Download Investigation on relative genome sizes and ploidy levels of

Indian Journal of Biotechnology
Vol 9, January 2010, pp 64-68
Investigation on relative genome sizes and ploidy levels of Darjeeling-Himalayan
Rhododendron species using flow cytometer
Kalyan Kumar De, Aniruddha Saha1*, Ranju Tamang and Bilok Sharma
Post Graduate Department of Botany, Darjeeling Government College, Darjeeling 734 101, India
1
Department of Botany, University of North Bengal, PO-NBU, Darjeeling 735 013, India
Received 28 November 2008; revised 15 April 2009; accepted 2 July 2009
The relative 2C genome size (in pg), ploidy level (X) and total number of base pair (in Mbp) of ten threatened, rare and
endangered Indian Rhododendron species of Darjeeling Hills (eastern Himalaya) were determined by using flow cytometer. Out
of the 10 species, 9 were diploid and their genome sizes (2C) ranged from 1.30 to 1.51 pg. But in one species, i.e., R. niveum,
the value of genome size was significantly high, i.e., 4.27 pg, appeared to be a natural hexaploid (6X). The cytometric genomic
data indicating the hexaploid nature of R. niveum can be correlated with some phenotypic characters, specifically the size of
leaf, size of flower and number of flower in truss. The reported phenotypic character of diploid species of R. niveum reveals that
leaf size varies from 9-12 cm in length and 3.5-5.5 cm in breadth, individual flower size varies from 2-2.5 cm in length and
2-2.5 cm in diameter and number of flower per truss is 18-23 in diploid species. However, the findings on the same phenotypic
characters of hexaploid R. niveum in the present study showed some differences from the diploid one. The leaf size of hexaploid
R. niveum varies from 15-20 cm in length and 5-7 cm in breadth, individual flower size varies from 3.5-4 cm in length and
3.5 cm in diameter and number of flower per truss is 25-30. Therefore, these differences in floral traits and leaf size may be
direct result of polyploidization itself. Thus, the morphological traits and the genomic information of Rhododendron species
may serve as a valuable database for Indian Rhododendron breeders.
Keywords: Flow cytometer, genome size, Rhododendron
Introduction
The genus Rhododendron (Family: Ericaceae),
comprising of 85 species in India, is mainly distributed
in the Himalayan region. Out of this, a total of
36 species occur in Darjeeling and Sikkim Himalaya
alone1,2. This genus is one of the most neglected groups
of plants in terms of scientific inquiry in India. The
Rhododendron flowers show wide range of colour,
shape and size in their wild forms. The plants are
mostly shrub or tree with flowers actinomorphic,
bisexual, pentameraous and hypogynous; corolla
gamopetalous; stamens obdioplostemonous and
inserted on a nectar secreting disc, free and usually not
epipetalous, pollen in tetrads; ovary many, seeds small
with endosperm, and frequently roots are associated
with mycorrhiza. Being among the first to colonize
wasteland, the Rhododendron plants help to prevent
soil erosion and allow regeneration of vegetation. The
decoction of leaf or dried flower petals of
________________
*Author for correspondence:
Tel: 91-353-2776367; Fax: 91-353-2699001
Mobile: 09832372105
E-mail: [email protected]
Rhododendron spp. have several medicinal uses, such
as, for the treatment of rheumatism, checking diarrhea
and blood desentry, and dissolving fish bones struck in
the throat. The horticultural values of Rhododendron
spp. are internationally known. The Rhododendron
spp. are conducive to inter- and intra-generic crosses
and, therefore, open to hybridization. In the western
countries, plant breeders and horticulturists have
further worked to produce a range of hybrids that is
needed to fully convert the aesthetics of Rhododendron
spp. in commercial advantages.
In breeding programmes, the information on ploidy
level and relative genome size is very useful.
Polyploidy can occur in nature through many ways.
Most of Rhododendron spp. have been reported to be
diploid with 2n =2X =263. However, polyploids occur
naturally, which includes triploids, tetraploids,
hexaploids, octaploids and decaploids. Thus, the
natural occurrence of polyploids in Rhododendron
can plays an important role in its breeding
programmes4,5 as polyploidy can influence the
characteristic of growth vigour or ornamental value,
i.e, increase in flower size, number of flowers in truss,
fragrance quality, variation of colours, etc.
DE et al: GENOME SIZES OF RHODODENDRON SPECIES USING FLOW CYTOMETER
The chromosomes in Rhododendron spp. are
difficult to view and count because of their small
size6,7. Light microscopy is, therefore, not always
accurate method for determining the ploidy level.
However, flow cytometer can provide a fast and
accurate determination of nuclear DNA content
(genome size) that is related directly to ploidy
level8-10. Flow cytometry has been successfully used
to determine the relative DNA content and ploidy
levels of Rhododendron spp.11-18. The objective of the
present investigation is to determine the relative
genome sizes (2C) and ploidy levels of ten threatened,
rare and endangered Rhododendron spp.1,2 of
Darjeeling Hills (Lat 27° 2′57″N; Long 88°15′45″E)
by using flow cytometer.
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Materials and Methods
The young and fresh leaf samples (1-2.5 cm length
and 0.5-1 cm breadth) of ten Rhododendron species,
such as, R. decipiens Lacaita (threatened), R. falconeri
Hook.f. (threatened), R. fulgens Hook.f. (rare), R.
grande Wight (threatened), R. maddenii Hook.f.
(rare), R. niveum Hook.f. (endangered), R. pendulum
Hook.f. (rare), R. setosum D.Don (threatened), R.
sikkimense U.C. Pradhan & S.T. Lachungpa
(endangered) and R. triflorum Hook.f. (threatened),
were collected from different altitudal ranges of
Darjeeling Hills starting from Batasia (2247 m asl) to
Sandakphu (3580 m asl). The phenotypic differences
among the studied species were also noted (Table 1).
Approximately 1 cm2 of newly expanded leaf of each
Table 1—Showing the phenotypic differences among the studied species of Rhododendron
Species
R. decipiens Lacaita
Phenotypic characters
Trees 4-15 m high, bark not peeling; leaves very large; flowers rose pink to purple crimson, wide campanulate,
rose pink fading to almost white lobes; corolla obliquely bell shaped, swollen on the side; stamens 10.
R. falconeri Hook.f.
Trees 4-15 m high; leaves very large, mat green very rugose and covered with dense rusty tomentum beneath;
flowers white to creamy yellow (rarely pink); corolla obliquely bell shaped, swollen on the side; seed-capsule erect.
R. fulgens Hook.f.
Medium to tall shrubs, usually 2-3 m high; leaves long, variously shaped, felted beneath,
oval to elliptic-oblong, shiny green above and with dense rusty tomentum beneath; flowers in
shade of red, mauve or rose pink, with large blackish nectar pouches at the base.
R. grande Wight
Trees 4-15 m high; leaves very large, glossy green above and covered below with their silvery white indumentum;
flowers bell shaped, white to creamy yellow; corolla obliquely shaped, swollen on the side; seed-capsule curved.
R. maddenii Hook.f.
Epiphytic and lithophytic shrub, above 1 m high, young shoots not bristly; flowers one to several, not fleshy,
tubular-scented, companulate, white tinged with pink, other than lemon; corolla, rotate, funnel shaped;
stamens 15-20, filaments glabrous; seed-capsule ovate, woody and valves not recurved to their bases.
R. niveum Hook.f.
Small trees or tall shrubs, trees 4-15 m high; leaves opaque and dull green, leaf petiole and young shoots not
bristly, underside always covered with thin silvery white or fawn tomentum; flowers in rounded truss, blood red
or smoky-blue to purple-mauve; calyx 2-3 cm long; corolla not swollen on the lower side; seeds in capsule.
R. pendulum Hook.f.
Temperate to alpine shrub, over 1 m in height, epiphytic, lithophytic or terrestrial; young growths covered
with thick felt or wooly hairs; leaves oblong to oblong-elliptic, rugose and glabrous above and covered with
dense brown woolly hairs beneath; flowers one to several, white tinged with reddish-pink yellow, not
fragrant, rotate; corolla much larger, rotate; seed-capsule ovate, tubular to funnel shape or tubular
companulate, woody and valves not recurved to their bases.
R. setosum D.Don
Plants usually 0.6-1 m tall, non-aromatic shrublets, alpine and shrublets usually not acceding 1 m in height;
leaves and young branches bristly scaly; flowers few in each cluster, usually 5 or less, bright purple pink with
very open and spreading corolla lobes.
R. sikkimense
Pradhan &Lachungpa
Medium to tall shrubs, usually 2-3 m high; leaves long, variously shaped, oblong-ovate, beneath leathery and
thick matt texture, dull yellowish green above and glaucous green, and covered with thin layer of silvery,
yellowish-brown tomentum beneath; flowers in shade of red, mauve or rose pink, in trusses.
R. triflorum Hook.f.
Temperate to alpine shrubs, terrestrial with strongly aromatic shrublets; leaves green, glabrous above and
glaucous, covered densely with small uniform scales beneath; flowers 1-3, yellow, erect, azalea-like, broadly
funnel-shaped, not fleshy, light yellow spotted with green on inside dorsal.
INDIAN J BIOTECHNOL, JANUARY 2010
66
species was finely chopped with a razor blade in a
petridish with 500 µL of nuclei extraction buffer
(Cystain Ultraviolet Precise P Nuclei Extraction Buffer,
Partec, Munster, Germany). The solution was filtered
using Partec Cell Trics disposable filters with a pore size
of 50 µm to remove leaf tissue debris. Nuclei were
stained with 1.5 mL 4,6-diamidino-2 phenylindole
(DAPI) staining buffer (Cystain Ultraviolet Precise P
Nuclei Extraction Buffer, Partec, Munster, Germany)
and incubated for 1 to 2 min at 24oC.
Fig. 1—R. niveum showing flowers and close up view of a flower
(inset)
The suspension containing stained nuclei were
analyzed using a flow cytometer (Partec PA-1, Partec,
Munster, Germany) to determine relative DNA
fluorescence. Ploidy and genome size were
determined by comparing mean relative florescence
of each sample with the 2C peak of diploid and an
internal standard of known genome size. Pisum
sativum L. ‘Citrad’ with genome size of 9.09 pg19,13
was used as an internal standard to calculate nuclear
DNA content [2C DNA content of the
sample = 9.09 × (mean fluorescence value of
sample/mean fluorescence value of standard)]. The
total number of base pair present in 2C nuclear DNA
of each sample of Rhododendron was calculated. The
standard 1 pg of DNA contains 980 Mbp20. Therefore,
the total number of base pair present in 2C nuclear
DNA was calculated by multiplying the total DNA
content obtained for each sample in pg by 980 Mbp.
Results and Discussion
Flow cytometric results, in the present study,
revealed that out of the 10 species, 9 species were
diploid and their genome size (2C) ranged from 1.30
to 1.51 pg (Table 2). However, in one species, i.e., R.
niveum (Fig. 1), the value of genome size (4.27 pg)
was significantly high. The high value of genome
indicated R. niveum to be a natural hexaploid (6X).
The genome size of diploid and hexaploid values
obtained in the present study on Darjeeling
Himalayan Rhododendron spp. correlate with the
generalized value of earlier reports3,22. Earlier reports
suggest that genome size (2C) for diploid
Rhododendron spp. in general range from 1.3 to
1.9 pg, whereas the value of hexaploid ranges from
4.2 to 4.6 pg. On the basis of chromosomal study, R.
maddenii was found to be either hexaploid or
octaploid. However, flow cytometer study in the
present investigation did not tally with earlier reports.
Table 2— Relative genome sizes (2C) and estimated ploidy levels determined by flow cytometer in Rhododendron spp. of Darjeeling Hills
Species
Rhododendron decipiens Lacaita
R. falconeri Hook.f.
R. fulgens Hook.f.
R. grande Wight
R. maddenii Hook.f.
R. niveum Hook.f.
R. pendulam Hook.f.
R. setosum D. Don
R. sikkimense Pradhan & Lachungpa
R. triflorum Hook.f.
Relative genome size (in pg)
Estimated ploidy (X)
Total no. of base pairs (in Mbp)
1.57 ± 0.08
1.40 ± 0.10
1.51 ± 0.06
1.51 ±0.11
1.34 ± 0.09
4.27 ± 0.06
1.40 ± 0.11
1.30 ± 0.10
1.39 ± 0.08
1.33 ± 0.10
2X
2X
2X
2X
2X
6X
2X
2X
2X
2X
1538.6 ± 78.4
1372.0 ± 98.0
1479.8 ± 58.8
1479.8 ± 107.8
1313.2 ± 88.2
4184 ± 58.8
1372.0 ± 107.8
1274.0 ± 98.0
1362.2 ± 78.4
1303.2 ± 98.0
DE et al: GENOME SIZES OF RHODODENDRON SPECIES USING FLOW CYTOMETER
Our cytometric data suggests that R. maddenii of
Darjeeling Hills is diploid rather than polyploid. On
the other hand, the 2C genome size in R. niveum was
consistently higher (4.27 pg) than the other diploid
species in which the genome size ranged from 1.30 to
1.51 pg. Thus, R. niveum was found to be a hexaploid.
The results obtained by flow cytometry are contrary
to the previous reports which described R. niveum to
be diploid on the basis of chromosome study23. Such
differences were also reported in several other species
of Rhododendron, such as, R. occidentale and R.
flammeum, based on the sampling of taxa from
diverse sources and geographical origins. Therefore,
we suggest that different ploidy levels may exist
under the same species.
In the present investigation, the cytometric
genomic data indicating the hexaploid nature of R.
niveum can be correlated with some phenotypic
characters of the species, specifically the size of
leaf, size of flower and number of flower in truss.
Literature survey24 on phenotypic character of
diploid species of R. niveum reveals that leaf size
varies from 9-12 cm in length and 3.5-5.5 cm in
breadth, individual flower size varies from 2-2.5 cm
in length and 2-2.5 cm in diameter and number of
flower per truss is 18-23. However, hexaploid R.
niveum, in the present study, showed some
differences in phenotypic characters from the
diploid one. The leaf size of hexaploid R. niveum
varies from 15-20 cm in length and 5-7 cm in
breadth, individual flower size varies from 3.5-4 cm
in length and 3.5 cm in diameter and number of
flower per truss is 25-30. These differences in floral
traits and leaf size may be direct result of
polyploidization.
Thus, the present study provides insights into
genetics, molecular evolution and reproductive
biology of Indian Rhododendron, which may serve as
a valuable database for Rhododendron breeding
programmes.
Acknowledgement
Authors appreciate the excellent technical
assistance provided by Professors Thomas G Ranney
and Jeff R Jones of the Mountain Horticultural Crops
Research Centre, North Carolina State University,
North Carolina, USA. They are also grateful to the
Department of Biotechnology, Government of West
Bengal,
Kolkata
for
financial
assistance
(Grant No. 105/JS-BT/07 dated 19.03.08).
67
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