Uses of Random Amplified Polymorphic DNA (RAPD) Markers in the Altitudinal
Diversity of Plagiochasma appendicualtum
Random amplified polymorphic DNA markers were used to determine the altitudinal
variation within and between Plagiochasma appendicualtum collected from
different altitude of Western Himalaya especially from Mussoorie region of India.
Findings of UPGMA cluster analysis and band frequency of all the nine accessions
were separated according to their altitudes supporting to their morphological
differences as well. Gene flow and spore dispersal plays an important role in
the polymorphism. Gene flow within P. appendiculatum growing on same
altitude is very high as compared to accessions collected from different gradient
of altitudes i.e., the genotypes collected from same altitude showing not so
much polymorphism compared to different altitude. It has been concluded that
the RAPD markers would be useful to characterize the altitudinal variation between
different accessions of P. appendiculatum and may be also valuable to
other bryophytes collected from various environmental condition.
Received: November 27, 2013;
Accepted: December 21, 2013;
Published: February 27, 2014
Bryophytes are the diverse group of land plants after the flowering plants
(Mishler, 2001) but due to complexity in their identification
and lack of literature from tropical areas, they have rarely been included in
biodiversity analysis (Pharo et al., 1999).
They are found from the tropics to the polar regions, from sea level to mountain
summits and are principle candidates for latitudinal and altitudinal revision
(Andrew et al., 2003). Several evocative studies
of bryophytes and their altitudinal zonation have been reported in South America,
in Puerto Rico along a transect of 200-1075 m by Fulford
et al. (1971), in the Sierra Nevada de Santa Marta in Colombia (Van
Reenen and Gradstein, 1983, 1984). Also in Northeastern
Peru (Gradstein and Frahm, 1987), Bolivia, Peru and
Columbia (Kessler, 2000), on Mt. Kinabulu, in the eastern
part of Borneo (Frahm, 1990) and in New Zealand (Frahm
and Ohlemuller, 2001; Pfeiffer, 2003) as well as
in Africa on Mount Kilimanjaro (Pocs, 1994).
Mussoorie is a city about 30 km from Dehradun located in Dehradun district,
Indian state of Uttarakhand. This hill station, situated in the foothills of
the western Himalaya ranges, is also known as the Queen of the Hills. Mussoorie,
with its green hills and varied flora and fauna, is notable for its unique geographical
location with varied topography and associated altitudinal diversity supporting
unique assemblage of biodiversity. Except for the coast and backwaters, almost
all habitats in Western Himalaya, from low altitude to the high altitude grasslands
and shoal forests, occur in Mussoorie, which forms the major part of the Himalaya
Plagiochasma appendiculatum is one of the important liverwort belongs
to the order Marchantiales under family Aytoniaceae. Lehman
and Lindenberg (1832) first described the species. Plagiochasma is
a thalloid liverwort represented by 30 species (Bischler,
1978), but in India only 10 species have been reported, viz., Plagiochasma
appendiculatum Lehm. et Lindb, Plagiochasma articulatum Kash., Plagiochasma
bicornutum Steph., Plagiochasma cordatum Lehm. et Lindb, Plagiochasma
cordotii Steph., Plagiochasma intermedium L. et Gott., Plagiochasma
martensii Steph., Plagiochasma nepalensis Steph., Plagiochasma
pauriana Udar et Chandra and Plagiochasma quadricornutum Steph. (Parihar
et al., 1994). Out of these taxa P. appendiculatum abundantly
grows in Mussoorie and also on other parts of India as well.
It is a monoecious plant and usually grows in moist places, on rocks surface,
soil covered rocks, walls of old buildings and show extra ordinary regeneration
(Mahabale and Bhate, 1945). Besides this, P. appendiculatum
also represents the maximum xerophytic habitat and can grow on comparatively
naked and exposed rocks (Kachroo, 1954). Ghate
and Chaphekar (2000) proved that this taxon could be used as a biotest for
water quality assessment. P. appendiculatum is significant taxon which
possesses antimicrobial property. Banerjee (2000), Kumar
et al. (2000) and Singh et al. (2006)
stated that in India, it is used by Gaddi tribes in Himachal Pradesh for the
treatment of cuts, wounds and burns. Genetic variation of P. appendicualtum
collected from different geographical conditions have been reported by using
RAPD markers (Soni et al., 2009). Under the same
study, RAPD markers were also stated the genetic diversity of P. appendicualtum
within and between populations.
Random amplified polymorphic DNA markers (RAPD) can be widely used as DNA fingerprinting
techniques (Williams et al., 1990, 1993)
that has been utilized in bryophytes to survey population genetic structural,
dispersal of spores, phylogeographic patterns and species relationship (Skotnicki
et al., 2000; Scotnicki et al., 2001;
Freitas and Brehm, 2001; Boisselier-Dubayle
and Bischler, 1994; Boisselier-Dubayle et al.,
1995). There were several reports related to RAPD genetic variation of various
species of Antarctic moss (Selkirk et al., 1997;
Skotnicki et al., 1997, 1998a,
P. appendiculatum is widely distributed in western, eastern Himalayas,
central India and south India and generally growing upto an altitude of 8000
ft from sea level. This species in known from the east part of the central and
south African continent Eritrea, Ethiopia, Kenya, Tanzania to Rhodesia, Zimbabwe
and South Africa (Perold, 1999; Wigginton,
2002). In Asia, it is widespread ranging from the southwest of the Arabian
Peninsula and Socotra Islant (Frey and Kurschner, 1988)
to the southern part of the Himalayas, Formosa, Philippines and Celebes (Bischler,
1979). Due to occurrence of such variable altitudinal range, present study
was planned out to determine altitudinal variation in P. appendiculatum
through random amplified polymorphic DNA marker.
MATERIALS AND METHODS
Field sampling: The samples of P. appendiculatum were collected
from various localities of Mussoorie e.g., Library road, Camels
back road, Kempty fall, Wood stock collage, Lal-Tibbs and Company garden (Fig.
1). Voucher specimens have been deposited at NBRI Bryophyte Herbarium, Lucknow,
India (Table 1). Each specimen has been identified by their
characteristic features through literature and authentic specimens available
in the NBRI Herbarium.
Plant identification: Thalli of Plagiochasma appendiculatum is
characterized by large, purplish green patches, thick, 20 mm long and 5 mm wide,
dichotomously branched and occasionally with adventitious shoots.
||Collection sites of P. appendiculatum from various
altitude of Mussoriee (Western Himalaya) India
|| Genotype collection of P. appendiculatum from various
localities of mussoorie, growing at different altitude
Lobes oblong, dorsal surface smooth margins undulate. Midrib distinct passing
into lamina, ventral surface purple. Scales in one row on each side of the midrib
purple, broadly lunate, body with 1 or 2 appendages reaches half way to the
margins, appendages large, usually hyaline, fan shaped, entire and occasionally
purple. Male receptacle horse-shoe shaped sometimes scattered on the thallus
or in acropetal order. Female receptacle sessile or stalked, usually with 2-5
lobes, situated on the thallus in row or scattered or some time at the basal
part of the plant. Spores yellowish brown and elaters bispirate. This species
is mainly separated from the other species on the basis of its scale structure
that has typical from shaped on broad lunate appendages (Table
||Morphological data of Plagiochasma appendiculatum collected
from geographical localities of Mussoorie (India)
Plant DNA preparation: Fresh and matured thallii of P. appendiculatum
were used to isolate the genomic DNA. All samples were carefully washed
and checked under the microscope to be sure that no possible contaminations
(microalgae, fungi) were left. DNA was extracted from 1 g of plant material
with some modification of several standard protocols (Doyle
and Doyle, 1990; Soni and Kumar, 2009). Fresh material
was crushed in liquid nitrogen and mixed with modified CTAB extraction buffer.
Add 1% α-mercaptoethanol, 2% proteinase K (20 mg mL-1) and 100
mg PVP (Sigma) and then incubated in a water bath at 68°C for 5 h, add Chloroform:
Isoamyl alcohol (24:1) to this mixture, followed by centrifugation for 10 min
at 12,000 rpm. The supernatant was transferred to a corex tube, add 0.7 Vol.
iso-propanol, mixed well, store in -20°C for 5 h. to precipitate the DNA.
Centrifuge 10 min at 12,000 rpm. Supernatant was discarded and the pellet washed
with a solution of 70% ethanol. Tubes were incubated at 37°C for 15 min
and resuspended the pellet in TE buffer. Purify the DNA followed by phenol/chloroform
method, then precipitate and washing as before. Finally, the aqueous phase was
discarded and the pellet was dried for 15 min in incubator at 37°C. Resuspended
the pellet in 100 μL TE buffer. This procedure recovered at least 800 ng
μL-1 genomic DNA which is good quality for RAPD analysis.
Primers and PCR: Decamer primers from OPERON Technologies (USA) were
screened on individual representative of the populations under study. Many of
the primer produced either complex banding pattern of non-reproducible and inconsistent
amplification products. Hence, only 45 primers scored good result out of 60
primers used for the subsequent analysis (Table 3). Reproducibility
of bands was assessed by replicating extraction of DNA and amplifications of
selected samples. Polymorase Chain Reaction (PCR) was carried out in 20 μL
volumes using DNA, dNTPs (2 mM of each of four nucleotides: Fermentas), 10X
Taq Buffer, 5 pmol primer, 1 unit Taq DNA polymerase (Bangalore Genei). PCR
conditions were initiated at 92°C followed by 44 cycles of denaturation
at 92°C for 1 min, annealing at 36°C for 1 min and extension at 72°C
for 1:30 min, followed by the final extension of 5 min. Amplified products were
separated in 1.4% agarose gel, stained with ethidium bromide and visualized
under ultraviolet (UV) light. The image of gel was taken by multiimager TM 3400
(Alpha Innotech Co.).
Data analysis: RAPD profiles were scored for each individual as discrete
characters (presence or absence of amplified products) across all individuals
from all populations and for each primer used. Relationships among and between
populations were evaluated via the unweighted pair-group method UPGMA (Sneath
and Sokal, 1973) and all analysis was performed by using NTSYS (Rohlf
et al., 1990). Jaccards coefficient was calculated by using
FREETREE program, a common estimator of genetic identity and was calculated
where, NAB is the number of bands shared by samples, NA
represents fragments in sample B. Similarity matrices based on these indices
were calculated. Similarity matrices were utilized to construct the UPGMA (Unweighted
pair group method to construct arithmetic average) dendrogram. Statistical stability
of the branches in the cluster was estimated by bootstrap analysis with 1,000
replicates, using the winboot software program (Yap and Nelson,
Altitudinal variation: Altitudinal variation was found in the genotypes
of P. appendiculatum. Genotypes M3, M4 and M5
and M7, M8 and M9 were collected from 4000
and 6000 ft, respectively. Similarly, genotypes M1 (7000ft), M6
(6800 ft) and M2 (6000 ft) showed variation related to their altitude.
Thus, these genotypes clearly separated on the basis of their collection site
(Fig. 2a) and by band frequency (Fig. 2b)
obtained by amplified bands.
RAPD analysis: Genomic DNA amplification of 9 accessions of P. appendiculatum
were carried out by using 30 random primers out of which 27 primers yielded
875 reproducible fragments and rest of the 3 primers were not given the amplification
(Fig. 3). All the chosen primers amplified across the 9 accessions,
with the number of amplified fragments ranging from eighteen (OPA-13) to fourty
(OPD-08 and OPAP-03) which varied from 300 to 3500 bp. Out of the 875 amplified
bands, 863 were polymorphic, with an average of 29 polymorphic fragments per
primer and rest of the 12 fragments were monomorphic (Fig. 3).
Percentage of polymorphism ranged from 93.5% (OPD-07) to a maximum 100% with
an average of 98.5% polymorphism (Table 3).
The Polymorphism Information Content (PIC) obtained by random amplified bands
were obtained with an average of 0.228, ranged from 0.000 to 0.62. Primers,
OPA-04, OPA-07, OPA-15, OPA-16, OPA-17, OPA-19 and OPD-10 gave highest PIC values
Cluster analysis: Dendrogram based on UPGMA cluster analysis all the
9 accessions of P. appendiculatum were clearly separated according to
their altitudinal sites. Genotypes MS1 and MS6 formed separate OUTs from other
genotypes according to their high altitude (Fig. 4). However,
MS1 and MS6 genotypes appeared to be closer to each other with similarity coefficient
of 1.000 and 1.149, respectively (Table 4). Group I consist
of genotypes MS3, MS4 and MS5, in which MS3 appeared to closer to M4 with similarity
coefficient of 0.511 and genotype MS5 make separate out group with less difference
similarity coefficient of 0.550. Similarly, genotype MS2 formed separate out
group but appeared close to Group I. In the same manner, Group II consist MS7,
MS8 and MS9 genotypes out of which MS7 and MD8 appeared close to each other
with similarity coefficient of 0.419 and MS9 formed a separate out group but
close to MS7 with similarity coefficient of 0.493. Therefore, all the genotypes
are separated in relation to their collection site and altitude.
||Graph representing the (a) Altitudinal variation and (b) Band
frequency obtained by amplified fragments
||RAPD profile of P. appendiculatum collected from different
altitude of Mussoorie. Lane M: EcoRI and Hind III double digested marker.
Lane 1: P. appendicualtum collected at 7000 ft; Lane 2: at 6000ft
; Lane 3-5: at 4000ft; Lane 6: at 6800ft; Lane 7-9: at 6400 ft
||UPGMA based Dendrogram showing the altitudinal variation and
genetic relation ship between nine accessions of P. appendiculatum
collected from various altitude of Mussoorie
|| Sequences, bands, fingerprints and calculated parameters
for the 30 RAPD primer used in Plagiochasma appendiculatum
|| Jaccard's similarity coefficient matrix followed by UPGMA
analysis of P. appendiculatum based on RAPD markers
The RAPD marker is simple and reproducible technique that allowing comparison
of genetic variation between wide range of bryophytes (Boisselier-Dubayle
and Bischler, 1989; Bischler-Causse and Boisselier-Dubayle,
1991). Bopp and Capesius (1996) reported that RAPD
is used for identification and gene sequencing of bryophytes. However, other
methods, such as isozymes and microsatellite have been reported to detect colonization
and dispersal in many bryophytes. (Cronberg and Natcheva,
2002; Fang et al., 1997; Stenoien
and Sastad, 1999; Rumsey et al., 2001).
The results obtained from the RAPD analysis indicated significant altitudinal
variation among the P. appendiculatum, as collected from their different
growing site (Fig. 3). UPGMA based dendrogram obtained by
RAPD analysis indicated that the genotypes of Group I viz., MS3, MS4 and MS5
comes closer to each other because of short geographical distance and different
altitudinal range. On the other hand, similar result has been found within genotypes
of Group II i. e., MS7, MS8, MS9. Similarly, genotype MS1 and MS6 are separated
according to their higher altitude i.e., 7000 and 6800 ft, respectively (Fig.
4). Soni et al. (2009) deescribed the morphological
and genetic variation within and between the genotypes of P. appendiculatum
due to growing at different altitude and habitat. These variation is due to
the gene flow within closely and far located genotypes. Low gene flow may be
one of the major cause of altitudinal variation in P. appendiculatum
because high gene rate flow within closly growing individuals occurs by environmental
factors indicating not so much vatiation. It means that gene flow takes place
from higher to lower altitude and it has been reported that water is essential
for the sexual reproduction by mean of dispersal of spores (Wyatt
and Anderson, 1984). Restricted gene flow can generally promote local adaptation
and genetic divergence between different microhabitats (Via
and Lande, 1985). The magnitude of gene flow between the habitats in the
current study is difficult to assess. Gamete dispersal distances are considered
highly restricted in bryophytes (Wyatt and Anderson, 1984).
Spore dispersal distances are probably orders of magnitude higher than gamete
There is an important role of substrate in bryophyte species diversity and
composition has been well established (Pharo and Beattie,
2002), but little is known about the effects of microhabitat and altitude
in bryophytes (Romero et al., 2006). Andrew
et al. (2003) did not find an overall pattern of bryophyte diversity
on different mountains in Tasmania and New Zealand. They considered that altitudinal
gradient may control community structure and diversity but suggested that factors
operating at smaller scales (moisture, microhabitats) should be studied to understand
the underlying mechanisms.
Water plays important role in the spreading of bryophyte propagules from higher
to lower altitude along short drainage channels indicating water dispersal where
at mixing of geographically divergent populations divided by numerous kilometers
and snow confirmed that wind also is imperative for long-range dispersal (Skotnicki
et al., 1999). Gene flow in bryophyte is caused during asexual reproduction
by dispersal of sperm and spores. All the views of sperm dispersal in mosses
and liverworts have reached to the same conclusion: sperm dispersal is very
short (Wyatt and Anderson, 1984). Even in large species
with splash cups, only rarely do sperms get dispersal more than 50 cm in species
without splash cups, fertilization typically occurs within a radius of 10 cm.
In the same manner P. appendiculatum growing on short distance having
high gene flow so less amount of genetic variation that is why genotypes collected
from same altitude comes closer to each other. It is therefore concluded that
the RAPD markers may be beneficial for the revealing of altitudinal variation
among the genotypes of P. appendiculatum collected from various environmental
The authors are grateful to Director, National Botanical Research Institute,
Lucknow India for encouragement and providing facilities and to Department of
Biotechnology (DBT), New Delhi for financial support.
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