating genetic diversity among closely related cultivars. Rice varieties originating from 19 countries in the Portuguese working germplasm collection were analysed for DNA profile using 24 SSR loci covering two loci per chromosome. Cluster analysis of the 178 accessions revealed three major groups, japonica, basmati, and indica . Many of the accessions included in this study are morphologically similar and lack pedigree information. Hence, identification of genetic distances among the accessions should improve their use in breeding programs. As a result of this study, genetically diverse parents can be identified, increasing the usefulness of germplasm collections by broadening the genetic base of rice varieties. Herrera et al. (2008) studied 11 Venezuelan rice varieties by 48 simple-sequence-repeat (SSR) markers to assess the genetic diversity. UPGMA-cluster-analysis based on genetic distance coefficients clearly separated all the genotypes, and showed that the Venezuelan rice varieties are closely related. Although the genetic diversity was low, SSRs proved to be an efficient tool in assessing the genetic diversity of rice genotypes. Thomson et al. (2009) investigated genetic diversity among Indonesian rice cultivars using 30 microsatellite markers. They showed genetic variations between the indica and japonica varietal groups. These variations were correlated with the field-level ecotypes also. The simple sequence repeat (SSR) marker analysis was done to determine the allelic diversity and relationship among thirty-five Asian cultivars of rice including 19 aromatic, 13 nonaromatic and 3 japonica type cultivars. Basmati rice varieties amplified different alleles at 15 of the SSR loci than those in the japonica and/ or indica rice varieties. A number of SSRs were identified that could be utilized to differentiate between basmati and other non-basmati rice varieties.Two major groups effectively differentiating the tall, late maturing and slender aromatic cultivars from the short statured, early, short bold and long bold non-aromatic cultivars. These results could be useful for monitoring purity, genotype identification and for plant variety protection. (Pervaiz et al., 2009).

  • Comparative study of different marker systems in genetic diversity analysis

Ravi et al. (2003) studied the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) and randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers. Qian et al. (2001) investigated genetic variation within and between five populations of Oryza granulata from two regions of China using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population, respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population, respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pair-wise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. These results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Fugang et al. (2003) studied to estimate genetic relationships of the AA-genome Oryza species, RAPD and SSR analyses were performed with 45 accessions, including 13 cultivated varieties (eight Oryza sativa and five Oryza glaberrima) and 32 wild accessions (nine Oryza rufipogon, seven Oryza nivara, three Oryza glumaepatula, four Oryza longistaminata, six Oryza barthii, and three Oryza meridionalis). It is also demonstrated from this study that both RAPD and SSR analyses are powerful methods for detecting polymorphisms among the different AA-genome Oryza accessions. However, the RAPD analysis provides a more-informative result in terms of the overall genetic relationships at the species level compared to the SSR analysis. The SSR analysis effectively reveals diminutive variation among accessions or individuals within the same species, given approximately the same number of primers or primer-pairs used in the studies.

  • Statistical tools to evaluate genetic diversity

Various statistical methods have been used by the geneticists and molecular biologists to evaluate diversity among crop populations. The use of principal component analysis (PCA) not only allows a number of comparisons between treatments, but also enhances the meaningfulness of these comparisons (Sneedon, 1970). PCA is a useful technique, which enables inter-correlations among variables. Additionally, a useful data reduction technique that removes interrelationship among variables (Broschat, 1979). By using PCA, not only the number of comparisons between treatment means is reduced but interactions among two or more variables may be pointed out by such analysis. In taxonomy, it can be used to express multidimensional inter-OTU (Operational Taxonomic Unit) distances in 3 or fewer dimensions, which can readily be conceptualized. Additional applications of this technique would certainly be found in fields of biological sciences, where it has been used extensively. Multivariate approaches have been used in analysis of genetic diversity of different crop species (Chandra et al. 2007).

Weltzien (1989) accounted for major proportions of the total variation among population of barley landraces. The landraces clustered into 9 distinct groups based on their similarity for all the traits. Each group showed a close association to specific geographic or environmental factors, indicating that adaptive processes are operating in the current agricultural systems. These results emphasized the need to thoroughly describe the locations from which the germplasm accessions originated. The populations were also grouped according to their similarity for qualitatively inherited morphological traits. Subsequent analysis of variance showed that this type of classification was as effective as geographic grouping in distribution of variance among and within groups, suggesting that germplasm collections could be arranged according to either geographic information or morphological similarity.

Perry and McIntosh (1991) evaluated soybean germplasm from 78 countries for 17 characters and worked out variation within and among all regions for most of the traits. The clustering of canonical means, grouped into four region: i) Africa and India, ii) Southwest Central Asia, iii) China, Europe, New World and Southeast Asia and iv) Korea and Japan. They further reported that cluster containing the Korean/Chinese accessions were the most diverse and suggested that plant germplasm from diverse geographical origin was another approach for building gene pool. Elings (1991) estimated phenotypic variation for days to heading, flag leaf length and width, plant height, awn and spike length, awn and spike colour, spikelets per spike and seed shriveling in 84 Syrian durum wheat landraces/populations. Multivariate patterns of variation were established through PCA to describe relationships between landraces groups and regions of collection. Agro-ecological site characteristics and plant traits were compared with respect to patterns of geographical variation. Grouping on the basis of landraces' groups proved more discriminative than on the basis of regions of origin. Landraces originating from sites characterized by favorable growth conditions tended to be later heading and to have longer spikes with long awns but fewer spikelets. The observed relation between favorable growth conditions in the regions of origin and smaller flag leaves may be caused by genotype x environment interaction. Among populations, variation was high and amounted to 96% of the total variation, whereas the remnant 4% was attributed to differences within populations and among lines (Sneedon, 1970). Smith et al. (1991) studied North African and Arabian alfalfa and found that the average linkage cluster and PCA resulted in classification of populations into six phenotypically distinct geographical groups. In another study, Smith et al. (1995) reported that Southern Arabian alfalfa accessions collected below 1000 masl were distinct from those collected at higher elevation. The most distinctive accessions were from low elevation Oases in Yemen and were extremely susceptible to frost damage. Significant regional variation reported the utility of these results for conservation and utilization of germplasm for crop improvement.

Pezzotti et al. (1994) evaluated 81 accessions of orchard grass using both univariate and multivariate analysis for six quantitative traits. A positive correlation was found between seed and dry matter yield, which normally were negatively correlated, whereas two PCs were able to explain 80% of the total variation. The patterns of morphological diversity were examined in relation to geographical origin of 157 accessions of wild Lupins angustifolius from the Aegean region using multivariate techniques (Clement and Cowling, 1994). They reported that genetic diversity was extremely large for most of the morphological traits, with a significant variation among localities in Greece and within and between collection sites for some traits. Thirteen groups were identified by hierarchical clusters analysis. Accessions from northern Greece grouped together as later flowering, shorter and smaller seed size, but some accessions from southern Greek Island were grouped with northern mainland types. Smith et al. (1995) conducted average linkage cluster and PCA, and reported utility of these results in preservation and utilization of germplasm. Knowledge on the pattern of variation for important morpho-agronomic traits is needed for a proper improvement and better exploitation of gene pool (Jain et al. 1975). Genetic relationship among 18 NERICA (New rice for Africa) was explored by using multivariate analysis (Semagn et al. 2006). This analysis provides a good evaluation of landraces by identifying those that should further be evaluated in depth (Rouamba et al. 1996). The phylogenetic relationship studied by Ahmed et al. (1997) indicated that first two canonical components contributed 85% variation in lentil genotypes. He further reported that cluster analysis on the basis of quantitative characters were phenotypically more distinct and exhibited higher breeding value. Although, cluster analysis grouped accessions together with greater morphological similarity, but cluster did not necessarily included all the accessions/ genotypes from same or nearby sites. The extent of diversity and relationship among fifty-two accessions of Brassica germplasm from Pakistan for thirty-five morphological traits were determined by using cluster and PCA (Rabbani et al. 1998a). The germplasm was separated into six groups. Landrace groups were associated with morphological differences among accessions with breeding objectives and horticultural uses. The germplasm collected from Pakistan showed relatively low level of phenotypic variation, which revealed that evaluated germplasm had a narrow genetic base. Although cluster analysis grouped accessions with greater morphological similarity, but did not necessarily include all the accessions from the same nearby sites.

  • Objectives

To meet the increasing demands for food supply, the human race has to significantly enhance crop productivity, for which fuller exploitation and utilization of genetic resources in crop species will provide many more opportunities. Serving as a vast genetic reservoir, landraces provide elite germplasm for improving crop varieties by transferring beneficial genes to the crops (Zhiping Song, 2005). In Pakistan research activities on rice are targeted for increase in yield, and resistance to disease and pest. In this regard mechanization of rice cultivation, adaptation of improved varieties and more recently, use of biotechnology for the incorporation of gene for disease resistance have come up in PARC (Anon., 2000a). Salt tolerance studies are also in progress, but no studies have been marked for grain quality evaluation of local rice genetic resources; though grain quality of some improved varieties was carried out (Ahmad & Akram, 2005). However, recently it was realized at national level in Pakistan that rice with better grain quality should be produced (Anon., 2000b). Germplasm is a vital source in generating new plant having desirable traits. It helps in increasing crop quality and production as well, that improve the level of human nutrition. It is stated that germplasm collection and conservation is meaningless if it is not evaluated for the traits of concern. The present research project was initiated with the following objectives:

  • Evaluate the extent of polymorphism in landrace genotypes of rice from Pakistan using morphological traits, biochemical and molecular markers.
  • Determine the level of genetic relatedness among improved varieties and local landraces at DNA level.
  • Study the association among various traits in rice.
  • Identify promising accessions having traits for future rice breeding program.

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