Poster Impatiens Shajitha
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Transcript of Poster Impatiens Shajitha
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 [email protected], 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-AACACCAGCTTTGAATCCAA-
3) (Steven et al.2006). 25µl PCR reaction was set by mixing 1 µl template DNA (25ng), 0.5 µl
each of forward and reverse primer (10µM), 2.5 µl of Taq buffer, 0.6 µl MgCl2 (25µM), 0.5 µl of
dNTP mix (10µM) and 0.5 µl of Taq DNA polymerase (3U/µl) 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 700-
1032bp. 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.
Baskar V.Taxonomic monograph on Impatiens L. (Balsaminaceae) of Western Ghats,South India
The key Genus for Endemism(2012),Centre for Plant Taxonomic Studies, Bangalore.
Clifton,R. 2000. Geraniales species checklist series: vol. 6. Geraniaceae Bentham and Hooker,
tribe VII Balsamineae. Dover: The Geraniaceae Group.
Grey Wilson,C. 1980a. Impatiens of Africa. Rotterdam: Balkema. 1980 b. Hydrocera triflora, its
floral morphology and relationship with Impatiens. Studies in Balsaminaceae. Kew Bulletin 34:
221–227.
Grey Wilson,C. 1980b. Impatiens of Africa. A. A. Balkema, Rotterdam 1980b. Hydrocera
triflora, its floral morphology and relationship with Impatiens. Studies in Balsaminaceae. Kew
Bulletin 34: 221–227.
Hooker, J. D. and Thompson,T.1859. Praecursores ad floram Indicam. Balsaminaceae. Bot. J.
Linn. Soc. 4: 106–157.
REFERENCES
Steven Janssens, Koen Geuten, Yong-Ming Yuan, Yi Song, Philippe Kupfer, Erik Smets.
Phylogenetics if Impatiens and Hydrocera(Balsaminaceae) using Chloroplast atpB-rbcL Spacer
Sequences. Systematic Botany(2006). 31(1):171-180.