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2390 J. Med. Plants Res. Table 1. Geographic localities and sample sizes of naturally distributed D. hatagirea. S/N Population name Population code Longitude Latitude Altitude (ft) Sample sizes 1 Bogdang P N34°48'.198 E77°02'.453 9240 ± 25.8 10 2 Skampuk Q N34°35'.238 E77°34'.481 10490 ± 17.4 15 3 Skurru R N34°40'.229 E77°18'.031 10295 ± 20.8 15 4 Hunder S N34°35'.043 E 77°28'.592 10357 ± 18.0 10 5 Turtuk T N34°50'.849 E76°49'.720 9240 ± 25.8 10 6 Tirith U N34°32'.378 E77°38'.481 10443 ± 26.9 10 7 Sumur V N34°31'.128 E77°34'.481 10120 ± 12.7 10 8 Changlung W N34°55'.884 E77°28'.276 10982 ± 39.7 10 9 Staksha X N34°55'.885 E77°28'.276 11081 ± 49.2 6 Table 2. List of primers used for RAPD amplification. Primer Primer sequence (5’ 3’) Total number of fragments amplified Percentage of polymorphic loci Resolving power S21 CAGGCCCTT C 597 100 12.44 S22 TGCCGAGCT G 545 100 11.35 S23 AGTCAGCCA C 341 100 7.10 S24 AATCAGCCA C 566 88.9 11.79 S25 AGGGGTCTT G 508 100 10.58 S26 GGTCCCTGA C 530 100 11.04 S27 GAAACGGGTG 423 100 8.81 S28 GTGACGTAG G 512 80 10.67 S29 GGGTAACGC C 362 100 7.54 S30 GTGATCGCA G 287 100 5.98 S31 CAATCGCCG T 485 100 10.10 S32 TCGGCGATA G 410 100 8.54 S33 CAGCACCCA C 558 100 11.63 S34 TCTGTGCTG G 455 100 9.48 S35 TTCCGAACC C 476 100 9.92 S36 AGCCAGCGA A 310 100 6.46 S37 GACCGCTTG T 459 100 9.56 S38 AGGTGACCG T 511 100 10.65 S39 CAAACGTCG G 380 100 7.91 S40 GTTGCGATC C 430 100 8.96 Total 9145 98.30 - each reaction before electrophoresis. After electrophoresis, the gels were documented on a gel documentation system (Alpha Innotech, Alphaimager, USA). Molecular size of the amplicon was estimated using 100 and 500 bp DNA ladders (‘Bangalore Genei.India’). Data analysis The resulting presence/absence binary data matrix was analyzed using POPGENE version 1.31 (Yeh et al., 1999). Jaccard’s similarity coefficient (J) was used to calculate genetic similarity between pair of individuals. The similarity matrix was subjected to cluster analysis by unweighted pair group method with arithmetic mean (UPGMA) using the SAHN clustering module and dendrogram was generated using the program NTSYS-pc software ver. 2.02 (Rohlf, 1992). POPGENE software version 1.31 was used to describe the structure of studied populations with their geographic location. Observed number of alleles (Na), effective number of alleles (Ne), Nei’s genetic diversity (H), Shannon’s information index (I), number of polymorphic loci (NPL) and percentage polymorphic loci (PPL) across were analyzed (Zhao et al., 2006). The non-parametric analysis of molecular variation (AMOVA) (Excoffier et al., 1992) was performed using squared Euclidean distances among all samples to partition the variation into two hierarchical levels; individual and population. GenAlEx software was used to calculate a principal coordinates analysis (PCA) that plots the relationship between distance matrix elements based on their first two principal coordinates (Peakall and Smouse, 2001). According to Prevost and Wilkinson (1999) the resolving power (Rp) of a primer is: Rp = Σ IB where IB (band informativeness) takes the value of: 1–[2* (0.5–P)], P being the proportion of the 96 genotypes
Warghat et al. 2391 Table 3. Summary of genetic variation statistics for all loci of RAPD among the Dactylorhiza populations with respect to their distribution. Markers Sampling sites Sample size H (mean + SD) I (mean + SD) PPL Bogdang 10 0.1184 ± 0.1706 0.1820 ± 0.2528 36.72 Skampuk 15 0.2346 ± 0.1647 0.3673 ± 0.2284 81.36 Skurru 15 0.1633 ± 0.1869 0.2499 ± 0.2704 51.41 Hunder 10 0.1925 ± 0.1791 0.2977 ± 0.2601 61.58 RAPD Turtuk 10 0.2216 ± 0.1909 0.3629 ± 0.2716 68.36 Tirith 10 0.2036 ± 0.1873 0.3109 ± 0.2689 62.15 Sumur 10 0.2068 ± 0.1884 0.3151 ± 0.2700 62.71 Changlung 10 0.1736 ± 0.1854 0.2666 ± 0.2679 54.80 Staksha 6 0.1375 ± 0.1881 0.2055 ± 0.2749 37.29 Mean 0.185767 0.284211 - H= Nei’s genetic diversity, I=Shannon’s information index, PPL= percentage of polymorphic loci. Table 4. Inter-population genetic distances calculated by Nei’s method. Population Staksha Changlung Sumur Tirith Turtuk Hunder Skurru Skampuk Bogdang Staksha **** 0.103 0.079 0.089 0.160 0.102 0.174 0.150 0.224 Changlung 0.118 **** 0.053 0.036 0.065 0.092 0.126 0.077 0.137 Sumur 0.095 0.068 **** 0.044 0.081 0.058 0.086 0.061 0.108 Tirith 0.105 a 0.051 0.060 **** 0.062 0.079 0.101 0.066 0.112 Turtuk 0.177 0.081 0.098 0.079 **** 0.090 0.118 a 0.051 0.129 Hunder 0.119 0.107 0.076 0.096 0.108 **** 0.084 0.067 0.135 Skurru 0.186 0.137 0.099 0.113 0.131 0.096 **** 0.042 0.072 Skampuk 0.166 0.092 0.077 0.082 0.068 0.084 0.053 **** 0.063 Bogdang a 0.236 0.148 0.121 0.124 0.142 0.148 0.080 0.075 **** Above diagonal values are Nei’s unbiased genetic distances, those below the diagonal are Nei’s genetic distances. a values in bold are maximum or minimum genetic distances. containing the band. Nei’s analysis of gene diversity among population (Nei, 1978) was carried out with counting total genetic diversity (Ht), within species diversity (Hs), genetic diversity between populations (Gst) and estimation of gene flow (Nm) from parameters. Fst index (Wright, 1951) was measured via this formula (Lynch and Milligan, 1994). RESULTS A total of 177 reproducible bands were produced using the 20 RAPD primers (8.8 Bands per primer) of which 174 were polymorphic (PPL= 98.30%). RAPD genetic diversity analysis revealed the highest values of Nei’s genetic diversity (0.23), Shannon information index (0.36) and polymorphic loci (81.36%) among accession from Skampuk population and lowest values of Nei’s genetic diversity (0.11), Shannon information index (0.18) and polymorphic loci (36.72 %) among accession from Bogdang population (Table 3). Nei’s (1978) classified levels of genetic distance at < 0.05 as low, between 0.05 and 0.15 as medium and > 0.15 as high. Thus, the Bogdang accession varied in narrow range while, the Skampuk accessions were more diverse. Pair-wise Nei’s distances (Nei’s, 1973) were calculated for all populations. The greatest inter-population average distance (0.23) was between Bogdang and Staksha. While, the corresponding least distance (0.05) was between Skampuk and Turtuk (Table 4). The dendrogram was obtained from UPGMA cluster analysis based on RAPD data was presented in (Figure 2). The dendrogram showed four main clusters (I, II, III and IV). The cluster I consisted of samples from populations Bogdang, Skampuk and Skurru. Cluster II consisted of samples of population Tirith, Sumur and Changlung. Cluster III consisted of Hunder and some populations of Tirith, Sumur, Skampuk, Skurru and Turtuk. cluster IV and consisted of Staksha and some population of Skampuk, Skurru, and Turtuk. Population genetic structure Genetic analysis of RAPD marker showed that the highest genetic identity (0.964) existed between
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Journal of Medicinal Plants Researc
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Figure 1. Chuanxiong Rhizoma (http:
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Table 1. Identification of main pea
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Table 3. Pharmacokinetic parameters
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active component study, many invest
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and 1185 kg ha -1 in the first site
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Table 1. Recommendation for use fer
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Sadanandan AK, Hamza S (1996c). Stu
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and it has been traced to South Ame
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Table 3. Results of phytochemical a
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(a) (b) (c) Figure 1. (a) Normal ce
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emedies in the south of Brazil. J.
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known fatty acids and terpenoids we
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Table 1. Renal function tests of ra
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Al-Radahe et al. 2271 Figure 3.nnnn
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namely disruption of the vascular e
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Zayachkivska OS, Konturek SJ, Drozd
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β-carotene were purchased from Sig
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Figure 1. Effect of ethanol concent
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Table 3. Climatic and soil factors
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content of polysaccharides. Phospha
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Serge et al. 2285 Table 1. Relative
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Percentage inhibition % of inhibiti
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Table 1. Result of phytochemical sc
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Table 3. Result of thin layer chrom
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Table 2. MIC of a compound 1 from c
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Zirihi et al. 2301 Figure 2. Termin
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Figure 5. T. catappa Linn. (Combret
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Figure 8. A. fumigatus: Aspect of c
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Figure 12. Sensitivity of A. fumiga
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Noormi et al. 2311 Table 1. Callus
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Table 2. Contd. NoC=No callus. 4.0
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Figure 2. Contd. at 2.0 mg/L yellow
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Table 1. Biochemical parameters in
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Figure 3. Bcl-xL reactions in inter
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ACKNOWLEDGMENTS The authors would l
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Figure 1. The first page of the boo
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Figure 3. The most widely included
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Figure 4. Lithographic print “St.
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Table 1. List of plants included in
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Table 1. Contd. Nedelcheva 2333 Cin
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Table 1. Contd. Nedelcheva 2335 Rhe
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14 7 1 15 1 35 17 16 Imported plant
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Page 153 and 154: Table 6. Results of ANOVA. Nie et a
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2460 J. Med. Plants Res. Department
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2466 J. Med. Plants Res. AOAC (2000
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2486 J. Med. Plants Res. components
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2490 J. Med. Plants Res. Inhibition
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2494 J. Med. Plants Res. can increa
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2500 J. Med. Plants Res. Flowering
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2524 J. Med. Plants Res. ACKNOWLEDG
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UPCOMING CONFERENCES 15th European
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