Genetic diversity of crop landraces requires every aspect of the diversity to be explored. It is necessary to mine new genes for crop improvement (Iannetta et al., 2007). Conservation of genetic diversity of landrace crops is an important issue because of the extent to which this diversity is declining after green revolution. In order to maintain, evaluate and effectively utilize germplasm, extent of genetic diversity must be measured. Only source of genetic diversity is the germplasm by which the plant breeders develop new cultivars (Baranger, 2004). In present investigation, diversity was measured at three different levels in rice (Oryza sativa L.) landraces obtained from all over Pakistan. Three levels of diversity include:
Germplasm collections exist to conserve the genetic diversity of crop species and their wild relatives (Williams, 1990). To facilitate efficient germplasm collection and management practices and to make the best use of germplasm resources for future breeding programs, it is essential to understand the extent and nature of genetic diversity and relationships among various germplasm collections. Such analysis is particularly useful in the characterization of individual landraces or cultivars, for the identification of cultivars, in detection of duplicates of genetic material in collections and as general guide in the selection of parents for hybridization program. The conserved germplasm supplied to plant breeders for use in the development of improved varieties for farmers ultimately produces the economic return on investment made in germplasm collection, preservation and evaluation (Vaughan and Jackson, 1995). In present study considerable level of diversity was observed in agro-morphological traits as reported by others (Yousaf et al., 2008; Sabu et al., 2009). Information from present study will be helpful in the study of genetic relationships among landraces, representation of genetic diversity, morphological evaluation and also its an effort to assess trait-gene association (Pervaiz et al., 2010). In the present study nine qualitative traits were observed in 177 rice landrace genotypes. Considerable diversity was observed for seed coat color, awn color, flag leaf angle and flag leaf shape during both years. Brown seed coat with white awn color was predominant in Pakistani rice germplasm. Allelic variation in qualitative traits found in present investigation could be utilized in breeding programs because genetically heterogeneous lines are highly stable as compared to homogenous ones. The qualitative traits are under genetic control of two or many alleles of a single gene with little or no environmental modifications to obscure the gene effects e.g. seed color, seed shape, awning, awn color etc, so no considerable change was observed in qualitative traits in next growing season. Seed coat color has relationship with mechanical resistance of the seed for different environmental stimuli. Pigmentation in pericarp is correlated with its hardness and dormancy (Han et al., 2009). Gene flow from red disease resistant wild rice to white susceptible commercial rice varieties is one of the examples. Pakistani rice germplasm has brown coat color dominance which could be utilized for further breeding programs for producing resistant varieties.
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Quantitative traits are economically important; those have high variability because they are also controlled by more than one gene and could be used for the crop improvement (Amurrio et al., 1995; Fall et al., 2003). In Pakistani rice landraces, basic statistics for both 2006 and 2007 showed high degree of variation for all these 18 quantitative traits. These traits are directly or indirectly yield contributing and genetically very important for the selection of high yielding genotypes. Subdividing the variance into its component assists genetic resources conservation, utilization and it enables planning for use of appropriate gene pool in crop improvement for specific plant attributes (Pecetti et al., 1992; Rabbani et al., 1998; Ahmad, 2004; Tar'an et al., 2005). Days to flower initiation ranged from 48 to 99 days during 2006, while in 2007 it ranged from 43 to 97 days. Days to maturity also showed maximum variance during both years 179.8 and 184.5, in 2006 and 2007 respectively. Our findings are also in conformity with the results of Zaman et al. (2005) who observed 96.33 to 121 days to heading in different varieties. Koutroubas et al. (2004) have also found 60.to 142 days in heading while maximum 170 days to maturity were reported in some Basmati lines. Zafar et al. (2004) found minimum value of days to heading was 72 days and maximum 171 days to maturity among landraces of rice from Pakistan. Days to heading in one study on Bangladesh rice landraces ranged from 78 days to 109 days (Bisne and Sarawgi, 2008). The variation for days to heading and maturity may be attributed to the material used and seasonal variations. Shah et al. (1999) stated that the opening of the spikelets depend primarily on the prevailing atmospheric temperature, the light intensity and other climatic conditions. Flag leaf area with its angle is the most important character in which maximum photosynthesis is occurred. Flag leaf area has the maximum contribution towards grain yield. The total leaf area of a rice population is a factor closely related to grain production (DeDatta, 1981). Flag leaf area in present study ranged from 33.3 to 130.2 cm square in 2006 and 26.2 to 128.6cm square with maximum variance 263.2 and 257.0 during 2006 and 2007, respectively. Alleles for medium and narrow flag leaf area were found to be dominant in present study.
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A breakthrough in plant breeding was attained with the development of semi-dwarf cultivars characterized by lodging resistance, nitrogen responsive and erect leaves. The success of the "Green Revolution" is directly related to the intensive use of these dwarf varieties (Hirano et al., 1992). The semi-dwarf plant type has been extensively utilized in the improvement of rice (Oryza sativa L.) cultivars throughout the world. In some regions of the world local farmers want tall rice varieties so they can feed its straw to cattle. However, tall varieties lodge when heavily fertilized, and significantly reduce yields. Mean plant height (156cm) in the Pakistani rice was almost same during both years. The maximum plant height was observed in accession 6636 and minimum plant height was exhibited by accession 6626 during both seasons. Reduction in plant height may improve their resistance to lodging and reduce substantial yield losses associated with this trait (Abbasi, 1995).
Panicle length is an important yield component and is given due consideration in rice improvement. Grain yield can be increased either by increasing the number of panicles (productive tillers) or the number of spikelets panicle-1 (panicle length) (Takeda, 1984). It ranged from 13.3 to 47.7 cm during 2006 with a mean value of 26.8 cm and coefficient of variation of 14%. In 2007, the landraces attained uniformity in panicle length, ranging from 12.4 to 48.5 cm with mean value of 24.9 cm and coefficient of variation was 17.0%. Our findings are also in conformity with the results of Zafar et al. (2004) who observed maximum panicle length of 29.6 cm in Pak3169. Iqbal et al. (2001) also observed 20.3 cm panicle length in accession Pak3167 and maximum 30.0 cm in accession Pak3078 and Pak3394. Abbasi et al. (1995) observed minimum panicle length of 23.1 cm in genotype DR46 and maximum 27.30 cm in DR39. Although it contributes positively yet maximum panicle length is not the only factor responsible for higher grain yield. It was also observed that DR39 had maximum panicle length but due to lower grain fertility exhibited lower grain yield. So panicle length alone does not determine the high grain yield as traits such as grain size, grain shape, higher numbers of tillers plant-1, longer panicles and greater number of grains panicle-1 ultimately contribute to higher grain yield (Akram et al., 1994). Kwon et al. (2002) also reported (22.3 to 26.5 cm) range of panicle length in 13 Tongil-type rice cultivars in Korea.
The largest variation was found for days to heading, days to maturity, flag leaf area, plant height, grain yield plant-1, biological yield plant-1 and 1000-grain weight. The variances for the said traits were 117.0, 179.8, 263.2, 216.4, 89.5, 3370.5 and 91.1, respectively in 2006 and 129.3, 184.5, 257.0, 211.6, 101.0, 2984.7 and 101.4, respectively in 2007. Relatively, a low level of variability was observed for flag leaf width, total and productive tillers plant-1, panicle length, branches panicle-1, harvest index and grain size.
Yield is a function of total dry matter and harvest index (Khush, 1996). Therefore, yield can increased either by enhancing the total dry matter (biomass) or the harvest index or both. Grain straw ratio of the rice crop helps the plant breeders while selecting the promising high yield genotypes for use in the breeding programs. Paddy grain length showed moderate variance during two years, 9.5% in 2006 and 9.2% in 2007 respectively. Maximum grain length was observed in 6505 and Super-basmati (Table 3.1). Paddy grain breadth ranged from 1.6 to 3.2 mm with a mean breadth of 2.3 mm and co-efficient of variance 15.2% during 2006, while in 2007 it ranged from 1.7 to 3.3 mm with a mean value of 2.3 and coefficient of variance of 14.8% which revealed moderate amount of diversity in grain breadth of landraces. Similar observations were reported by Koutroubas et al. (2004) on observing 0.77 standard deviation using 299 rice lines for grain length. Grain length ranged from 6.7 to 9.73 mm with 9.04% coefficient of variance and 0.71 standard deviation was reported by Zafar et al. (2004).
Grain breadth ranged from 1.6 to 3.2 mm with 15.2 % in 2006, while it ranged from 1.7 to 3.3mm with co-efficient of variance 14.8 which revealed that moderate amount of diversity in grain breadth of genotypes. Our findings are also in conformity with the results of Bhat and Gowda (2005) who reported grain breadth of 1.9 mm in Sanna bhatta landrace and maximum 3.0 mm in 'Bili Kagga' cultivar. Koutroubas et al. (2004) also reported 2.98 mm grain breadth in rice lines 'SHSS53' and 'SR113' out of 298 lines. Akram et al. (1995) reported 1.53 mm grain breadth in IR62871-344-5-2 variety. Sagar et al. (1988) studied minimum grain breadth of 1.6 mm in 'DR82' as well as maximum 3.00 mm in 'JP5' rice varieties.
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The relationship between plant characteristics is useful for selecting yield improving traits. Correlations in phenotypic traits may reflect underlying biological processes that are of significant evolutionary interest. Correlation may be the result of genetic, functional, physiological or developmental relationships among characters (Wagner and Schwenk, 2000). A single trait can have a direct effect on the fitness of an organism, the covariation among multiple phenotypic characters arising from these types of relationships (Stearns, et al., 1991).
Simple correlation coefficients between the means of 18 morpho-physiological characters were calculated for 177 rice genotypes for the two years, separately (Table 3.4 and 3.5). Generally, low level of correlation was observed between different characters. However, of 171 combinations of traits, measurements of some functionally related traits, such as flag leaf morphology (flag leaf length, flag leaf width, flag leaf area), traits associated with heading (days to heading and maturity) and traits related to yield (grain length, width and 1000 grain weight, etc.) showed significant correlation with each other during both growing seasons. For example, days to heading had highly significant correlation with the component trait such as days to maturity. Similarly flag leaf length had strong positive association with its component traits, flag leaf width as well as flag leaf area. Highly positive correlations were also observed between heading and maturity time, paddy grain length and paddy grain length breadth ratio, flag leaf size and plant height. There was also significant positive association between grain yield and its component traits such as, total tiller plant-1, productive tiller plant-1, etc. It was also observed that landraces from northern hilly areas e.g Swat, Malakand, possessed smaller and broad grains. This may attribute to the fact that grain length decreases with increase in altitude (Siddiqui et al., 2007). Though the grain length decreased in high altitude the increase in breadth compensated for grain volume to accumulate the grain weight, as it showed an increasing trend with increase in altitude, though other factors are also involved in the grain filling. These results suggest that Pakistan rice accessions possess a distinct correlation in terms of altitudinal distribution for grain morphology. A positive correlation of grain breadth and seed weight with altitude increase and a negative correlation of grain length and length to breadth ratio was found with increasing 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. However, there was least variation for seed colour. The prevailing diversity for grain type (shape and size) and pericarp colour has distinct correlation to its geographical distribution in terms of altitude. Consumer preference is another factor which may affect the choice of farmers for short grained rice in northern areas and long grained rice in southern areas of Pakistan.
Different clustering analyses and PCAs scattered plot revealed that there is a lot of genetic diversity among the studied genotypes and also found that quantitative traits can also be used as a maker in breeding program but there was no geographic association because the genotypes from one place enter into more than one clusters. Conversely, genotypes from different geographic origins were relatively unique and tend to be clustered as was determined not only by environmental difference but also by genetic factors. Although principal component analysis grouped genotypes together with greater morphological similarities, the clusters did not necessarily include all the landraces from the same or nearby sites. The landraces were grouped according to their morphological similarity and not due to geographical origin. Gupta et al. (1991) reported that genetic diversity in mustard was not related to geographical distribution of the germplasm, as lines from different geographic regions were pooled in the same cluster. Similarly, Amurrio et al. (1995) observed that grouping patterns of pea landraces did not reflect geographical origin.
Variance of heritable quantitative traits provides an estimation of genetic diversity within and between collecting sites that is expressed by the diversity in peas (Amurrio et al., 1995; Ghafoor et al., 2005). High degree of variation was observed for most of the traits that indicated the scope of direct selection to develop high yielding cultivars from present material. Although selection improves the cultivars, but simultaneously narrows down the gene pool, therefore conservation is important for future use of genetic resources (Rabbani, 1998).
Present study on agro-morphological diversity concludes that the rice landraces of Pakistan have significant variation even with same local name. Evaluation at field level provides evidence that heterogeneity in some quantitative traits is not a very rare phenomenon. Ecological and environmental heterogeneity are directly correlated with genetic variation within a country instead of geographical distribution (Li and Rutger, 2000) and it may be due to the changing environmental conditions during 2006 and 2007 in experimental field area of Islamabad.
4.2 Proteomic diversity in Pakistani Rice landraces
In present investigation, maximum 25 bands were observed in accession 6685, while 24 in accessions 6532, 6542 and 6695. Minimum 17 bands were observed in accession 6757 and 18 in accessions 6613 and 6655. Habib et al. (2000) evaluated 15 genotypes of rice using SDS-PAGE, showed a total of 32 bands. Sharief et al. (2005) reported maximum17 bands in basic, registered, certified and commercial seed of rice 'Giza 177' cultivar using SDS-PAGE and also observed minimum 12 bands in commercial seed of Sakha 101 using SDS-PAGE. This variation in number of polypeptide subunits may be due to differences in genotypes used, gel percentage, preference of major subunits for evaluation and size of the gel.
No significant association of geographical location and variation in seed protein profile was observed. The landraces from different geographical zones were grouped together in same clusters. This shows that expression profile is least affected by environment reported by various studies on rice as well as other cereal crops (Sanni et al., 2008). Lower genetic distance (similarity 0.81) may be attributed to the same genetic background, traditional framing practices and consumer preference (Javaid et al., 2004).
Our study revealed that storage protein polymorphism in rice can be used to discriminate rice accessions on the basis of amylose contents (high or low on the basis of presence or absence of Wx gene product in protein banding profile) and glutinous rice varieties from non-glutinous ones on the basis of presence of acidic and basic peptide subunits of glutelin. In present study one japonica variety Kinmaze also showed absence of Wx gene product in addition to other twenty-one Pakistani landraces. Pakistani germplasm is not japonica type because Kinmaze clustered with few landraces and showed no 100 percent similarity with any accession. Majority of the accessions grouped with indica type check variety IR36, with 100 percent similarity, which clearly indicates that rice landraces of Pakistan are almost all indica type. So protein-banding profile can be used to differentiate japonica and indica subspecies of Oryza sativa. Variation in 13 and 16 kDa prolamin peptide subunits is limited to Asian rice (Hilu and Sharova, 2002) and our study is again a confirmation of this conclusion, 16 kDa peptide subunit showed considerable polymorphism in Pakistani germplasm.
Another important thing observed in Pakistani germplasm accession's protein banding profile, was that four accessions (6755, 6756, 6757, and 6758), which showed no acidic and basic glutelin peptide subunit, took least days to heading and maturity (data based on morphological studies). This gives a clue to a correlation between glutelin markers and days to heading and maturity in rice landraces. Further study is required to link protein markers with agronomic traits.
Two novel peptide markers (52kDa and 32 kDa peptide bands) that were observed in Pakistani germplasm need further exploration. It may be concluded that hybridization between accessions from two groups (one with all bands and other with missing band is suggested to be conducted with the expectation that missing or extra band might be linked with some agronomic traits.
In the present study 173 Pakistani landraces were evaluated at protein expression level. SDS-PAGE separated the whole landraces into two groups i.e. indica type and japonica and concluded that majority of Pakistani rice landraces are indica type. In the study Wx gene product, glutelin subunit showed significant level of polymorphism, while low level of variation was recorded in prolamin peptide subunit. It was also identified that Pakistani rice landraces have important genes, which need further study by using Serial Analysis of Gene Expression.
4.3 Genomic diversity in Pakistani landraces
4.3.1 Genetic diversity based on Random DNA Makers
A clear and thorough knowledge of the extent of genetic variation and inter and intra-specific relationship is essential for devising strategies to efficiently utilize and maintain the rice germplasm conservations. In the investigation reported here, RAPD markers were used to examine the relationships among seventy two landraces collected from different locations within the country along with three commercial cultivars. Use of RAPDs as a tool to study the genetic diversity and relationships among the different cultivars have previously been reported (Chalmers et al., 1992; Landry et al., 1994; Neeraja et al., 2002; Rabbani et al., 2008). A considerable level of genetic variability was observed among landraces; cultivars 'Super-basmati' and 'IR6' shared limited number of fragments with majority of the landraces. Also lower numbers of bands were common among landrace genotypes and japonica type check variety (JP5) demonstrating that Pakistani landraces are closer to indica varietal group. However, the origin of Basmati varieties is still controversial. Kovach et al. (2009) reported three distinct genetic subpopulations of rice including tropical japonica, temperate japonica and Group V ("Basmati" and "Sadri" varieties) are from japonica varietal group.
RAPD assay interferes in true diversity analysis by producing alleles from both homozygous and heterozygous conditions leading to reduced diversity estimates. However some modifications in RAPD procedure allow calculation of the genetic parameters (Lynch and Milligan, 1994). Similarity index for landraces varied from 0.67 to 0.90 with an average value of 0.78. Similar levels of diversity among various panels of rice genotypes under different analysis have been reported previously (Davierwala et al., 2000; Ren et al., 2003). However, relatively higher similarity value (90%) in comparison with 25 to 77.5% (Raghunathachari et al., 2000) could be the result of reduced intra-specific variations in Pakistani rice due to the common ancestors and the selection for few selected traits as compared to Indian scented rice germplasm.
DNA fingerprinting helped in grouping of rice landraces at subspecies level. In cluster analysis, most of the Pakistani landraces fell into a close sub-group corresponding to Super-basmati. Fifty of the seventy-five landraces, which have similar characteristics for various morphological traits, were grouped into the upper portion of the dendrogram. This may lead to conclusion that majority of Pakistani landraces are Basmati and indica type. Ren et al. (2003) also reported that RAPD based dendrogram supported the clustering of five distinct groups with exceptions. This diversity analysis also differentiated most of the landraces, which shared same local name but different accession numbers e.g. Jhona-129 ( 6620), Jhona-145 (6626), Jhona-101 (6745) collected from different geographical locations. Medium grained Jhona-129 (6620) and Jhona-101 (6745) were grouped with Super-basmati, while short grained Jhona-101 (6745) was grouped with JP5 japonica type variety. Similarly Rohru-414 (6593) and Rohru-150 (6578) were entirely different in banding pattern. Studies previously conducted used RAPD markers to group the long-grain basmati cultivars into a single cluster, whereas the other short-grained rice landraces fell into a different group (Rabbani et al., 2008). They reported a low level of variability among Pakistani basmati rice varieties using RAPD markers.
It is concluded that the Pakistani rice landraces possess considerable variation in their genetic base. The study showed that most of the Pakistani landraces genetically resembled with Super-basmati and IR6. Genetic makeup of a crop species is not affected by environment and only expression profiles can be the targets of environment proved in present study as the landraces collected from different geographical zones grouped together on the basis of RAPD banding profiles. The landraces with wide genetic distance can be used as parents to exploit heterosis in future rice breeding programs. The rare or unique alleles observed can further be employed for marker assisted selection programs. The primers which proved more informative can be converted to sequence tagged sites (STS) and sequence characterized amplified regions (SCAR) for amplification of specific alleles which could be further utilized in rice genome analysis. Also the information gained from clustering behavior of landraces can be useful to design strategies for their management in the gene bank.
4.3.2 Genetic diversity analysis based on specific markers among rice cultivars and landraces.
One main cause of eradication of plant genetic resources has been the adaptation of narrowly based advanced varieties for intensive cultivation practices. Variation in landraces is helpful for broadening the crop gene pool (Frankel and Soule, 1981). Diversity exploration among plant landraces made necessary by the failure of the green revolution to be sustained. Little was known about the relationship between Pakistani agronomic crop cultivars in general and rice landraces in particular on the basis of SSR analysis. Here, 35 microsatellite markers were used to assess the genetic diversity of 75 genotypes of rice including three check varieties Super-basmati (Indica type, aromatic), IR6 (Indica type, Non-aromatic) and JP5 (Japonica type, non-aromatic). The results indicated significant genetic variation among the rice landraces of Pakistan. Microsatellite assays identified a number of alleles that were shared among the Super-basmati and some landraces. A close relationship between 'Super-basmati' and a group of twelve landrace genotypes (16%) was observed. That phenomenon could be a favour towards strong discrimination power of some of the DNA markers. However, the similarity coefficients of these landraces ranged from 0.47 to 0.75 compared with Super-basmati rice. These landraces also shared many morphological and agronomic traits with Super-basmati (indica type) that strengthen the supposition of close relationship between them. 'Check variety 'IR6' (indica type and non-aromatic) shared limited number of fragments with 54% of landraces (shown in group II of dendrogram), suggesting a close association. Similarity coefficients of 'IR6' with landraces of this group ranged from 0.25 to 0.86.
The numbers of alleles detected by microsatellite markers varied from 2 to 13 in rice varieties as well as in landraces with an average of 4.5 alleles per locus and were similar to other reports in basmati and non-basmati rice varieties (Siwach et al., 2004; Neeraja et al., 2005; Herrera et al., 2008). The 2.0-5.5 alleles per SSR locus for various classes of microsatellites were similar to those reported by Cho et al. (2000) using a different set of rice germplasm. However, the average numbers of alleles detected here were significantly higher than those reported in Indian aromatic rice varieties by some researchers (Nagaraju et al., 2002; Singh et al., 2004; Joshi and Behera, 2006). This could be due to inclusion of several landraces of diverse origin in this study. In contrast, the average numbers of alleles noticed in present study were lower than those reported previously (Ni et al., 2002; Jain et al., 2004; Xu et al., 2004; Lu et al., 2005; Brondani et al., 2006; Jayamani et al., 2007; Thomson et al., 2007). Those reports had an average of 6.8, 7.8, 11.9, 6.6, 14.6, 7.7 and 13.0 alleles per locus. They used rice subspecies, Indian quality rice germplasm; US rice genetic resources, traditional varieties of Brazilian rice, a diverse collection of Portuguese rice and an Indonesian rice germplasm, respectively. The inconsistency among reports might be due to the genotypes used and selection of microsatellite primers with scorable alleles.
The polymorphism information content (PIC) values were quite high and varied (range 0.124 to 0.836 in landraces and 0.157 to 0.897 in rice varieties) considerably among SSR loci. The PIC values observed in this study were comparable to those reported in some studies (Jain et al., 2004; Saini et al., 2004; Siwach et al., 2004; Lu et al., 2005; Jayamani et al., 2007; Thomson et al., 2007), but higher than those reported by Singh et al. (2004) and Joshi and Behera (2006). However, this study report lower PIC values compared to those described by Xu et al. (2004) and Brondani et al. (2006), who observed an average PIC value of 0.73 and 0.74 for the world collection and traditional varieties of Brazilian rice, respectively. This difference might be linked with selection of different markers and more diverse set of varieties used.
Lower bootstrap values were observed at some node points in dendrogram in present investigation as compared to Herrara et al. (2008). The reason may be the sample size in present investigation we used 75 genotypes and in previous one only eleven genotypes were studied. The cluster analysis based on similarity coefficients places 75 rice genotypes into two major groups at 0.34, while at 0.40 four clusters are formed. Most of the basmati-type landraces fell into the same group along with Super-basmati check variety. Cluster analysis also grouped most of the basmati landraces from different districts of Punjab together indicating that they are genetically similar with each other and have common ancestors. These rice landraces might share basmati parents in their pedigree. A similar study conducted by Kobayashi et al. (2006) using 18 microsatellite markers, grouped 23 rice landraces into two groups, one small cluster of two indica cultivars, while other of japonica type landraces.
The microsatellite markers used in this study were well distributed amongst the 12 chromosomes, and were located in both coding and non-coding segments of the genome (Cho et al., 2000; Temnykh et al., 2000). Only three markers were monomorphic, while remaining 32 gave polymorphic alleles. RM241 located at chromosome number 4 (106.2-106.2Cm) gave five polymorphic alleles with PIC value of 0.831 in Pakistani landraces, while this marker was monomorphic when used previously by Kobayashi et al. (2006) in analyzing genetic diversity of an old Japanese landrace, 'Echizen'. This is a co-localized marker linked to some quantitative traits as well as qualitative traits e.g. 1,000 grain weight, awn length, biomass yield, chlorophyll contents and hull color, and flour color (www.gramene.org/db/qtl). This showed that Pakistani landrace germplasm is heterogeneous at the genomic level. Some landraces with similar morphologies in field studies proved to be diverse by DNA marker analyses.
Amplification of microsatellite markers RM70 and RM72 resulted in 13 and 6 polymorphic alleles with size ranges from 128 to 167bp and 151 to 200bp, respectively. Separate cluster analysis of these two markers showed groups of late, early and very early maturing landraces (not included in this work). Therefore, earliness, an agronomically important trait, may be linked to these markers. There is no clear evidence for this from the literature except that microsatellite loci RM72 is present as a neighboring marker at chromosome 8 (69-69Cm) with co-localized markers (RM483, RM404, RM617 and RM44) that were linked to days to heading in rice (www.gramene.org/db/qtl.). Another evidence is that the QTL (Quantitative Trait Locus) for days to heading (Hd-4 and Hd-5) are located on chromosome 7 and 8, respectively (Lin et al., 1998). Further analysis will be required to prove this hypothesis.
Rice landraces are reported to be heterogeneous and include different genotypes within the population (Fukuoka et al., 2000). Microsatellite markers proved to be a useful tool for clarifying the genetic diversity among landrace genotypes. Ecogeographical adaptation of landraces was also reflected in DNA profiles at specific loci. Three cold tolerant landraces from Malakand, Swat and Dir (Cluster-1) were grouped together showing similarity at genomic level and difference from other landraces. The reason for this grouping might be the same genetic background with limited out-crossing and farmer choice/priority in these hilly areas.
The results based on 32 microsatellite markers analysis on thirty five rice varieties showed a clear division of cultivars into aromatic and non-aromatic groups. A close relationship between 'Basmati-370' and 'Basmati-Pak' could be due to small differences at the DNA level between the two varieties. In addition, these varieties were morphologically similar in agronomic traits, which supported a close relationship between them. However, there exist sufficient variations at molecular level among all rice varieties.
Here microsatellite analysis was an efficient tool for diversity analysis, and differentiation of rice landraces on the basis of different traits. Overall results show that Pakistani landrace germplasm of rice is not japonica type. The few landraces which grouped with JP5 in the SSR-based analysis of aromatic and quality rice implied a long, independent and complex pattern of evolution for basmati germplasm. The present investigation further indicated that genetically basmati type rice was different from that of coarse/non-aromatic and japonica type. In addition, marker-based identification and differentiation of basmati rice may help to maintain the integrity of this high quality product to the benefit of both farmers and consumers. The microsatellite assay generated cultivar-specific alleles in some of the genotypes screened; these may be used as DNA fingerprints for cultivar identification. This would be of enormous assistance for the establishment and defense of proprietary rights and the determination of cultivar purity.
4.4 Comparative study of diversity at morphological, biochemical and molecular levels
Traditionally genetic diversity of genotypes was assessed based on differences in range of expressions of morphological and agronomical characters. Currently, a variety of molecular techniques are available for measuring genetic diversity. The most common ones are RFLP, RAPD, AFLP and SSR. All of them detect polymorphism by assaying subsets of the total amount of DNA sequence variation in a genome. However, they differ in principle, application, type and amount of polymorphism detected and cost and time requirements (Karp et al., 1998). Consequently, comparison of the efficiency of different types of molecular markers is vital and carried out by several investigators on a number of plant species. In present study, genetic variation in Pakistani rice landraces was measured at three different levels. Each marker has its own importance and detects variability at moderate to high level. Extent of variation may be correlated with the specificity of the marker. Morphological markers detect variation at phenotypic level, considerable variation was observed during both years. Among molecular and biochemical markers, molecular or DNA based markers were more efficient and gave clear variation among the genotypes. Protein markers based clustering reveals the grouping at 0.81 similarity coefficient value, while DNA based markers gave least similarity values. Microsatellite markers gave variation at 0.34 similarity coefficient value, while random markers grouped genotypes at 0.67. This also confirms the specificity of the marker is more closely related to the genetic variability. Several investigators compared the efficiency of morphological, biochemical and molecular markers for genetic diversity assessment in different crop species. The molecular marker technique RAPD was more effective compared to morphological traits for classifying Chilean common bean landraces into the Andean and Mesoamerican gene pools (Johns et al., 1997). Bahrman et al. (1999) while assessing the genetic diversity of 26 winter barley varieties using molecular, biochemical and morphological markers, observed higher differentiation levels between varieties using DNA markers compared to biochemical and morphological markers. Briard et al. (2002) studied the genetic diversity of wild seakale (Crambe maritime L.) using morphological and RAPD markers and observed little relation between results of morphological and molecular classification. Lage et al. (2003) evaluated the genetic diversity in synthetic hexaploid wheat parents using AFLP and agronomic traits and reported non-significant correlation between genetic distances obtained from AFLP and agronomic data. Hamza et al. (2004) studied the genetic diversity of 26 Tunisian winter barley cultivars using SSR markers and morphological traits and reported significant correlation between the two diversity measures and correspondence of clusters constructed using morphological and SSR data. Jacoby et al. (2003) and Rotondi et al. (2003) reported similarity in clustering of genotypes using molecular and morphological characters in Solannum and olive genotypes, respectively. Maguire et al. (2002) did comparative analysis of the genetic diversity in the mangrove species using AFLP and SSR and reported the congruence between AFLP and SSR data sets, which suggested that either method or a combination was applicable to assess genetic diversity of mangroves. Uptmoor et al. (2003) compared the efficiency of RAPDs, AFLPs and SSRs for analysis of genetic relatedness of sorghum landraces from South Africa and reported that RAPD and AFLP similarity indices were highly correlated (r = 0.81), while the Spearman's rank correlation coefficient between SSRs and AFLPs (r = 0.57) and RAPDs and SSRs (r = 0.51) were relatively low.
Suggestions/ Researchable questions
Morphological evaluation of rice landraces collected from different ecological zones of Pakistan was performed under normal soil and water conditions in homogeneous environment. Although the nature and atmospheric conditions in different regions of Pakistan are very diverse, the germplasm should also be characterized for the salinity, drought and heat tolerance. Agronomic performance of these landraces should be evaluated under such conditions for selection of extreme parents to broaden the genetic base of rice suitable for particular regions of Pakistan.
Selected novel protein markers can be used for diversity analysis at peptide level, germplasm classification and could be explored further by using two dimensional gel electrophoresis and Serial Analysis of Gene Expression.
From the selected novel RAPD markers, Sequence Characterized Amplified Region (SCAR) makers can be developed for improving rice MAS breeding program.
Selected microsatellite markers which differentiate landraces into different phenotypic classes could also be used in further MAS breeding program.