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Aquaculture 318 (2011) 389396

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Growth and agarose characteristics of isomorphic gametophyte (male and female)

and sporophyte of Gracilaria dura and their marker assisted selection
Vishal Gupta, Ravi S. Baghel, Manoj Kumar, Puja Kumari, Vaibhav A. Mantri, C.R.K. Reddy , Bhavnath Jha
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Bhavnagar 364021, India

a r t i c l e

i n f o

Article history:
Received 7 February 2011
Received in revised form 4 June 2011
Accepted 7 June 2011
Available online 15 June 2011
Keywords:
Agarose
Chromosome
Gracilaria dura
Marker assisted selection
Life cycle stage

a b s t r a c t
The characteristics of agarose and growth for three isomorphic life phases of G. dura with their bio-molecular
marker assisted selection have been described in this study. The tetrasporophyte showed superior quality of
agarose over gametophytes and recorded growth rate was highest for females. The genetic relatedness
studied with ISSR markers showed quadratic line of correlation between these phases (R2 = 1). Their genetic
diversity determinants as percentage of polymorphic loci (PPL), average heterozygosity (He) and Shannon's
Weaver index (I) were 55.55%, 0.5 0.07 and 0.33 respectively. The cytological analysis for chromosome
count revealed 8 chromosomes in haploid gametophytic thallus (N) and 16 for diploid tetrasporophyte (2N)
together with genetic structure analysis conrmed to their sexual mating behaviour. Their marker assisted
selection based on ISSR generated characteristic band of 430 bp specic to male, 860 bp for female and two
bands of 800 and 1600 bp for tetrasporophytic thallus from primer A. Similarly ISSR primer E also generated
bands specic to male, female and tetrasporophytes while others gave bands specic to either of life phase.
Interestingly, endogenic ABA content was signicantly higher for haploid gametophytes (female more than
male) than diploid tetrasporophyte while no signicant difference was observed in IBA content. Thus the
study described not only the features of three life phases of G. dura but also reliable biomarkers for
differentiating such isomorphic life phases which could be benecial for the selection of cultivar and in
breeding programmes.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Among the red algae, genus Gracilaria constitutes important
agarophytes with more than 150 species reported across the world
(Byrne et al., 2002). The development of improved processing
technologies for agar extraction has increased the annual harvest
rate of this genus. The agar industries throughout the world consume
over 72,300 dry tonnes of agarophytes annually to produce ca.
9600 tonnes of agar worth of US$173 million (Bixler and Porse,
2011). Of this, Gracilaria alone accounts for about 80% of the world's
agar market with a value of US$ 138 million (Bixler and Porse, 2011).
The increasing demand for agar worldwide coupled with short
supplies of raw material from wild stocks has led to the development
of viable methods for their large scale cultivation in the sea (Peng et al.,
2009). The cultivation of Gracilaria for commercial purposes is being
carried out in several countries including Chile, China and Taiwan and
on pilot scale in Namibia, Venezuela, Mexico and India.
Gracilaria dura (C. Agardh) J. Agardh from the Indian waters has
been reported to produce superior quality agarose (1%
gel N 1900 g cm 2) suitable for biotechnological applications (Meena
Corresponding authors. Tel.: + 91 278 256 5801/256 3805x614; fax: + 91 278 256
6970/256 7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0044-8486/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2011.06.009

et al., 2007). Like other tropical seaweeds the limited distribution

coupled with short lifespan prevented its commercial utilisation thus
underlie the need for their cultivation. Further, selection of cultivars
or life phases with superior traits of growth and phycocolloid will
ascertain the economical viability of resources.
The genus Gracilaria has a characteristic Polysiphonia type life cycle
with an alternation of isomorphic gametopyhtic (male and female)
and tetrasporophytic generations (Engel et al., 2004). Moreover,
isomorphic life phases with high resemblances in their morphological
features have frequently misled the identication. Thus, segregation
of life phases at early stages of their development is desired for
cultivar selection, breeding and other biotechnological interventions
aimed at genetic improvement.
In higher plants, several physiological processes are controlled
with synchronised hormonal signals (Stirk et al., 2009). The reported
implication of plant growth regulators (PGRs) seems to be very
promising since some of these compounds can be used as potential
markers for segregating the isomorphic life phases of species such as
G. dura. Along with the PGRs, the adoption of molecular sex-linked
markers offers additional tools for differentiation of life phases of
species even at their juvenile stages dispensing the long wait till the
development of reproductive bodies (Sim et al., 2007).
The RAPD based molecular markers have been extensively
employed for determination of sex in higher plants (Chaves-Bedoya

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V. Gupta et al. / Aquaculture 318 (2011) 389396

and Nuez, 2007). Such studies are sparsely investigated for

seaweeds. Sex and phase linked markers have been evaluated in
Gracilaria lemaneiformis (Bory de Saint-Vincent) Greville using
different PCR-RAPD universal operon primers (Li et al., 1998).
Martinez et al. (1999) determined the markers specic to male and
female thalli of Gracilaria gracilis (Stackhouse) M. Steentoft employing
RAPD. Similarly, in Gracilaria changii (Xia et Abbott) Abbott, Zhang et
Xia, RAPD marker has been identied for differentiating the males
from female and tetrasporophytes in natural populations (Sim et al.,
2007). Most recently, SCAR marker has been generated specic to
female gametophyte of brown alga Saccharina japonica (Liu et al.,
2009). Also, these studies provide baseline information for the
delineation of life phases, identication of sex specic markers and
their inter-phasic genetic structure. Inter simple sequence repeat
(ISSR) exploits the abundance of highly variable SSR sites to generate
a complex banding prole with consistent reproducibility, reliability
and dominancy and hence make this markers superior to RAPD
(Ammiraju et al., 2001; Souframanien and Gopalkrishnan, 2004). The
genetic diversity among the sexually reproducing population determined by dominant markers (ISSR) depends on the frequency of
homologous alleles (Andreakis et al., 2009).
The present study demonstrated the potential of three life phases
of G. dura for agarose yield and gel strength along with their
respective growth rates. The genetic proling of three life phases
was also carried out using ISSR technique. Further, the ISSR loci were
assessed for marker assisted selection of different life phases. Also the
distinctness of three isomorphic life phases was conrmed with their
chromosome count.
2. Materials and methods
2.1. Sample collection and determination of agarose yield and gel
strength
The biomass of G. dura was collected from Veraval (latitude 20.55
N and longitude 70.20 E) along the West coast of India during low tide
in March 2008. The plants were immediately transported to the
laboratory in cool packs under low temperature. The collected
biomass was segregated according to the life cycle phase upon critical
observations of either morphological (cystocarps in case of female) or
anatomical structures (spermatangial conceptacles in case of male
and cruciate tetraspores in case of tetrasporophyte) (Fig. 1). The
native agarose was prepared from three life stages following the
protocol described by Meena et al. (2007). Briey, dry G. dura samples
were soaked in tap water for 1 h at room temperature and then
treated with 10% aqueous NaOH at 85 C for 2 h, washed with water to
remove excess alkali. The residue was then autoclaved in water. The
extract thus obtained was vacuum ltered over a Celite bed. Filtrate
was thereafter squeezed to obtain agarose which was then dried and
ground. Yield of agarose was calculated on the dry weight basis of
seaweed containing nil moisture. The gel strength measurements

were done on a Gel Tester (Kiya Seisakusho, Ltd., Tokyo, Japan).

Intrinsic viscosities [] were determined at 32 C using an Ostwald
viscometer (Prasad et al. 2005). The 13C spectra were recorded for the
extracted agarose at 80 C on Bruker-Avance II 500 at 125 MHz. The
spectra were acquired for 2% agarose in D2O. 13C chemical shifts were
referenced to internal standard DMSO (39.4 ppm). The peaks were
assigned to the polymeric structural units as that of the convention of
Freile-Pelegrn and Murano (2005).
2.2. Growth experiment
Since signicant differences were recorded in agarose properties,
the gametophyte (male and female) and tetrasporophyte plants of G.
dura were then evaluated for their growth potential. The ephemeral
distribution of this species resulted in segregation of cultures as three
males, nineteen females and seven tetrasporophytes which were
assigned with accession numbers (Table 2) and maintained in unialgal
culture following the method described by Mantri et al. (2009). The
accessions were maintained at 24 1 C under daylight white
uorescent lamps of irradiance 15 mol photon m 2 s 1 with a
12:12 h light: dark photoperiod. The medium was replenished once in
a week. Five healthy fragments of about 5 cm in length were excised
from each accession and cultured for 24 days to evaluate their growth
performance. The biomass increase was measured every eighth day.
The relative growth rate (RGR) of the fragments was calculated by
= ln (W2 / W1) n 1 100 where is the relative growth rate
(expressed in % per day); W2 and W1 are nal and initial biomass
weight, respectively; and n is the number of days in the test (Mantri et
al., 2009). The mean RGR was presented with SD.
2.3. Hormonal analysis
Abscisic acid (ABA), Indole-3-acetic acid (IAA), Indole-3-butyric
acid (IBA), standards (purity N99%) were purchased from SigmaAldrich (USA). The stock solution of the above standards (1 mg/ml)
was individually prepared in methanol and stored at 40 C.
Working concentration of individual standard was obtained by
diluting each with methanol. A mixture of all the standards of
working concentration was also made to analyse the peak separation
and assignment of individual peak to the respective standard.
Hormone extraction and their enrichment by dispersive liquidliquid
microextraction (DLLME) were done by the method of Lu et al.
(2010). Chromatographic separation was performed on the Waters
alliance HPLC system coupled with photodiode array detector
(Waters 2996). A Luna-C18 reversed-phase column (5.0 m,
4.6 150 mm, Phenomenex, USA) maintained at 35 C was used as
the separation channel. The mobile phase was composed of methanol:
0.6% acetic acid (60:40 v/v%) and the ow rate was set as 1.0 ml/min.
The injection volume was 50 l for each analysis. UV detection was
carried out at three different wavelengths as 208, 254 and 265 nm.

Fig. 1. Habit of female gametophyte (A), male gametophyte (B) and tetrasporophyte (C) of Gracilaria dura. Scale bars = 0.65 cm.

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V. Gupta et al. / Aquaculture 318 (2011) 389396

The calibration curves were constructed in the concentration range of

0.8100 g/ml for all the standards selected in the study.
2.4. DNA extraction
The accessions of gametophytes and tetrasporophytes that
showed higher growth rates were further studied for the genetic
variation analysis. Genomic DNA from the each accession was
extracted by modied cetyltrimethyl ammonium bromide (CTAB)
method. Thallus were blotted with tissue paper, homogenised in
liquid nitrogen and mixed with preheated (65 C) CTAB buffer
consists of [2% CTAB (w/v), 1.4 M NaCl, 100 mM TrisHCl (pH 8.0),
50 mM ethylenediaminetetraacetate (EDTA 2Na), 50 mM sodium
sulphite and 1% PVP] and incubated at 65 C in water bath for 1 h
with occasional gentle mixing. The cooled mixture was extracted
twice with equal volume of chloroform: isoamyl alcohol (24:1),
centrifuged at 12,000 g for 5 min and upper aqueous layer was
collected. The recovered aqueous layer was treated with RNase A
(10 g) for 30 min at 37 C. DNA was precipitated with isopropanol
and pelleted by centrifugation at 12,000 g. The DNA pellet was
washed by mixture of ethanol (76%) and sodium acetate (0.2 M). A
second wash was given with mixture of pure ethanol and 10 mM
ammonium acetate and the resulting pellet was air dried and nally
rinsed with pure ethanol. The nal DNA pellet thus obtained was
dissolved in Tris EDTA (10 mM, pH 8.0). The purity of the extracted
DNA was checked by OD260/OD280 ratio and by agarose gel
electrophoresis.
2.5. PCR amplication and electrophoresis
PCR amplications were carried out in 25 l reactions containing
10 mM TrisHCl (pH 9.0), 0.5 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs,
0.2 M primers, 1 U Taq DNA polymerase (Sigma-Aldrich, USA) and
2025 ng template DNA. A total of 20 ISSR primers were screened for
amplication. Amplication was carried out in Bio-Rad thermal cycler
(USA). The ISSR programming involved an initial denaturation step of
3 min at 94 C followed by 45 cycles at 94 C for 40 s, respective
primer annealing temperature for 45 s and 72 C for 1.5 min, with
nal extension step at 72 C for 5 min. The amplicons were size
fractionated by electrophoresis on 3% agarose gel and bands were
visualised under UV light after ethidium bromide staining.
2.6. Band prole reproducibility
Two replicates of DNA extraction were performed for each
accession of gametophyte and tetrasporophyte. ISSR amplication
was also performed in duplicate to access the reproducibility and
consistency of amplication prole.
2.7. ISSR band scoring
Assuming two alleles per locus, the fractionated ISSR proles were
scored for each accession as discrete characters based on the presence
or absence of amplied bands. The estimators of genetic diversity as
percentage of polymorphic loci (PPL), Polymorphism information
content (PIC) and Shannon indices of diversity (I) were calculated
(Verma et al. 2009) considering the population inbreeding and in
HardyWeinberg equilibrium. All pair-wise comparisons of genetic
similarity among the three phases of G. dura was measured using
Dice's coefcient of similarity (Nei and Li, 1979) as follows:
Sab = 2Nab / (Na + Nb), where Nab is the number of pair-wise shared
bands, Na and Nb are the number of bands present in individual a and
b. A genetic similarity (GS) matrix was thus constructed based on
SIMQUAL module of software package NTSYS-pc, version 2.02e
(Applied Biostatistics Inc., USA). The GS matrix was then used to
construct the dendrogram using unweighted pair group method of

391

the arithmetic averages (UPGMA). The allele frequencies, as an

estimate of genetic diversity among inter-phasic population of G. dura
was calculated based on the frequency of the null allele (i.e., the
number of individuals without the band) treating each band as an
individual character. The frequency of dominant allele was determined from null allele by pi = 1 qi where qi represents the
frequency of the null allele, and pi represents the frequency of the
dominant allele. Phenotypic gene diversity was calculated as He = 1
pi2 qi2. Estimates of He were averaged per locus.
Regression analysis was used to investigate the linear, quadratic
and cubic association between the haploid gametophytes and diploid
tetrasporophyte phases of G. dura and their respective band frequency
considering correlation coefcient, R 2, values as signicant of
variables.

2.8. Cytological analysis

The young growing apical tips of gametophytes of male, female
and tetrasporophytic plants of G. dura were excised and xed in
formaldehyde and glacial acetic acid (1:1) for approximately 24 h.
Afterwards, tissues were hydrolysed in 1 N HCl for 510 min at 60 C
and rinsed twice with cold distilled water to remove excess acid from
material. A drop of stain carbol fuchsin stain (Qualigens Fine
Chemicals, India) solution was added to material on a slide and
heated till the stain solution began to boil. Then the material was
covered with a cover slip and excess stain solution was blotted off by
placing the slide between the sheets of lter paper and squashing
gently. The stained slides were examined under microscope at 100
(Olympus BX60, Japan). Chromosomes were count in 100 nuclei of
each gametophyte and tetrasporophyte.

3. Results
3.1. Agarose yield and gel strength
The yield (%) of agarose from natural population of male, female
and tetrasporophyte was 12.5 0.81, 18.5 0.32 and 19 0.21
respectively. The apparent viscosity of agarose was highest with
tetrasporophyte as 55 0.81 cP followed by 44 0.42 cP in female
gametophytes 36 0.5 cP in male. The gel strength was recorded
highest for tetrasporophytes (1800 8.76 g cm 2) than female
(1500 7.85 g cm 2) and male (1200 10.57 g cm 2) gametophytes (Table 1).
The 13C NMR spectra of the polysaccharide extracted from three
life phases of G. dura have 12 signals as characteristics of agarose. The
signals at 102.4, 71.9, 83.3, 68.7, 74.5 and 62.5 ppm correspond to
3-linked -D-galactopyranosyl units and the signals at 99.6, 70.8, 81.1,
78.4, 76.6 and 70.1 ppm correspond to 4-linked 3,6-anhydro--Lgalactopyranosyl units. The additional signals at 72.7 and 79.7 ppm
are proposed to 4-O-methyl--L-galactopyranosyl which is attached
to the C6 of some of the 3-linked -D-galactopyranosyl unit of the
agarose from G. dura. Weak signal of chemical shift at 60 ppm was
observed for 6OMe and 2OMe (Supplementary Fig. 1). The additional
signal at 103.6 was there for all the three life phases.

Table 1
Agaraose characteristics of the different life cycle stages of G. dura.
Agarose

Male gametophyte

Female gametophyte

Tetrasporophyte

Yield (%)
Gel strength
(g/cm 2)
Viscosity (cP)

12.5 0.81
1200 10.57

18.5 0.32
1500 7.85

19 0.21
1800 8.76

44 0.42

55 0.81

36 0.5

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V. Gupta et al. / Aquaculture 318 (2011) 389396

corresponding values for IBA were 1.67 0.73, 1.42 0.05 and 1.31
0.33 respectively.

3.2. Growth experiment

All the fragments of different phases of G. dura incubated under
laboratory conditions produced numerous apical tips (buds) from the
periphery of the cut fragments within two weeks. Signicant
variations were recorded in the growth pattern of all the accessions
studied (Table 2). The RGR varied in the range (1.19 1.10 to 9.63
3.04) in female gametophytes, (2.73 3.58 to 9.31 3.49) in male
and 1.57 2.27 to 3.12 0.26 in tetrasporophyte. Among the
accessions studied, relatively 52.63% of the accessions showed RGR
more than their average RGR in the female gametophyte followed by
42.85% in tetrasporophyte and 33.33% in male gametophytes. Thus
female accessions could be considered to show higher RGR than male
and tetrasporophyte accessions. The highest RGR was observed for the
accession CSMCRICCMA5027 (9.63 3.04%) among the female gametophytes, CSMCRICCMA507 (9.31 3.49) in male gametophytes and
CSMCRICCMA5014 (3.12 0.26) in the tetrasporophytes.
3.3. Identication and quantication of phytohormone
For the three life phases, HPLC analyses revealed the presence of
the auxin i.e. indole-3-butyric acid (IBA) and abscisic acid (ABA) with
a retention time identical to that of their authentic standards. These
hormones were detected at an absorbance wavelength of 265 nm. The
characteristic separation of IBA and ABA standard gave the retention
time of 4.95 and 3.91 min respectively (Supplementary Fig. 2). The
linear range for the targeted compound was derived with regression
equations calculated as follows: IBA, y = 74,274 x 72,997
(r 2 = 0.99); ABA, y = 26,547 x 10,400 (r 2 = 0.995) where y and x
denote the peak area and the concentration (g/ml) of the analytes
respectively. The limit of detection (LOD) for IBA and ABA was found
to be 0.8 and 0.2 g/ml respectively. The analyses resulted with an
endogenic ABA content of 17.68 1.6, 7.39 0.125 and 0.37 0.57 for
female, male gametophyte and tetrasporophyte respectively and the

Table 2
The accession number identiers assigned to G. dura isolates including identication of
the life cycle phases and growth performance.
Life cycle phases

Accession numbers

Relative growth rate (%)

gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
gametophyte
sporophyte
sporophyte
sporophyte
gametophyte
sporophyte
gametophyte
gametophyte
gametophyte
gametophyte
sporophyte
sporophyte
gametophyte
sporophyte
gametophyte
gametophyte
gametophyte

CSMCRICCMA501
CSMCRICCMA502
CSMCRICCMA503
CSMCRICCMA504
CSMCRICCMA505
CSMCRICCMA506
CSMCRICCMA507
CSMCRICCMA508
CSMCRICCMA509
CSMCRICCMA5010
CSMCRICCMA5011
CSMCRICCMA5012
CSMCRICCMA5013
CSMCRICCMA5014
CSMCRICCMA5015
CSMCRICCMA5016
CSMCRICCMA5017
CSMCRICCMA5018
CSMCRICCMA5019
CSMCRICCMA5020
CSMCRICCMA5021
CSMCRICCMA5022
CSMCRICCMA5023
CSMCRICCMA5024
CSMCRICCMA5025
CSMCRICCMA5026
CSMCRICCMA5027
CSMCRICCMA5028
CSMCRICCMA5029

6.91 2.34abcd
2.22 0.67ef
5.70 0.65bcde
2.59 0.21ef
4.49 0.86cdef
4.58 1.56cdef
9.31 3.49ab
4.52 0.86cdef
2.86 0.94ef
3.77 0.52cdef
2.47 0.36ef
2.84 1.61
4.48 1.06cdef
3.12 0.26ef
1.83 1.49f
1.65 1.26f
2.73 3.58ef
1.57 2.27f
2.25 0.74ef
7.03 2.57abc
1.19 1.10f
4.83 1.49cde
3.50 0.85def
6.99 2.0abcd
1.41 0.79f
3.06 0.55ef
9.63 3.04a
5.66 1.46bcde
7.18 2.18ab

The mean values superscript with different letters in the column showed signicant
difference at p b 0.01 (Mean SD).

3.4. Band prole reproducibility

No variation was observed in the position and number of bands
that were selected for scoring although subtle changes in relative
band intensities were observed. No major differences were there
between independent scorings of any gel for any of the selected band
position (Fig. 2).
3.5. ISSR data analysis
A total of 20 ISSR primers were analysed of which eight primers were
screened for generating highly reproducible and clear polymorphic
bands. The PIC value for the primers studied ranged from 0.08 to 0.85
with an average value of 0.368. A total of 354 scorable and repeatable
DNA fragments were generated from male, female and tetrasporophytic
thallus of G. dura with size ranging from 150 to 1600 bp (Table 3). An
average ISSR locus frequency was 14.7 loci per primers. The estimates of
phenotypic diversity as allele frequency, expected average heterozygosity (He) and Shannon's index (I) were 0.607 0.37, 0.5 0.07 and
0.33 respectively for all the three types of thallus of G. dura (Table 4). The
pair-wise average polymorphic loci per primer among the three types of
thallus were found to be 6.37 between male and female, 4.37 between
male and tetrasporophyte and 5.5 between female and tetrasporophyte.
The pair-wise estimation of phenotypic diversity showed the similar
diversity indices (I) as 0.37 between female and tetrasporophyte and
0.36 for both malefemale and maletetrasporophyte. The average He
for malefemale is 0.48 compared to 0.44 for femaletetrasporophyte
and 0.41 for maletetrasporophyte (Table 5). The generated similarity
matrix revealed maximum genetic similarity of 0.65% between male and
tetrasporophyte, 0.58% between femaletetrasporophyte and the
lowest of 0.52% for malefemale (Table 6).
A dendrogram was generated based on unweighted pair group
method with the arithmetic averaging (UPGMA) using software
NTSYS-pc version 2.0 for all the three thallus types of G. dura. The

Fig. 2. Electrophoresis of amplied fragments of ISSR markers for the selected

accessions of male (Lane 2, 3), female (Lane 4, 5), and tetrasporophyte (Lane 6, 7) of G.
dura. Lane 1 = 123 bp DNA ladder. Arrowheads show phase specic bands for
respective genotypes of G. dura.

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V. Gupta et al. / Aquaculture 318 (2011) 389396
Table 3
ISSR primers and details of polymorphism generated per primer among the three
phases of G. dura.
S.
no

Primer

1
2
3
4
5
6
7
8

A
B
D
E
F
G
H
I

Sequence
(CA)6 GG
(CA)6 AC
(CA)6 GT
(GA)6 GG
(GA)6 cm3
(GT)6 cm3
(AG)8 T
(AG)8 C

Annealing temp.
(C)

TB

55
50
55
55
41
52
42
50

29
9
11
13
13
12
11
19

PL
23
3
7
11
1
2
1
17

PPL
(%)

PIC

79.3
33.3
63.6
84.6
7.7
16.6
9.09
89.47

0.502
0.258
0.85
0.502
0.097
0.16
0.08
0.502

Size range
(bp)
1501600
4001400
250650
250950
1751200
3001000
2001100
2001100

TB: Total no. of bands; PL: Polymorphic loci; PPL (%): Percentage of polymorphic loci;
PIC: Polymorphic information content.

bootstrap support by considering the 1000 replicates resulted into

two clades with male and tetrasporophyte in one cluster and female
constituting the other clade. The genetic distance between the
gametophytes (male and female) was found to be more than the
pair-wise combination of tetrasporophyte with either of
gametophyte.
Further, the mean frequency of present band among the three life
phases, male gametophyte, female gametophyte and tetrasporophyte
was 0.69, 0.7 and 0.72 respectively. The regression analysis indicated a
quadratic association between different phases (r 2 = 1) (Fig. 3). Thus,
the generated regression equation (Pbf = 0.005 x 2 0.005 x + 0.69)
could give an inference about the frequency of present band for a
particular genotype with haploid or diploid ploidy.
3.6. Phase specic ISSR markers
Of the 5 primers conrmed for polymorphism, ISSR primer A
[(CA)6 (GG)] and ISSR primer E [(GA)6 (GG)] generated bands linked
specically to sex and life phases in life history of G. dura. ISSR primer
A generated two bands of 800 and 1600 bp specic to tetrasporophyte
and a single band of 430 bp for male and 860 bp for female
gametophyte (Fig. 2). The ISSR primer E generated bands of 700,
400 and 350 bp corresponding to male, female and tetrasporophytes
respectively. The other ISSR primers I [(AG)8 C], D [(CA)6 GT] and F
[(GA)6 cm 3] generated bands of 1150, 650 and 1100 bp that were
specic to only male, female and tetrasporophyte respectively.
3.7. Chromosome count
Cells that were undergoing division in thalli of gametophytic and
tetrasporophytic plants were investigated for chromosome count
using carbol fuchsin stain. The highly contracted chromosomes were
observed at the end of prophase or at early stage of metaphase.
Chromosomes were clearly differentiated with dark red colour over
the contrast of cytoplasm. Haploid chromosome number of 8 was
observed in each gametophytic thalli of male and female and 16 in
diploid tetrasporophyte (Fig. 4).
Table 4
Detailed estimation of genetic diversity in G. dura population.
Components

Results

No. of primers
No. of markers
Polymorphic markers
Percentage of polymorphism
Marker Index (MI)
Average allele frequency
Average heterozygosity (He)
Shannon's Weaver diversity index (I)

8
118
65
55.55%
2.99
0.607 0.37
0.5 0.07
0.33

393

Table 5
Pair-wise genetic polymorphism in the triphasic population of G. dura as determined by
ISSR marker.
Members

PL

PPL (%)

Average allele frequency

He

MXF
MXT
FXT

51
35
44

43.22
29.66
37

0.61 0.3
0.64 0.4
0.63 0.4

0.48
0.41
0.44

0.36
0.36
0.37

4. Discussion
The study detailed the growth behaviour and agarose characteristics of three isomorphic life phases of G. dura. Further the biomarkers
for phase differentiation were identied with the ISSR analysis as well
as with their hormonal complementation. G. dura has been reported
as a source of quality agarose with a yield of 2025% and
benchmarking gel strength of 1% gel N 1900 g cm 2 (Meena et al.,
2007). Whyte et al. (1981) reported that the gel strength varies
among the life cycle stages of Gracilaria. The higher yield and superior
agarose characteristics from tetrasporophyte over the male and
female gametophyte investigated here is in agreement to the report
of Durairatnum and Nascimento (1985) on G. cylindrica. However, in
G. bursapastoris the vegetative plants showed higher gel strength than
carposporic and tetrasporic plants, with no signicant differences in
yield and intrinsic viscosities (Marinho-Soriano et al., 1999). Hoyle
(1978) concluded no variation in agar yield and gel strength from
gametophyte and tetrasporophyte of G. bursapastoris and G.
cornopifolia.
The inuence of environmental cues on growth of Gracilaria is well
documented, but their effect on respective life cycle phases is not well
studied (Kain and Destombe, 1995). In general under different
environmental conditions, the growth rate for most of the Gracilaria
species has been reported in the range of 510% day 1 (MarinhoSoriano et al., 2002). In the present study laboratory grown
tetrasporophytes reported an average RGR of 3.0 1.88% day 1. The
highest RGR was recorded for female accessions compared to male and
tetrasporophyte (Table 2). In the present study highest growth rate of
9.31 3.49% day 1 recorded for female gametophyte which was comparable to the one reported for G. lemaneiformis (6.913.1% day 1)
(Zheng and Gao, 2009), G. tenuistipitata (68.5% day 1) (Skriptsova and
Nabivailo, 2009), G. arcuata (11.73% day 1), G. incurvata (11.18% day 1),
G. textorii (8.13%day 1), G. lichenoides (11.69%day 1) and G. foliifera
(10.62% day 1) (Raikar et al., 2001). However, Baru et al. (2010)
reported growth rate of G. tenuistipitata as high as 21.1% day 1 for
gametophytes while 24.01% day 1 for tetrasporophyte. The higher
growth rate in females could be attributed to the fact that selective
propagation inhibits their reproductive maturity as a result the growth is
vegetative only. Zhang and van der Meer (1988) also observed highest
growth rate of female gametophyte in Gracilariopsis lemaneiformis (as
Gracilaria sjoestedtii). A similar conclusion was also drawn by Choi et al.
(2006) from their eld cultivation experiments on G. verrucosa and
G. chorda in Korea.
The present study revealed that tetrasporophytes yielded better
quality agarose over the haploid gametophytes. Also, Mantri (2009)
reported for the natural dominance of tetrasporophytic phase along
Veraval coast, India during a period of May to June. Therefore the
sustainable utilisation of natural resources could be attained by

Table 6
Genetic similarity matrix of G. dura gametophytes and tetrasporophyte based upon the
Dice coefcient of similarity.

Male
Female
Tetrasporophyte

Male

Female

Tetrasporophyte

1.00
0.52
0.65

1.00
0.58

1.00

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V. Gupta et al. / Aquaculture 318 (2011) 389396

Fig. 3. Distribution of triphasic ploidy levels and band frequency of ISSR markers in G.
dura and the best t equation of quadratic line.

selective harvesting of tetrasporophytes during this time to obtain

quality agarose with higher yield. Moreover, cultivation of selected
germplasm with traits of superior quality agar/agarose will further
enhance the economic viability of farming of this seaweed.
About the signalling of phytohormones, ABA has been postulated for
the development of haploid plants (Imamura and Harada 1980). The
loss of capacity for embryogenesis by the inhibition of ABA synthesis
pathway has also been reported in Pennisetum sp. (Rajasekaran et al.,
1987) and barley (Van Bergen et al., 1999). Therefore, higher content of
ABA analysed in haploid gametophytes of G. dura over the diploid
tetrasporophyte could be considered as biomarker for distinguishing
haploid from diploid thalli. Auxins mainly IAA has been established as an
endogeneous plant hormone in variety of plant species including
seaweeds (Stirk et al., 2009). The other auxin IBA showed higher
stability and regulate the growth of adventitious root more efciently
compared to that of IAA (Ludwig-Mller, 2000). It has also been studied
that IBA is effective at a concentration where IAA was ineffective
(Ludwig-Mller, 2000) and thus revealed its independent auxin effect.
Thus the amount of IBA detected in three life phases of G. dura could be
correlated with their growth rates.
The measured differences in growth rate, agarose yield and gel
strength among gametophyte and tetrasporophyte of G. dura
necessitate the development of methods for selecting particular life
forms in their juvenile state for successful farming method. Previous
studies on Gracilaria have mainly employed the dominant marker as
RAPD to delineate the species lacking distinct morphological
characters (Gonzlez et al., 1996; Lim et al., 2001). Furthermore, the
sex linked genetic markers generated in earlier studies in seaweeds
were specic to a single phase of the life cycle. Martinez et al. (1999)
described 430 bp and 620 bp RAPD amplicons specic to male and
female gametophytes respectively in G. gracilis. The RAPD technique
was further evaluated to have a male discriminatory marker with

amplicon size of 716 bp in G. changii (Sim et al., 2007). Most recently

the SCAR marker of 494 bp specic to female gametophyte was
generated for the brown alga Saccharina japonica (Liu et al., 2009). The
ISSR primer A (CA)6 GG as well as ISSR primer E (GA)6 GG in this
study generated highly differentiated sex-linked marker for all the
three phases in the life history of G. dura. ISSR primers I, D and F
investigated could differentiate male, female or tetrasporophyte as in
the case of RAPD markers (Martinez et al., 1999; Sim et al., 2007).
The ISSR primers employed in the present study were 3 anchored
with nucleotide base to ensure their binding at one end of SSR motifs
giving higher average PIC value that in turn supported the reliable
estimation of genetic diversity. Also the ISSR based analysis of
regression showed the quadratic relation (R 2 = 1) between different
phases. The similar trend was also observed in population of Cynadon
with increasing ploidy levels (Gulsen et al., 2009). Thus higher
diversity obtained in G. dura is also due to the diversity in ploidy
among the three life phases.
Sexual inheritance studies have been conducted in a limited
number of species such as Gracilaria tikvahiae, Palmaria palmata,
Devaleraea ramentacea, G. gracilis, G. verrucosa and demonstrated the
Mendelian mode of segregation (van der Meer, 1990). Previous reports
on cytological analysis and nuclear division studies have clearly
demonstrated different chromosomal count with the alternation of life
phases (Tseng and Sun, 1989). It has also been concluded that
Gracilariales poses high level of inter/intra specic variability of
chromosomal count (Godin et al., 1993). Kapraun et al. (1993)
characterised four species of Gracilaria with constant chromosome
count (1N = 24). Conversely, Godin et al. (1993) reported anomalous
number of chromosomes (1N = 17 1 and 2N = 33 3) in Gracilaria
verrucosa. The chromosome staining in the present study clearly
showed for the alternation in generation in the life history of G. dura
with haploid phase containing chromosome number 8 (1N) and
tetrasporophyte, the diploid phase with two sets of chromosomes
numbering 16 (2N). Also, the morphology of the two sets of
chromosome in the tetrasporophyte is quite identical to those of the
single set of male and female thalli contrasting to the different
morphology observed by Kapraun et al. (1994) in Gelidiella acerosa.
The evolution in Gracilaria may attributed to be a complex process of
polyploidy and aneuploidy as earlier studies showed different
chromosome counts with 1N = 17 in G. verrucosa (Godin et al.,
1993) and 25 in different species of Gracilaria (Kapraun et al., 1993).
The higher genetic distance among the male and female
gametophytes over gametophyteterasporophyte reveals together
with their chromosome count evidenced for their mode of sexual
reproduction. Similar results were also reported for an apomictic
plant species Hieracium pilosella, (Houliston and Chapman, 2004) and
also in red seaweed Gracilaria gracilis (Engel et al., 2004). In addition,
higher polymorphism of 43.22% in malefemale over the other pairs
studied also support for their different genetic identity with sexual
compatibility. All ISSR primers used for genetic diversity analysis have
recorded higher diversity coupled with 989% polymorphic bands and

Fig. 4. Chromosomes of different thallus types of G. dura. (A), haploid male (N = 8), (B) female gametophyte (N = 8) and (C) diploid tetrasporophyte (2N = 16). Scale = 300 m.

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V. Gupta et al. / Aquaculture 318 (2011) 389396

Shannon's weaver index as high as 0.32 compared to previous reports

on seaweed genetic diversity thus supported for their sexual
inheritance (Wang et al., 2008).
About the sexual differentiation in seaweeds so far an additional
sex chromosome has been reported only in Wrangelia argus (Rao,
1971) and Aglaothamnion oosumiense (Chah et al., 2004). Although,
van der Meer (1990) suggested that there are two alleles (mt f or mt m)
at single locus that determines the sexual phases in seaweeds rather
than a separate sex chromosome. Like in monoecious higher plants e.g.
Cucumis sativus, genes designated as F, M and A control the expression
of sex (Pierce and Werner, 1990). Another plant studied for sex
determination, Cariaca papaya has similar allelic sex determining
mechanism but controlled by a single gene (Storey, 1938). Also G. dura,
with no additional sex determining chromosome can be postulated to
have gene controlled or allelic expression of sexual traits.
In conclusion, industrial viability of a resource G. dura was
described by studying the characteristics of its life phases for their
growth, agarose yield and gel strength. The investigation of biomarkers as hormonal content as well as the markers associated with
sex or life phase screened would help in selection of strains early in
their developmental stages, thus augment its industrial utility.
Furthermore, the genotyping system for G. dura developed in the
present study can be used efciently for gene tagging, linkage analysis
and gene mapping of markers associated with the traits studied.
Supplementary materials related to this article can be found online
at doi:10.1016/j.aquaculture.2011.06.009.
Acknowledgements
We would like to thank Dr. Kamlesh Prasad for the characterisation
of agarose. The nancial support received from CSIR (NWP-018) is
gratefully acknowledged. Mr Manoj and Miss Puja would like to thank
CSIR, New Delhi for awarding the Senior and Junior Research
Fellowships respectively. We would like to thank the section editor
of Aquaculture and reviewers for their valuable comments and
suggestions while reviewing this manuscript.
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