MOLECULAR PHYLOGENETIC RELATIONSHIP AMONG ENDEMIC BALSMS OF WESTERN GHATS USING CHLOROPLAST atpB-rbcL SPACER SEQUENCES

Shajitha PP1, Danesh NR1, Jomy Augustine2, Linu Mathew1,* 1 School of Biosciences, M.G. University, Kottayam, Kerala, India, 686560 2 Department of Botany, St. Thomas college, Pala, Kerala, India *corresponding author, Email ID linumathew@mgu.ac.in, Contact number: 9447505690 INTRODUCTION Balsaminaceae are annual or perennial herbs with flowers that exhibit a remarkable diversity. The family consists of more than 1,000 species (Grey-Wilson 1980a; Clifton 2000), but only two genera are recognized. Impatiens is a very large genus of over 1,000 species, while Hydrocera triflora is the only member of the genus Hydrocera. Impatiens usually has three free sepals and always five petals, four of which are united into two lateral pairs (Steven et al.2006). The genus Impatiens contains over 210 species in India, more than half of which occur in the Western Ghats in South India. At least 103 species of Impatiens are endemic to the Western Ghats including the new species (Bhaskar., 2012) . Some Impatiens species are of horticultural importance, e.g., the popularly grown flowers, the Balsam, I. balsamina, the Busy Lizzie, I. walleriana, and the New Guinea Hybrid Impatiens, I. hawkeri. Impatiens is a well-known example of a taxonomically difficult group (Hooker et al., 1859; Grey Wilson, 1980b). Here we conducted molecular phylogenetic study with chloroplast atpB-rbcL sequence data and propose a preliminary reconstruction of the intrageneric relationships in Impatiens. Key Words:Impatiens, chloroplast atpB-rbcL spacer, N-J Analysis, PCR

MATERIALS AND METHODS Taxon Sampling and out group choice We sampled genus Impatiens of family Balsaminacea. Twenty two species of Impatiens were collected from Munnar,Vagamon hills and Wayanad and identified by Dr.Jomy Augustine, Taxonomist, St.Thomas college Pala, Kerala, India. We took sequences of Hydrocera triflora

(DQ 147896.1), Marcgravia maguirei (DQ 147896.1), Marcgravia umbeiiata(DQ 147897.1), Impatiens campanulata (DQ 147822.1) as out group from GENBANK. Molecular Protocols DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted using plant DNA isolation kit (Sigma Aldrich, St. Louis, USA). Quality and quantity of DNA was estimated by Agarose Gel Electrophoresis (0.8%). Specific primers for the atpB-rbcL spacer in Balsaminaceae were synthesized. IMP-atpB (5-ACATCTAGTACCGGACCAATGA-3) and IMP-rbcL (5-AACACCAGCTTTGAATCCAA3) (Steven et al.2006). 25l PCR reaction was set by mixing 1 l template DNA (25ng), 0.5 l each of forward and reverse primer (10M), 2.5 l of Taq buffer, 0.6 l MgCl2 (25M), 0.5 l of dNTP mix (10M) and 0.5 l of Taq DNA polymerase (3U/l) and volume made with milliQ water. PCR conditions were as follows 3min. initial denaturation at 95oC and 35 cycles of 30 sec.denaturation at 95oC, 30 sec. primer annealing at 55oC and 30sec.extention at 72oC with a final extension of 72oC for 10 minutes. (Agilent sure cycler 8800, Agilent Technologies). The PCR product was visualized by 1.5% AGE with 100 bp ladder.The PCR product directly sequenced (Scigenome Labs Pvt Ltd., Cochin). Sequencing of amplicon with forward and reversed primers was carried out in ABI 3730xl cycle sequencer. Sequence alignment The obtained atpB-rbcL sequences were initially aligned with Clustal W applying the default parameters and edited with Bio Edit 7.0.0.(Hall,1984) Neighbor-joining (NJ) analysis A distance analysis applying NJ optimality criteria (Saitou & Nei, 1987) was conducted on reduced data matrices by using MEGA Version 5.05 (Tamura K. et al., 2011). Bootstrap values were obtained from 1000 replicate NJ analysis. RESULS AND DISCUSSION The length of the unaligned atpB- rbcL fragments of the ingroup taxa range from 7001032bp. The full atpB-rbcL data set including out group and ingroup taxa consisted 848 characters. Out of 226 were variable 128 were parsimony informative 95 were single tons. Percentage of T, C and A were 36.1, 13.3, 35.7 respectively and AG dinucleotide were 15 percentage.

Phylogeny inferred from atpB- rbcL region of South Indian Impatiens Sister genus Hydrocera formed a separate clade with 100% boot strap support showing the monophyly of Impatiens. It was found that Impatiens goughi is closely related to Impatiens viscosa with boot strap of 96%. This clade showed relation to I. viscida. I. gughi, I. viscosa and I. viscida formed a clade with boot strap of 98%.Similarly I. uncinata and I. cordata formed a clade with boot strap support of 94%. I. wightianae and I. muculata formed a clade with boot strap of 98%. I. dasysperma and I. flaccida formed a single clade with boot strap of 87%. These two species shows very similar morphological characters. The molecular analysis also shows the close relation of these two species. I. scabriusculata and I. mysorense formed a clade with boot strap of 88% and I. leucida and I. gardneria formed a clade with boot strap of 86%.I. herbicola shows a close similarity to I .ligulata with boot strap of 100%. It is observed that the traditional taxonomy based on morphology was reproduced also in the atpB-rbcL based phylogeny. The relatedness of I. dasysperma and I. flaccida requires further investigation using additional sequence information namely ITS, matK, trnL-trnF spacers to unequivocally proove the taxonomic status of these two species of Impatiens.

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