Evolutionary Trend in Passiflora Species Revealed by RAPD-PCR, Gene Specific ITS and rbcl Markers

Abstract

Background: The present study represents a preliminary analysis of genetic diversity among Passiflora species using amplified genotypic data of specific ITS and rbcL sequences and non-specific RAPD-PCR markers for investigation of the molecular phylogeny. Methods: The PCR-RAPD uses ten primers for polymorphic DNA, which are compiled on NTSYS software to construct dendrogram.  The gene specific ITS and rbcL primers are used for specific amplification from genomic. The amplified ITS and rbcL markers assembled using Maximum Parsimony (MP) and Maximum likelihood (ML) methods. The BLAST, CLUSTAL W, and MEGA 6.0 have been used to conclude final genetic relation tree. Results: The PCR-RAPD primers translate 133 random amplified polymorphic DNA. NTSYS dendrogram placed P. vitifolia from Ramdaspeth and Shankar Nagar Nagpur, India in same clade (similarity coefficient 0.609) confirming same origin  Nagpur India. Moreover, P. foetida from England is not coming in same clade with Indian P. foetida showing geographically intra-specific variation. In addition, the change in a constructed tree was observed with respect to change in phylogeny methods MS/ML. The ITS MP consensus tree is supported by strong 100 bootstrap value, clusters P. vitifolia (HNI and RNI) and P. foetida (UAI and UGI) in equivalent clade. However, no single species have been recovered using rbcL in MP and ML method. Thus, it is inference that rbcL have tendency to differentiate Passiflora species not allowing clustering around same species in same clade and the ITS region having parsimony informative sites that provide valid resolution and identification at inter-intra species level. Conclusions: The evaluation of properties of RAPD indicates 100% PCR success, sometimes with low rate of amplification. The ITS region found to be best for identification at inter-intra species, On the contrary, rbcL region is good to distinguished inter species, making it best as local DNA barcode for marking a Passiflora species in phylogenetic community.