Journal of Life Sciences Volume 6 Number (7)
J LS
Journal of Life Sciences
Volume 6, Number 1, January 2012 (Serial Number 45)
Contents
Molecular Biology and Medical Biochemistry
1 Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics of a NaCl-Tolerant Potato Cell Line
Filipa Queirós, José M. Almeida, Domingos P.F. Almeida and Fernanda Fidalgo
9 The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence from Mitochondrial DNA Sequences
Anglv Shen, Chunyan Ma, Yong Ni, Zhaoli Xu and Lingbo Ma
14 Parameters Analysis of Gastric Motility Signals in Time Domain and Frequency Domain
Zhangyong Li, Likun Xu and Zhui Xu
20 Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for Application of Bioequivalence Study
Yahdiana Harahap, Krisnasari Dianpratami, Mahi Wulandari and Rina Rahmawati
28 Rotation Thromboelastography for Assessment of Hypercoagulation and Thrombosis in Patients with Cardiovascular Diseases
Antoaneta Dimitrova-Karamfilova, Yuliana Patokova, Tania Solarova, Irina Petrova and Gencho
Natchev
36 New Silver Nanosensor for Nickel Traces. Part II: Urinary Nickel Determination Associated to Smoking Addiction
María Carolina Talio, Marta O. Luconi and Liliana P. Fernández
Physiology
41 Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya
Omotade Oloyede, Daniel Roos and Joao Rocha
48 Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice
Ikram-ul-Haq, Ghulam Yasin, Mumtaz Hussain and Ali Mohammad Dahri
55 Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress
Sibanarayan Mohapatra and Surjendu Kumar Dey
61 The Potentials of Locally Available Fruits Rich in Iron to Mitigate Iron Deficiency Anemia in Least Developing Countries (LSD)
Abdulkadir A. Egal and Wilna H. Oldewage-Theron
68 Feeding Habits of the Red Porgy Pagrus pagrus (Linnaeus, 1758) from Benghazi Coasts, Libya
Mohammad El-Mor
74 Nesting Activity and Conservation Status of the Hawksbill Turtle ( Eretmochelys imbricata) in Persian Gulf
Seyyed Mohammad Bagher Nabavi, Ruhollah Zare and Mahdieh Eftekhar Vaghefi
Interdisciplinary Researches
80 Economic Feasibility of Simultaneous Production of Pine Sawlogs and Meat Goats on Small-Sized
Farms in Alabama
Brandi Broughton, James O. Bukenya and Ermson Nyakatawa
91 Constructing a Model of Digestion in a Primary School Using a Theatrical Performance
Maria J. Gil-Quílez, Begoña Martínez-Peña, Milagros De la Gándara, Marta Ambite and Marian Laborda
99 Creationism and Evolution Views of Brazilian Teachers and Teachers-to-Be
Ana Maria de Andrade Caldeira, Elaine S. Nicolini Nabuco de Araujo and Graça S. Carvalho 110
Research on Behavior of Governing Gene/Epigene Networks as a Problem of Cellular Automata
Identification
Rustem Tchuraev 114 Nymphaeaceae Salisb. and Trapaceae Dumort. Families in the Collection of O.V. Fomina
Botanical Garden
Tatyana Mazur, Nikolai Didukh and Anna Didukh
Journal of Life Sciences 6 (2012) 1-8
Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics of a NaCl-Tolerant Potato Cell Line
Filipa Queirós 1, 2 , José M. Almeida , Domingos P.F. Almeida and Fernanda Fidalgo 1. Biology Department, Faculty of Science, University of Porto, Porto 4169-007, Portugal
2. BioFIG-Center for Biodiversity, Functional and Integrative Genomics, Plant Molecular Biology & Biotechnology Lab, University of Porto, Porto 4169-007, Portugal 3. Faculty of Science, University of Porto, Porto 4169-007, Portugal 4. CBQF, College of Biotechnology, Portuguese Catholic University, Porto 4200-072, Portugal
Received: November 18, 2010 / Accepted: January 17, 2011 / Published: January 30, 2012.
Abstract: Salinity is a serious threat to agricultural production. Potato (Solanum tuberosum) is an important food crop characterised for having low to moderate salinity tolerance. Tissue cultures may be relevant to improve salt tolerance in potato through selection of salt-tolerant cell lines and subsequent regeneration of plants. In this work, the authors used the random amplified polymorphic DNA (RAPD) markers to investigate the occurrence of genetic polymorphism in a potato calli line tolerant to 150 mM NaCl. Out of 40 primers screened, eight generated polymorphic patterns that distinguished salt-tolerant line from the control. Although the macroscopic appearance was similar in both lines, ultrastructural study revealed alterations in salt-grown cells. These showed that plastids less differentiated with a lower number of grana had more and larger starch grains than control cells. In conclusion, RAPD analysis revealed that NaCl-adapted line is a somaclonal variant and the ultrastructural study showed changes essentially at the plastids.
Key words: Salt tolerance, potato, callus tissue, polymorphism, molecular marker, RAPD markers, ultrastructure.
1. Introduction billion by the year 2050 [5], which will increase the pressure for agricultural production from saline soils.
Soil salinity is one of the major environmental Developing salt-tolerant crops has been a highly
stresses causing a significant loss of productivity in desirable scientific goal but has had little success to
world agriculture, especially in irrigated lands [1]. It date [6]. Potato (Solanum tuberosum L.) is the most
has been claimed as a considerable threat to food important non-cereal food crop and it has a low to
provisions of world population resulting from limited moderate tolerance to salinity. Attempts to enhance the
growth and low harvestable yield of major crop species salt tolerance in potato and other crops through
[2, 3]. Worldwide, more than 800 million hectares of conventional breeding methods have had limited
land are estimated to be salt affected [4]. The success, warranting the use of biotechnological
imperative to develop plants capable of growing in approaches as a rapid means for the production of
saline agricultural systems is growing. The world’s salt-tolerant genotypes. Appropriate strategies may
population is expected to increase to more than 9 include the use of tissue culture techniques through in
vitro selection of salt-tolerant cell lines and subsequent Corresponding author: Fernanda Fidalgo, assistant professor, research field: biochemistry and plant physiology.
regeneration of whole-plants with improved salt E-mail: ffidalgo@fc.up.pt.
2 Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
tolerance [7, 8]. Besides those cell lines are a useful
0.5 mg·L -1 benzylaminopurine and 2 mg·L -1 tool to study the mechanisms of plant salt tolerance at
2,4-dichlorophenoxyacetic acid (2,4-D) (multiplication the cell level.
medium), on which it was cultivated for 28 days. Calli In this sense, we have previously selected a potato
grown for six successive subcultures (of 28 days each) calli line adapted to grow on 150 mM NaCl [9] and
in the multiplication medium were designed as changes in several physiological and biochemical
non-adapted calli line (control). The selection of parameters were induced by salinity, with some being
150 mM NaCl-tolerant calli was carried out by involved in salt tolerance [10]. Although salt-tolerant
progressively growing non-adapted callus tissue on line displayed a macroscopic appearance similar to the
media with increased concentrations of NaCl. Callus control, it is known that plant cells adapted to salinity
tissue was firstly subcultured on medium containing undergo also were ultrastructural modifications, which
50 mM NaCl for 4 weeks, and then on medium could contribute for their ability to grow in saline
supplemented with 100 mM NaCl for the same period, environment [11]. Thus, in this work we report that the
before being transferred to 150 mM NaCl. This ultrastructural changes induced by salinity to find out
NaCl-tolerant cell line was obtained after four those that may have an adaptive value for potato callus
successive subcultures on medium supplemented with tissue. Furthermore, the possibility of the occurrence of
150 mM NaCl.
somaclonal variation, a common phenomenon in plant
2.2 DNA Extraction and RAPD Analysis tissue cultures [12], which can be enhanced when
tissue cultures are exposed to the stress-inducing Total DNA from frozen calli (100 mg) samples was factors, led us to analyse if the salt tolerance displayed
extracted using the AxyPrep Multisource Genomic by the cell line that is based on physiological
DNA Miniprep Kit (Axygen Biosciences, USA) adaptation or genetic variation. Several PCR-based
according to the manufacturer’s instructions. DNA technologies have been developed to assay genetic
concentration of each sample was quantified by a polymorphism at the DNA level. Among these, random
biophotometer and DNA quality was checked in 0.8% amplified polymorphic DNA (RAPD) markers have
agarose gel.
proven to be efficient in detecting genetic variation in For RAPD assays, the amplification conditions were various plants [7, 13-15]. Therefore, in this work,
optimized by varying concentrations of template DNA, RAPD technique was applied to detect the occurrence
primer, and Mg 2+ ion. A total of forty arbitrary decamer of genetic polymorphism in potato calli in response to
primers (Kits OPB and OPE) obtained from Operon salt condition, and thereby the DNA-based markers
Technology (Alameda, USA) were used for were determined.
polymerase chain reaction (PCR) amplification and
2. Materials and Methods
identification of polymorphic markers. The PCR reaction mixture, in a final volume of 25 µL, consisted
2.1 Callus Culture of 1× PCR buffer (10 mM Tris-HCl, pH 8.4; 50 mM The procedures for induction and subculture of
KCl), 3 mM MgCl 2 , 200 µM of each dNTPs, 1.5 units callus tissue, and for the selection of a 150 mM
of Taq DNA polymerase, 7.5 pmol of primer and 30 ng NaCl-tolerant cell line have previously been described
of genomic DNA. The PCR amplification was in detail [9]. Briefly, potato callus tissue (Solanum
performed in a thermocycler (MyCycler, Bio-Rad) tuberosum L. cv. Désirée) was induced from young
under the following conditions: a first denaturing cycle leaves. After induction, the callus tissue was at 94 ºC for 1 min, followed by 45 cycles at 92 ºC for 1 transferred to Lam medium [16] supplemented with
min, 35 ºC for 1 min, 72 ºC for 2 min and a final cycle
Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
at 72 ºC for 10 min. After amplification, PCR The same micrographs were used for quantification of products (15 µL) were mixed with 2.5 µL of 6×
average number of starch grains.
loading dye solution (0.25% bromophenol blue, 0.25%
3. Results and Discussion
xylene cyanol and 40% sucrose, w/v) and separated in 1.8% agarose gels, in 1× TAE buffer (40 mM
3.1 RAPD Analysis
Trizma-base, 1 mM EDTA and 0.115% acetic acid In order to analyse if the salt-grown callus line is glacial) at a constant voltage of 70 V. The amplified
physiologically adapted or whether it is a variant line, a DNA bands were stained with ethidium bromide and
comparison of RAPD patterns of control and visualized under a UV transilluminator. A 1 kbp DNA
salt-tolerant calli was carried out. RAPD assay detects ladder was used as a molecular standard. Gels were
nucleotide sequence polymorphisms in a DNA analysed with the Quantity One software (Bio-Rad) to
amplification-based assay using only a single short detect and estimate molecular weight of the bands.
primer of arbitrary nucleotide sequence. The major Primers that gave clear and consistent polymorphic
advantage of this assay is that there is no requirement amplification products were tested three times with
for DNA sequence information, besides its simplicity DNA samples of control and salt-tolerant calli
and cost-effectiveness [17, 18]. A total of forty extracted from independent experiments, using similar
arbitrary primers were used in this research to amplify reaction conditions to assure the reproducibility of
the DNA extracted from the two calli lines, of which RAPD patterns. Polymorphism in RAPD profiles
eight primers generated reproducible polymorphic included disappearance of a normal band and/or
banding patterns. The analysis of RAPD profiles of appearance of a new band in comparison to control, and
both calli lines obtained with these primers revealed only polymorphic bands amplified in three replicates
that sixteen bands ranging from 482-2,211 bp in size were scored.
were polymorphic (Table 1). Polymorphisms were due to the loss and/or appearance of new amplified
2.3 Electron Microscopy and Morphometric Evaluation fragments in the tolerant line compared with control.
Callus tissue was fixed in 2.5% (v/v) glutaraldehyde Thus, eleven bands were absent and the remaining five followed by 2% (w/v) osmium tetroxide, using
were present in calli grown on the presence of 150 mM Na-piperazine-N,N’-bis
(2-ethanesulfonic acid) NaCl (Table 1). Among the primers screened, none (Na-PIPES) buffer (pH 7.2), dehydrated in acetone and
generated polymorphic bands that were unique to the embedded in Epon 812. Ultra thin sections were cut
salt-tolerant calli, having OPB series resulted in the and contrasted with uranyl acetate and lead citrate, and
greatest number of RAPD bands. viewed using a Zeiss EM C10 transmission electron
Examples of RAPD profiles generated by using the microscope (Zeiss, Göttingen, Germany).
selected primers are shown in Fig. 1. PCR Quantification of cellular structural changes induced
amplification with primers OPB-04 and OPB-18 by the salt was performed. To assure random pictures
resulted in the appearance of two bands that were for morphometric studies, five blocks were chosen
unique to the salt-tolerant calli (850 and 1,095 bp, from control and salt-tolerant calli and 250 respectively) and a loss of three bands that were micrographs were taken at random for each situation
detected in the control (Fig. 1, Table 1). Similarly, in and magnified 12,000×. Morphometric determinations
salt-tolerant calli two bands of about 566 and 1,296 bp of fractional volume (Vv) of starch/plastid were
were absent in comparison to control when amplified performed by measuring the area of the starch and
with primers OPE-02 and OPE-19, respectively, plastids using the Image J software from NIH Image.
occurring only a new band (2,211 bp) (Fig. 1, Table 1).
4 Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
Table 1 RAPD polymorphic bands generated by eight primers from Operon series and separated by agarose gel in control and salt-tolerant calli.
Primer code Sequence 5’ to 3’
Salt-tolerant calli OPB-01 GTTTCGCTCC 1,980
Molecular weight (bp)
Control calli
OPB-04 GGACTGGAGT 850
OPB-09 TGGGGGACTC 580
OPE-02 GGTGCGGGAA 566
OPB-18 CCACAGCAGT
OPE-06 AAGACCCCTC 1,242
OPE-14 TGCGGCTGAG 1,804
OPE-19 ACGGCGTATG 2,211
: indicates appearance of a new band; : disappearance of a normal band.
Fig. 1 RAPD profiles of control (C) and salt-tolerant calli (ST) of S. tuberosum obtained with primers from OPB and OPE series (Operon Technology Inc.).
Each sample was analyzed in duplicate to assure the reproducibility between the profiles. The polymorphic bands that were selected as RAPD markers which discriminated the two calli lines are marked in each of the patterns generated by the primers indicated on the top of the gels; the arrowheads point to bands that are present in control but absent in salt-tolerant line, while arrows indicate the appearance of extra bands in tolerant line that are missing in control pattern. The brace represents an area where are detected differences in banding pattern between the two calli lines. In each case a 1 kbp DNA ladder is included as a molecular weight marker (M).
These polymorphic bands and the remaining generated arbitrary primers was useful in detecting DNA by the primers listed in Table 1 (data not shown) were
polymorphism between control and salt-tolerant calli, selected as RAPD markers since they differentiated
suggesting that this one is a somaclonal variant. RAPD genotypically the salt-tolerant line from the control.
markers were also used for genotypic assessment of These results confirmed that RAPD analysis using
potato plants regenerated from a salt-tolerant callus
Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
line and only one polymorphic band generated by one cultured in vitro. Electron microscopic analyses of primer showed that these plants were somaclonal
potato line tolerant to NaCl showed that salinity did not variants [7]. Beyond the identification of somaclonal
affect the structural integrity of the cells and did not variants in micropropagated plants, RAPD approach
cause disruption of cell organelles, which is consistent has been successfully used to characterize the genetic
with the macroscopic aspect of callus tissue grown diversity in a variety of plant species as well as to
under saline condition (Fig. 2). Besides no differences differentiate cultivars [19, 20]. Based on RAPD
was observed in the plasma membrane, when endoplasmic markers, genetic distances can be estimated to establish
reticulum and mitochondria of tolerant cells compared phylogenetic relationships among the species, as was
with control (Figs. 3A and 3B). However, some demonstrated within the genus Solanum [21] or Vigna
changes were detected in plastids from salt-tolerant cell [22]. According to various authors, the analysis of
line (Fig. 4). Round-shaped plastids were more genetic differences and discrimination of genetic
common in tolerant cells, which appeared less relationships between taxa would be of great differentiated than those in the control cells, displaying significance for designing breeding strategies [22, 23].
a reduced membrane system and a lower number of In fact, our potato cell line grown under salinity is a
grana (Figs. 4A-4D). The fact that plastids in control somaclonal variant that can be used to regenerate plants
tissue have appeared with a more developed lamellar with improved salt tolerance, which could be an
system, led us to admit that the salt presence in culture alternative genetic resource for potato breeding
medium restricted the differentiation process. In fact, programmes.
under unfavourable growth conditions, the formation
3.2 Ultrastructural Analysis The ultrastructure of calli grown in 150 mM NaCl
was also investigated to gain a better understanding of the response of potato callus tissue to high salinity. The ultrastructural changes induced by salinity provide useful information as to the underlying mechanism of
salt tolerance [11]. However, there are few reports on
Fig. 2 Aspect of control callus tissue (A), and salt-tolerant
the effects of salinity on cell ultrastructure for tissues
callus cultivated in medium with 150 mM NaCl (B).
Fig. 3 Ultrastructural aspects of control and salt-tolerant cells. (A) Normal organization and structure of control cells with numerous peroxisomes containing paracrystalline inclusion (arrows) and mitochondria (arrowheads) (bar: 1 µm). (B) Region of vacuolated cells showing well preserved cytoplasm and organelles such as plastids containing starch (st), mitochondria (arrowheads) and peroxisomes (arrows); no modifications in membranes structure were observed (bar: 1 µm).
6 Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
Fig. 4 Ultrastructural aspects of plastids from control and salt-tolerant cells. (A) Plastid from control callus tissue showing developed grana (arrow) with some plastoglobuli associated (arrowhead) and the presence of vesicles at the periphery of the organelle (bar: 0.5 µm). (B) Aspect of the plastids from salt-tolerant tissue with a spherical shape, showing a reduced lamellar system and a high number of starch grains (bar: 1 µm). (C) Region of control cell displaying a plastid devoided of starch but with grana well organized (arrowhead) and various vesicles arranged along inside of the plastid envelope (arrows) (bar: 0.5 µm). (D) Detail of plastid from salt-tolerant tissue showing the lower level of organization than control cells but with a well preserved lamellar system and a huge starch grain (bar: 0.5 µm).
of grana is prevented because of inhibition of protein from tolerant and control calli indicated an increase in synthesis, as well as the formation of plastid ribosomes
starch content of the tolerant cells, aspect that was and chlorophyll accumulation [24, 25]. On the other
analysed through morphometric determinations. hand, the vesicles present in the stroma of the plastids
Although the morphometric analysis of electron of the control cells (Figs. 4A-4C) were absent in
microscope images of plastid profiles have revealed plastids from tolerant cells (Figs. 4B-4D); these
that salt condition did not affect the plastid size, it structures seem to be related with lipid transport within
increased the size of starch grains in the plastid. In fact, the organelle, participating in thylakoid formation and,
the fractional volume of starch relative to plastid size therefore, be involved in the differentiation of the
was higher in tolerant line, which indicates a higher plastids [26].
starch accumulation (Table 2). Besides, the average Additionally, the plastids from NaCl-tolerant cells
number of starch grains per plastid was higher in showed a less compact stroma with large starch grains
tolerant cells than that observed in control (Table 2). when compared to control material. As illustrated in
Unlike control cells where plastids had small starch Fig. 4D, the stroma region of the organelle was
granules or were devoided of starch inclusions partially occupied by starch grains, resembling the
(Figs. 3A-3C), more and larger starch grains could morphology of amylochloroplasts. Electron
be seen in salt-exposed cells (Figs. 3B-3D). The microscopy observation of numerous plastid profiles
accumulation of starch under salt conditions was also
Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
Table 2 Fractional volume (Vv) of the plastids and of the starch, and number of starch grains per plastid in control and salt-tolerant callus tissue.
Culture conditions
Number of starch grains/plastid Control calli
Vv plastid ( m 2 )
Vv starch/plastid
12.65 ± 0.22 a 0.12 ± 0.01 a 1.02 ± 0.16 a Salt-tolerant calli
11.19 ± 0.95 a 0.25 ± 0.02 b 2.41 ± 0.23 b The data are mean values ± standard error. Values in the same column with different superscript letters are significantly different at 5%
level according to Student’s t-test.
observed in NaCl-acclimated Citrus cell line and in microscopy and Filipa Queirós acknowledges the salt-tolerant wheat cultivars [27, 28], in contrast with
support by grant SFRH/BD/18037/2004 from NaCl-stressed potato plants where starch content
Fundação para a Ciência e Tecnologia (Portugal). decreased and the thylakoid membranes swelled [29].
References
As salinity has both ionic and osmotic effects, probably [1] T. Horie, J.I. Schroeder, Sodium transporters in plants: the accumulation of starch in our tolerant line may be Diverse genes and physiological functions, Plant
related with the osmotic adjustment process and, Physiology 136 (2004) 2457-2462. [2] M. Tester, R. Davenport, Na + tolerance and Na consequently, contribute to the adaptation of cells to + transport salinity. The acclimation of plant cells grown under in higher plants, Annals of Botany 91 (2003) 503-527.
[3] S. Sengupta, A.L. Majumder, Porteresia coarctata (Roxb.) salinity requires the production and accumulation of
Tateoka, a wild rice: A potential model for studying osmotically active solutes of low molecular mass that
salt-stress biology in rice, Plant, Cell and Environment 33 lead to the osmotic adjustment of the cell, but also the
(2010) 526-542. [4] N.L. Teakle, S.D. Tyerman, Mechanisms of Cl - transport
accumulation of the sugar reserve in the form of starch contributing to salt tolerance, Plant, Cell and Environment [30].
33 (2010) 566-589.
E.G. Barrett-Lennard, T.L. Setter, Developing saline
4. Conclusions
agriculture: Moving from traits and genes to systems, Data of the present study confirm the potential of Functional Plant Biology 37 (2010) iii-iv. [6] S. Schubert, A. Neubert, A. Schierholt, A. Sümer, C. Zörb,
RAPD technology as a reliable and sensitive screening Development of salt-resistant maize hybrids: The method to discriminate genotypically the salt-tolerant
combination of physiological strategies using line from the control. DNA fingerprinting by RAPDs
conventional breeding methods, Plant Science 177 (2009) 196-202.
showed that our potato cell line grown under salinity is [7] S.J. Ochatt, P.L. Marconi, S. Radice, P.A. Arnozis, O.H.
a somaclonal variant, which paves the way for the Caso, In vitro recurrent selection of potato: Production and regeneration of potato plants with improved salt
characterization of salt tolerant cell lines and plants, Plant tolerance. Based on ultrastructural study we conclude
Cell, Tissue and Organ Culture 55 (1999) 1-8. [8] Y. Miki, M. Hashiba, S. Hisajima, Establishment of salt
that the major alterations caused by salinity occurred stress tolerant rice plants through step up NaCl treatment
essentially at the plastids level, which consisted in a in vitro, Biologia Plantarum 44 (2001) 391-395. less developed membranous system associated with an
F. Queirós, F. Fidalgo, I. Santos, R. Salema, In vitro increase of starch amount. The accumulation of starch selection of salt tolerant cell lines in Solanum tuberosum L., Biologia Plantarum 51 (2007) 728-734.
observed in salt-tolerant cells might be related with the
F. Queirós, N. Fontes, P. Silva, D. Almeida, M. Maeshima, osmotic adjustment process.
H. Gerós, et al., Activity of tonoplast proton pumps and Na + /H + exchange in potato cell cultures is modulated by
Acknowledgments
salt, Journal of Experimental Botany 60 (2009) The authors acknowledge the technical assistance of 1363-1374.
H. Miyake, S. Mitsuya, Md.S. Rahman, Ultrastructural Rui Fernandes (Instituto de Biologia Molecular e
effects of salinity stress in higher plants, in: A.K. Rai, T. Celular, Porto, Portugal) in transmission electron
Takabe (Eds.), Abiotic Stress Tolerance in Plants,
8 Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics
of a NaCl-Tolerant Potato Cell Line
Springer, The Netherlands, 2006. (Solanaceae) as revealed by RAPD markers, Current [12] P.J. Larkin, W.R. Scowcroft, Somaclonal variation–A
Science 90 (2006) 711-716.
novel source of variability from cell cultures for plant [22] H.K. Dikshit, T. Jhang, N.K. Singh, K.R. Koundal, K.C. improvement, Theoretical and Applied Genetics 60 (1981)
Bansal, N. Chandra, et al., Genetic differentiation of Vigna 197-214.
species by RAPD, URP and SSR markers, Biologia [13] N.T. Nguyen, R.E.A. Moghaieb, H. Saneoka, K. Fujita,
Plantarum 51 (2007) 451-457.
RAPD markers associated with salt tolerance in Acacia [23] M. Santalla, J.B. Power, M.R. Davey, Genetic diversity in auriculiformis and Acacia mangium, Plant Science 167
mung bean germplasm revealed by RAPD markers, Plant (2004) 797-805.
Breeding 117 (1998) 473-478.
[14] L. Venkatachalam, R.V. Sreedhar, N. Bhagyalakshmi,
A.F. Abdelkader, H. Aronsson, K. Solymosi, B. Böddi, C. Genetic analyses of micropropagated and regenerated
Sundqvist, High salt stress induces swollen prothylakoids plantlets of banana as assessed by RAPD and ISSR
in dark-grown wheat and alters both prolamellar body markers,
In Vitro Cellular & Developmental transformation and reformation after irradiation, Journal Biology-Plant 43 (2007) 267-274.
of Experimental Botany 58 (2007) 2553-2564. [15]
D. Chakrabarty, S.K. Datta, Application of RAPD markers [25] J. Feierabend, U. Schrader-Reichhardt, Biochemical for characterization of γ-ray-induced rose mutants and
differentiation of plastids and other organelles in rye assessment of genetic diversity, Plant Biotechnology
leaves with a high-temperature-induced deficiency of Reports 4 (2010) 237-242.
plastid ribosomes, Planta 129 (1976) 133-145. [16] S.L. Lam, Plantlet formation from potato tuber discs in
[26] M.X. Andersson, A.S. Sandelius, A chloroplast-localized vitro, American Potato Journal 54 (1977) 465-468.
vesicular transport system: A bio-informatics approach, [17] S.V. Tingey, J.P. del Tufo, Genetic analysis with random
BMC Genomics 5 (2004) 1-8.
amplified polymorphic DNA markers, Plant Physiology [27] A.L. Ferreira, M.E. Lima-Costa, Growth and ultrastructural 101 (1993) 349-352.
characteristics of Citrus cells grown in medium containing [18] J.G. Williams, M.K. Hanafey, J.A. Rafalski, S.V. Tingey,
NaCl, Biologia Plantarum 52 (2008) 129-132. Genetic analysis using random amplified polymorphic
[28] S. Salama, S. Trivedi, M. Busheva, A.A. Arafa, G. Garab, DNA markers, Methods in Enzymology 218 (1993)
L. Erdei, Effects of NaCl salinity on growth, cation 704-740.
accumulation, chloroplast structure and function in wheat [19] S. Betal, P. Roy Chowdhury, S. Kundu, S. Sen
cultivars differing in salt tolerance, Journal of Plant Raychaudhuri, Estimation of genetic variability of Vigna
Physiology 144 (1994) 241-247. radiata cultivars by RAPD analysis, Biologia Plantarum
F. Fidalgo, A. Santos, I. Santos, R. Salema, Effects of 48 (2004) 205-209.
long-term salt stress on antioxidant defence systems, leaf [20]
E. Schiliro, S. Predieri, A. Bertaccini, Use of random water relations and chloroplast ultrastructure of potato amplified polymorphic DNA analysis to detect genetic
plants, Annals of Applied Biology 145 (2004) 185-192. variation in Pyrus species, Plant Molecular Biology
[30] T.H. Chen, N. Murata, Enhancement of tolerance of Reporter 19 (2001) 271a-h.
abiotic stress by metabolic engineering of betaines and [21] A.K. Singh, M. Singh, A.K. Singh, R. Singh, S. Kumar, G.
other compatible solutes, Current Opinion in Plant Kalloo, Genetic diversity within the genus Solanum
Biology 5 ( 2002) 250-257.
Journal of Life Sciences 6 (2012) 9-13
The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence from Mitochondrial DNA Sequences
Anglv Shen, Chunyan Ma, Yong Ni, Zhaoli Xu and Lingbo Ma East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China
Received: July 22, 2011 / Accepted: August 18, 2011 / Published: January 30, 2012.
Abstract: Japanese butterfly ray Gymnura japonica from twinspot butterfly ray G. bimaculata based on a pair of white spots behind eyes or not, which was not reliable. To clarify the taxonomic status of G. japonica and G. bimaculata, the nucleotide variation between the two butterfly rays was examined using mitochondrial DNA sequence comparisons. Approximately 585 bp of 16S ribosomal RNA (rRNA) and 1,128 bp cytochrome b (cyt b) genes were sequenced from representatives of two butterfly rays species in East China Sea. The results showed that there were the same sequences of 16S rRNA gene between two butterfly rays; six sites were variable among two butterfly rays of cyt b genes, the proportion of polymorphic loci was 0.53%, and two haplotypes were defined which genetic distance was 0.5%. Combined with the morphological character and the analysis of mtDNA sequence indicated that twinspot butterfly ray G. bimaculata was a synonym of Japanese butterfly ray G. japonica.
Key words: 16S rRNA, cyt b, morphological character, taxonomic status, butterfly ray.
1. Introduction morphology [6]. Isouchi had reported that the butterfly ray species, G. bimaculata is a junior synonym of G.
The Gymnura genus (van Hasselt, 1823) (order japonica [4], but lacked molecular evidence. It is Myliobatiformes, family Gymnuridae) includes eleven necessary to use new technology at the molecular species, of which three are found in China, Gymnura level, in order to understand the relationships between bimaculata (Norman, 1925), G. japonica (Temminck similar species; some studies have presented data on et Schlegel, 1850) and G. poecilura (Shaw, 1804) and molecular variation among genera in this family [6]. only two are found in the East China Sea (G. In this study, DNA sequence comparisons of two bimaculata and G. japonica) [1-3]. However, only mitochondrial genes, cyt b and 16S rRNA, were used few studies, using mainly morphological and to clarify the phylogenetic relationship between the anatomical characteristics, have explained the Japanese butterfly ray G. japonica, and the twinspot taxonomic status of these fishes [1-5]. Chu and Meng
butterfly ray G. bimaculata.
(2001) relied on the presence or absence of a pair white spots behind the eyes as a primary diagnostic
2. Materials and Methods
marker to distinguish between the Japanese butterfly
2.1 Morphometric Analysis
ray (G. japonica) and the twinspot butterfly ray (G. bimaculata) [3]. However, intraspecific variation and
One specimen of G. japonica and one specimen of sexual dimorphism may complicate taxonomic
G. bimaculata were collected from the Fujian designations, particularly among species with similar
province, the East China Sea (25 2337.44N, 119 2607.98E). The specimens were deposited at the East China Sea Fisheries Research Institute (ERFI). All
Corresponding author: Lingbo Ma, Ph.D., professor, research field: molecular biology. E-mail: specimens arrived at the laboratory in 95% ethanol, malingbo@vip.sina.com.
10 The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence
from Mitochondrial DNA Sequences
and samples of muscle were taken from each twinspot butterfly ray was also wide (Tables 1 and 2, individual. Morphology measurements followed the
Fig. 1b); the disc width was 2.21 times larger than the protocol of Chu and Meng [3].
disc length. The snout length of the twinspot butterfly ray was 0.19 times the disc length, and shorter than
2.2 Molecular Analysis the interorbital width. The tail length of twinspot
Total genomic DNA was extracted from muscle butterfly ray was 0.59 times the disc length. One spine tissue using standard phenol/chloroform techniques [7].
and seven black bands were observed on the tail. The Amplification reactions of the mitochondrial cyt b and
bands proximal to the spine were merged, and the two 16S rRNA genes followed the protocol of Cheng et al.
bands distal from spine were observed on a ring (Fig. [8]. Primer sequences used for cyt b gene 3b). The other characteristics of the twinspot butterfly
amplification were L14724 and H15915 [9], and ray were similar to those of the Japanese butterfly ray primer sequences for 16S rRNA gene amplification
(Tables 1 and 2).
were 16SAR and 16SBR [10]. These four primers were used for sequencing the corresponding gene
3.2 Molecular Analysis
segments. PCR products were purified in 1.5-2.0% The 16S rRNA gene sequences were corrected
agarose (Biowest) and sequenced using BigDye and aligned, and 586 bp consensus sequences
Deoxy Terminator Cycle Sequencing Kit (Applied were obtained. The sequences of 16S rRNA gene of Biosystems) with an automated DNA sequencer two butterfly rays were the same, and they had a shared (Applied Biosystems 3130) following the
manufacturer’s instructions.
Table 1 Anatomical measurements of the Japanese butterfly ray and the twinspot butterfly ray.
2.3 Data Analysis Japanese Twinspot butterfly ray
butterfly ray Genetic distance was estimated by calculating
66-3709 ( ♀) 66-3703 ( ♂) pairwise distances between populations and taxa using
270 the Kimura two-parameter model (Kimura 1980) in
Total length (mm)
Disc length (mm)
MEGA version 3.1 [11]. 369
Disc width (mm)
Head length (mm)
3. Results
Tail length (mm)
Snout length (mm)
10 6 As shown in Tables 1 and 2, Fig. 1a, the disc of the
3.1 Morphometric Analysis
Eye diameter (mm)
Interorbital width (mm)
11 Japanese butterfly ray was wide; the disc width was 9
Spiracle length (mm)
2.16 times larger than the disc length. The snout of the
Table 2 Ratios of anatomical feature measurements of the
Japanese butterfly ray was short (35 mm), the snout Japanese butterfly ray and the twinspot butterfly ray. Japanese
Twinspot length was 0.19 times the disc length, and shorter than
butterfly ray butterfly ray interorbital width (36 mm). The eye diameter of the
66-3709 ( ♀) 66-3703 ( ♂) Japanese butterfly ray was shorter than the spiracle
Disc width/Disc length 2.16 2.21
0.76 0.78 length. The tail length of Japanese butterfly ray was
Head length/Disc length
Tail length/Disc length
0.19 0.19 bands were observed on the tail. Proximal to the spine,
0.59 times the disc length. One spine and eight black
Snout length/Disc length
0.05 0.04 the bands were merged, and there were seven bands
Eye diameter/Disc length
Interorbital width/Disc length 0.20
0.06 0.05 on a ring distal from the spine (Fig. 2). The disc of the
Spiracle length/Disc length
The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence
from Mitochondrial DNA Sequences
Fig. 3 Images of the dorsal (a) and ventral (b) sides of the
twinspot butterfly ray.
Fig. 1 Images of the Japanese butterfly ray (a), the
twinspot butterfly ray (b), and the butterfly ray (c).
haplotype (Fig. 4a). The average nucleotide frequencies of thymine (T), cytosine (C), adenine (A),
and guanine (G) were 26.5%, 22.1%, 31.6% and 19.8%, respectively. The AT content (58.1%) was higher than CG content (41.9%). The results of sequence comparisons showed that cyt b fragment is 1,128 bp in length, including six sites that were variable among the two butterfly rays (Fig. 4b). The proportion of polymorphic loci was 0.53%, and two haplotypes (SB, RB) were defined. cyt b sequences included no insertions or deletions (indels). The average nucleotide frequencies were: 27.6% T, 34.2%
C, 26.1% A, and 12.1% G. The AT content (53.7%) was higher than CG content (46.3%). The genetic distance between the haplotypes was 0.5%.
4. Discussion
Japanese butterfly ray from twinspot butterfly ray based on whether a pair white spots behind eyes [3].
But, a specimen was collected at Xinhua bay, Fujian
Fig. 2 Images of the dorsal (a) and ventral (b) sides of the
Japanese butterfly ray.
province, China, in Sept. 2008, and this butterfly ray had
12 The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence from Mitochondrial DNA Sequences
(a)
(b)
Fig. 4 Nucleic acid sequences obtained and aligned from the two butterfly rays for 16S rRNA (a) and cyt b (b) genes.
only one spot after eyes and the size of spot was small to distinguish between the Japanese butterfly ray and (Fig. 1c). Isouchi reported there were a pair white spots
the twinspot butterfly ray. Based on these characters, on disc or only one spot on disc, and even no spot on
the difference of two butterfly rays may be due to the disc on the two butterfly rays [4]. Therefore, the
intraspecific variation. In addition, in this study, presence or absence of a pair white spots behind the
Japanese butterfly ray was a female, and all bands were eyes could not serve as the primary diagnostic marker
taken on a ring in the tail (Fig. 2), twinspot butterfly ray
The Taxonomic Status of Gymnura bimaculata and G. japonica: Evidence
from Mitochondrial DNA Sequences
was a male, 2-3 bands were taken on a ring in the tail
China Sea, Science Press, Beijing, China, 1963. [3] Y.T. Chu, Q.W. Meng, Fauna Sinica, Cuclostomata and
(Fig. 3), it was the same phenomena that could be Chondrichthyes, Science Press, Beijing, China, 2001. observed from Fig. 2c. According to these characters,
[4] T. Isouchi, Butterfly ray Gymnura bimaculata, a junior the difference of the two butterfly rays may be due to synonym of G. japonica, Jpn. J. Ichthyol. 23 (1977)
242-244.
the sexual dimorphism. Sexual dimorphism was widely [5] Y.T. Chu, Q.W. Meng, A Study of the Lateral-Line Canals in butterfly rays [6], which had been supported the
System and That of Lorenzini Ampullae and Tubules of Elasmobranchiate Fishes of China, Shanghai Scientific
results in this study. and Technical Publishers, Shanghai, China, 1980.
Different mitochondrial genes have different [6] W.D. Smith, J.J. Bizzarro, V.P. Richards, J. Nielsen, F. evolutionary rates, their analytical capabilities are also
Márquez-Farías, M.S. Shivji, Morphometric convergence and molecular divergence: The taxonomic status and
different, and the same gene in different species may evolutionary history of Gymnura crebripunctata and have different analytical abilities. The 16S rRNA gene
Gymnura marmorata in the eastern Pacific Ocean, J. Fish. is a relatively conserved mitochondrial gene marker Biol. 75 (2009) 761-783. [7] C.Y. Ma, Q.Q. Cheng, Q.Y. Zhang, P. Zhuang, Y.L. Zhao, with a low evolutionary rate compared to cyt b [12].
Genetic variation of Coilia ectenes (Clupeiformes: Thus, 16S rRNA should exhibit less genetic variation
Engraulidae) revealed by the complete cytochrome b sequences of mitochondrial DNA, J. Exp. Mar. Biol. Ecol.
between closely related species than cyt b [8]. The
385 (2010) 14-19.
evolutionary rate of the cyt b gene is greater than that of [8] Q.Q. Cheng, C.Y. Ma, H.P. Cheng, Q.Y. Zhang, 16S rRNA, so it is a very useful molecular marker for Mitochondrial DNA diversity of Coilia mystus (Clupeiformes: Engraulidae) in three Chinese estuaries, understanding the phylogenetic relationships at the
Environ. Biol. Fish. 83 (2008) 277-282. species-level and at the higher order level [6, 7, 13-16].
[9] W.H. Xiao, Y.P. Zhang, H.Z. Liu, Molecular systematics In this study, the mutation rate of 16S rRNA and cyt b of Xenocyprinae (Teleostei: Cyprinidae): Taxonomy, biogeography, and coevolution of a special group fragment was 0% and 0.53%, respectively, which
restricted in East Asia, Mol. Phylogenet. Evol. 18 (2001) showed a very close genetic relationship between the
163-173. [10] S.R. Palumbi, Nucleic acids II: The polymerase chain
two butterfly rays and confirmed that the 16S rRNA reaction, in: D.M. Hillis, C. Moritz, B.K. Mable (Eds.), was a more conserved gene.
Molecular Systematics, 2nd ed., Sinauer Associates, In conclusion, the morphological characteristics and Sunderland MA, 1996, pp. 205-247. [11] S. Kumar, K. Tamura, M. Nei, MEGA 3: Integrated the analysis of mtDNA sequences provide evidence
software for molecular evolutionary genetics analysis and that the twinspot butterfly ray, G. bimaculata, is a
sequence alignment, Brief. Bioinform. 5 (2004) 150-163.
A. Meyer, Evolution of mitochondrial DNA of fishes, in: junior synonym of Japanese butterfly ray, G. japonica. P.W. Hochachka, P. Mommsen (Eds.), Molecular Biology
Frontiers, Biochemistry and Molecular Biology of Fishes,
Acknowledgments
Elsevier Press, Amsterdam, 1993, pp. 1-38. [13] J. Briolay, N. Galtier, R.M. Brito, Y. Bouvet, Molecular
The authors thank J. Zhou, M.B. Luo, J.J. Chen, S.H. phylogeny of cyprinidae inferred from cytochrome b DNA Liu, Y.C. Bai, Y.M. Bi, H.J. Zhang, et al., for
sequences, Mol. Phylogenet. Evol. 9 (1998) 100-108. [14] A.P. Apostolidisa, Z. Mamurisb, C. Triantaphyllidis,
assistance with sample collection, and are grateful to Phylogenetic relationships among four species of Mullidae
the freshmen who worked on this project. This work (Perciformes) inferred from DNA sequences of was supported by the Special Research Fund for the
mitochondrial cytochrome b and 16S rRNA genes, Biochem. Syst. Ecol. 29 (2001) 901-909.
National Non-Profit Institutes (East China Sea [15] L. Zhao, J. Zhang, Z.J. Liu, S.M. Funk, F.W. Wei, M.Q. Xu, Fisheries Research Institute, No. 2008M04).
et al., Complex population genetic and demographic history of the Salangid, Neosalanx taihuensis, based on cytochrome
References b sequences, BMC Evol. Biol. 8 (2008) 201-228.
[16] J. Zhang, M. Li, M.Q. Xu, T. Takita, F.W. Wei, Molecular [1] Y.T. Chu, Fishes of the South China Sea, Science Press,
phylogeny of icefish Salangidae based on complete mtDNA Beijing, China, 1962.
cytochrome b sequences, with comments on estuarine fish [2] Y.T. Chu, C.L. Zhang, Q.T. Cheng, Fishes of the East
evolution, Biol. J. Linn. Soc. 91 (2007) 325-340.
Journal of Life Sciences 6 (2012) 14-19
Parameters Analysis of Gastric Motility Signals in Time Domain and Frequency Domain
Zhangyong Li, Likun Xu and Zhui Xu Biomedical Engineering Research Centre, Chongqing University of Posts and Telecommunications, Chongqing 404100, China
Received: June 13, 2011 / Accepted: July 22, 2011 / Published: January 30, 2012.
Abstract: In order to assess gastric motility, a new noninvasive method was addressed. Firstly, bio-impedance and stomach electric signals were recorded from the healthy control group and the pathologic stomach group. Wavelet transform was used to remove the influence of the heart activity signals. By analyzing and processing the two signals of the time domain and frequency domain, we get the corresponding parameters of the two groups. According to all the parameters, several verification tests have been carried out, from the result of the statistics, we can find that in both time and frequency domains, impedance signal and synchronize EGG (electrogastrogram) have some similar features. However synchronize EGG cannot be totally instead by gastric motility, especially in morbid state, EGG is not correspondence to impedance signal. The gastric contraction or gastric emptying is a complex procedure including electrical and mechanical activity. Electrical impedance (EIP) and the synchronous EGG should be analyzed together. In conclusion, the parameters have the value to evaluate gastric motility.
Key words: Gastric motility, signals, wavelet transform, time domain, frequency domain.
1. Introduction motility is still a challenge in the biomedical engineering and different ways will be explored [6].
Motility (or contractions) is one of the most critical Bioelectric impedance can be considered as a physiological functions of the human gut. Without powerful tool in diagnosis and medical research. Main coordinated motility, digestion and absorption of advantages of using bioelectric impedance are its dietary nutrients could not take place [1, 2]. noninvasive nature, low cost and ease of operation. Impairment in gastric motility results in delayed There are many applications using bio-impedance emptying of the stomach and leads to symptoms of signals for different pathological conditions, but its use nausea, vomiting, abdominal pain or discomfort, and so in assessment of gastric motility need to be explored in on [3, 4].
detail [7].
EGG (electrogastrogram) is a non-invasive and Gastric motility, stomach movement or emptying is convenience method used for the assessment of gastric
a complex course from electrical activity to mechanical motility [5]. Unfortunately, it only reflects frequency contraction and conduction. It begins from the of myoelectrical activity and falls to provide enough electrical activity of smooth muscle, and then develops information about gastric mechanical movement. Other gastric corpus and pylorus. In gastric active phase, such methods such as radionucleide scintigraphy, as contraction, peristalsis, the form and bulk of intracavitary pressure measurement, barostat checking, stomach change greatly, so the impedance of it changes gastroscopy, etc., fail to deliver a cost effective, simple evidently. The change regularity of the impedance to use and repeated use. The assessment of gastric corresponds to gastric motility. In order to investigate
Corresponding author: Zhangyong Li, Ph.D., professor, the complex course of electrical and mechanical
research field: medical signal processing. E-mail: activity of the gastric motility, including rhythm, li9547@yahoo.com.cn.
Parameters Analysis of Gastric Motility Signals in Time Domain and Frequency Domain
transmission, gastric emptying and the influences of less than 100 kHz. The stomach, additionally, being a them, it is necessary to extract gastric motility
muscular bag with a small volume in the fasting state information of both electrical and mechanical (approximately 50 mL) and becoming larger when a
activity [8]. meal is ingested, can be studied by electrical The bio-impedance method is a noninvasive, high
impedance (EIP). EIP is considered as the collection of sensitive technique to obtain the electrical feature
data in the form of electrical impedance measurements information corresponding to the gastric motility. Stain
at a suitable sampling rate, using surface electrodes et al. used bio-impedance technique to detect body
from the gastric area [10].
composition after gastric bypass and biliopancreatic EIP measurement system used in the present work diversion. Pereira et al. examined the impact of
generates an alternating current of 50 kHz, which can important weight loss on insulin inhibition of its own
be adjusted in intensity from 1 to 4 mA. The sampling secretion during experimentally induced rate of the data collection is 5 Hz and a multiplexed hyperinsulinemia under bariatric surgery. Murphy
system of electrodes is used to extract gastric compared the effects of tramadol and morphine on
impedance and synchronization EGG. gastric emptying in man [9].
2.2 Analysis Process
The fundamental frequency of the gastric impedance signal is about 0.05 Hz. The frequency may have
2.2.1 Data Acquisition
sudden changes over time, especially in abnormal Electrical impedance signals around abdomen of situation. When healthy control group (CG) and