Genetic Diversity of wild Viola species from Mid Hills of Himachal Pradesh using Morphological and Molecular Markers

Abstract

xii newlineABSTRACT newlineViola (family Violaceae) is one of the important wild medicinal plant genus in the Himalayan region composed of about 600 species. Members of this genus are mostly native of India, China and Bhutan with most of the species found in the temperate northern Hemisphere: Hawaii, Australasia, Andes. Out of the 35-species reported from India, seven species of Viola (V. canesencs, V. pilosa, V. odorata, V. canescens, V. kashmiriana and V. betonicifolia and V. biflora) have been recorded from the Himachal Pradesh. The distribution of these species is still not clear from the state as all the districts are not being studied by researchers. Addition to this inter and intraspecific genetic diversity of populations of wild Viola species has been rarely explored from the Himachal Pradesh. The study of genetic diversity is necessary because it plays an important role in adaptation, natural selection and conservation of wild plant species. Therefore, present study deals with the analysis of genetic diversity of wild Viola species from mid hills of Himachal Pradesh using morphological (qualitative and quantitative) and molecular (ISSR) markers. newlineSamples [three plants of each species from one village (twelve plants of one species/ district)] for the present study were collected from five districts (Mandi, Shimla, Solan, Sirmaur and Kullu), situated in mid hills of Himachal Pradesh. Total four species of wild Viola (V. odorata, V. pilosa, V. canescens, V. kashmiriana) were observed and collected from the selected sites. Different qualitative characters studied were habit, external shape, stem, leaf characters (leaf shape, margins, base and apex), flower characters (calyx, corolla, androecium, gynoecium) and fruit type. Results showed non-significant intraspecific morphological differences between the studied species of Viola w.r.t. qualitative traits, whereas observed interspecific differences didn t have shown any marked qualitative difference in accordance with earlier records. Quantitative characters which were used to study variation in Viola species were PL, MSL, PRL, NLP, LL, LW, LaL, NL, PeL, PiL, LA, LaL/W %, PeL/NL %, perimeter, AS and SF. Present study showed significant interspecific and intraspecific morphological variations in quantitative characters of the studied wild Viola species which were could be due to the variations in ecological and environmental conditions of different sampling areas. newlineThree wild Viola species (V. pilosa, V. canescens and V. kashmiriana) were selected for molecular analysis. A set of five ISSR primers (ISSR-A, ISSR-B, ISSR-C, ISSR-D and ISSR-E) were used to observed interspecific genetic diversity between V. pilosa (collected from Kullu and Solan district), V. canescens and V. kashmiriana (from Kullu district). Results of interspecific genetic newlinexiii newlinediversity observed total 32 bands with five primers out of which 25 were polymorphic with 78.125 % average polymorphism and remaining were monomorphic. Whereas, intraspecific diversity was observed in the populations of V. pilosa collected from three villages of district Solan (Kumarhatti, Bajhol, Kandaghat) and Kullu (Putai, Kullu vaishnodevi, Kashawri) and V. canescens collected from three villages (Putai, Kullu vaishnodevi, Kashawri) of Kullu district. Results showed total of 23 bands in genotypes of V. pilosa of Kullu district. Out of which, only five were observed to be polymorphic with average polymorphism of 21.73 %. Highest polymorphism (60 %) was observed with ISSR-A primer and lowest (16.67 %) was observed with ISSR-C primer. Similarly, in V. pilosa (collected from Solan district) five primers have amplified total 18 bands out of which four were polymorphic with 22.22 % polymorphism. Primer ISSR-A showed highest polymorphism (80 %) and other remaining primers were observed with 0 % polymorphism. The comparative analysis of V. pilosa of Solan and Kullu district showed total 26 bands (12 polymorphic) having 46.14 % polymorphism. The highest polymorphism (75 %) was observed with the ISSR-B and lowest polymorphism (16.67 %) was observed with primer ISSR-E. V. canescens genotypes of Kullu district characterised with total 21 bands, out of which seven were polymorphic. The average percentage polymorphism was 36.37 %. The highest polymorphism (83.33 %) was observed with primer ISSR-B and least (42.85 %) was observed with primer ISSR-E. Results hence proved the importance of ISSR-A and ISSR-B primers in the analysis of intraspecific genetic diversity hence these primers can be further applied to observed genetic polymorphism between the populations of V. pilosa and V. canescens collected from other districts. newlineResults of cluster analysis between three wild Viola spp. (V. pilosa, V. canescens, V. kashmiriana) showed more similarity between the V. canescens and V. pilosa as compared to the V. pilosa and V. kashmiriana or V. canescens and V. kashmiriana. All these results showed that due to reproductive isolation, all Viola species are originated into different species and successfully adapted to their particular environment. Molecular analysis showed significant genetic diversity between the species, therefore, proves adaptation of the Viola species in their respective habitat. Non-significant genetic diversity among the population of V. pilosa and V. canescens proves the absence of cross breeding in Viola spp. newlineKeywords: Viola, Genetic diversity, Quantitative character, Qualitative character, ISSR newline