Results and discussion
ISSR fingerprinting of 19 primers generated 270 scorable fragments, out of which 252 were polymorphic (93.33 % polymorphism) (Table 1). The primers viz., UBC 808, 810, 840, 841, 855, 862, 881 and 900 exhibited 100 percent polymorphism. The number of polymorphic fragments for each primer varied from 7 to 19 with an average of 13.26 polymorphic fragments (Fig. 1). ISSR banding pattern exhibited a maximum of 133 fragments in S. japonica followed by C. pareira var. hirsuta (132 fragments) and C. peltata (119). A maximum of eight unique bands were observed in C. pareira var. hirsuta while screening with the primer UBC 851. Interestingly, the number of unique bands in C. peltata was less compared to other plants. But in S. japonica, six and seven unique bands were observed while screening with the primers UBC 890 and UBC 900, respectively. Here also, the total number of unique bands was highest in C. pareira var. hirsuta (Table 2).
Table 1 Total number of bands and percentage of polymorphism amplified by 19 ISSR primers
Sl. no. Primer Annealing temperature Total number of bands Number of polymorphic bands Number of monomorphic bands Polymorphism (%)
1 (ACTG)4 49 11 10 1 90.91
2 UBC 807 50 9 8 1 88.89
3 UBC 808 52 17 17 0 100
4 UBC 810 50 16 16 0 100
5 UBC 825 50 14 13 1 92.86
6 UBC 840 54 17 17 0 100
7 UBC 841 54 12 12 0 100
8 UBC 842 54 10 8 2 80
9 UBC 847 52 16 13 3 81.25
10 UBC 851 54 16 14 2 87.5
11 UBC 854 56 13 12 1 92.31
12 UBC 855 52 19 19 0 100
13 UBC 857 54 15 14 1 93.33
14 UBC 859 54 8 7 1 87.50
15 UBC 862 55 13 13 0 100
16 UBC 873 52 12 9 3 75
17 UBC 881 59 19 19 0 100
18 UBC 890 59 20 18 2 90
19 UBC 900 59 13 13 0 100
Total 270 252 18 93.33
Fig. 1 Representative ISSR profiles of Patha plants using primers a (ACTG)4, b UBC 840, c UBC 847, d UBC 857, e UBC 881 and f UBC 890. M represents 1 kb ladder, lane 1Cissampelos pareira var. hirsuta, lane 2Cyclea peltata and lane 3Stephania japonica
Table 2 Primerwise ISSR banding pattern of Patha plants
Primer Cissampelos pareira var. hirsuta  Cyclea peltata  Stephania japonica
TB PB MB UB TB PB MB UB TB PB MB UB
(ACTG)4 6 5 1 3 4 3 1 1 6 5 1 3
UBC 807 4 3 1 0 7 6 1 4 3 2 1 1
UBC 808 9 9 0 5 4 4 0 1 9 9 0 5
UBC 810 7 7 0 4 10 10 0 5 6 6 0 0
UBC 825 9 8 1 6 6 5 1 1 6 5 1 1
UBC 840 9 9 0 3 10 10 0 3 7 7 0 2
UBC 841 3 3 0 2 4 4 0 4 6 6 0 5
UBC 842 4 2 2 0 7 5 2 2 7 5 2 2
UBC 847 9 6 3 5 7 4 3 2 9 6 3 3
UBC 851 11 9 2 8 7 5 2 1 6 4 2 1
UBC 854 5 4 1 2 7 6 1 3 6 5 1 4
UBC 855 9 9 0 7 6 6 0 1 10 10 0 5
UBC 857 9 8 1 5 7 6 1 1 8 7 1 1
UBC 859 4 3 1 3 4 3 1 1 4 3 1 1
UBC 862 6 6 0 4 6 6 0 3 5 5 0 2
UBC 873 7 4 3 4 5 2 3 2 6 3 3 3
UBC 881 9 9 0 6 8 8 0 3 9 9 0 3
UBC 890 9 7 2 5 7 5 2 3 12 10 2 6
UBC 900 3 3 0 3 3 3 0 2 8 8 0 7
Total 132 114 18 75 119 101 18 43 133 115 18 55
TB number of total bands, PB number of polymorphic bands, MB number of monomorphic bands, UB number of unique bands
Morpho-anatomical studies of roots of C. pareira var. hirsuta, C. peltata and S. japonica clearly indicate the significant differences among themselves (Hullatti and Sharada 2007). Hullatti and Sharada (2010) reported the HPTLC and HPLC fingerprinting of Patha plants and their studies clearly indicate the significant differences among the three plant materials. They concluded that all the studied parameters clearly indicate that roots of C. pareira var. hirsuta are the genuine source of Patha as it fulfills the ayurvedic claims of this drug. The present investigation confirms that there is no genetic relationship among C. pareira var. hirsuta, C. peltata and S. japonica. The high level of polymorphism and unique fragments detected in ISSR profiles authenticate that there exists a wide genetic disparity among the selected plants. The findings of the present study substantiate the reports on the applicability of ISSR markers to authenticate the herbal medicinal materials from its adulterants (Shen et al. 2006; Wang 2011). This investigation is the first report on the study of genetic relationships of Patha group of plants using ISSR markers.