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Molecular characterization of dunaliella salina and chlorella vulgaris fusant using 18SrDNA gene (Article)

,

Genetics Laboratory, Biology Department, Faculty of Mathematics and Natural Sciences, Diponegoro University, Jl.Prof. Soedarto, SH Tembalang, Semarang, 50275, IndonesiaMarine laboratory. Faculty of Faculty of Fisheries and Marine Science, Diponegoro University, Jl. Prof. Soedarto, SH

Tembalang, Semarang, 50275, Indonesia

AbstractProtoplast fusion was found to be an efficient method in improving carotenoid production from fusant ofcarotenogenic microalgae D. salina and C. vulgaris . Molecular characterization is needed for identifying thedominant parental genome in the fusant using ribosomal DNA sequences. The research was carried out by analyzingthe gene encodes for 18S rDNA of fusant and determining relationship of fusant with D. salina and C. vulgaris species from GenBank. Quantitative analysis showed that C. vulgaris was not remarkably dominant in fusant with 84 % similarity compare to D. salina with 82 % similarity. The result indicated that the fusant gained

both character from their parents. © 2016 Penerbit UTM Press. All rights reserved.

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Author keywords18SrDNA Chlorellla Dunaliella Protoplast fusion

Lee, Y.K., Tan, H.M. Interphylum Protoplast Fusion and Genetic Recombination of the Algae Porphyridium cruentum andDunaliella spp (1988) Journal of General Microbiology, 134, pp. 635-641.  .

 

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Lu, Y., Kong, R., Hu, L.

(2012) World Journal of Microbiology and Biotechnology, 28 (4), pp. 1827-1830.  . doi: 10.1007/s11274-011-0963-4

Santiago, C.M. Protoplast Fusion A New Tecnique for Genetic Manipulation and Breeding of Industrial Microorganisme. I.C (1982) Biotech, 5, pp. 435-440.

 

Pulz, O., Gross, W.

(2004) Applied Microbiology and Biotechnology, 65 (6), pp. 635-648.  . doi: 10.1007/s00253-004-1647-x

Borowitzka, M.A., Borowitzka, L.J. Dunaliella (1988) Micro-Algal Biotechnology.  . Borowitzka, M. A. (ed.), Cambridge: Cambridge University Press

 

Kusumaningrum, H.P., Zainuri, M. Optimization and Stability of Total Pigments Production of Fusan from Protoplast Fusion of MicroalgaeDunaliella and Chlorella in vivo. Attempts on Production of Sustainable Aquaculture Natural Food (2014) International Journal of Marine and Aquatic Resource Conservation and Co-Existance, 1 (1), pp. 1-5.  .

 

Guedes, A.C., Malcata, F.X. Nutritional Value and Uses of Microalgae in Aquaculture (2012) Aquaculture.  . Muchlisin, Z. A, Rijeka: InTech

 

Guedes, A.C., Amaro, H.M., Malcata, F.X.

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(2009) Biotechnology and Biotechnological Equipment, 23, pp. 892-895. doi: 10.1080/13102818.2009.10818566

Zainuri, M., Kusdiyantini, E., Widjanarko, Soedarsono, J., Yuwono, T. Preliminary Study on the Use of Yeast Phaffia rhodozyma as Pigment Source on The Growth of Tiger Shrimp(Penaeus monodon Fabricius) (2003) Indonesian Journal of Marine Sciences, 8 (1), pp. 47-52.

 

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Kusumaningrum, H.P., Zainuri, M. Detection of Bacteria and Fungi Associated with Penaeus monodon Postlarvae Mortality International Journal Procedia Environmental Sciences, 23, pp. 329-337.  .

 

Kusumaningrum, H.P.; Genetics Laboratory, Biology Department, Faculty of Mathematics and Natural Sciences,Diponegoro University, Jl. Prof. Soedarto, SH Tembalang, Semarang, Indonesia; email: © Copyright 2016 Elsevier B.V., All rights reserved.

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78:4–2 (2016) 61–68 |www.jurnalteknologi.utm.my | eISSN 2180–3722 |

Jurnal

Teknologi

Full Paper

MOLECULAR CHARACTERIZATION OF

Dunaliella salina AND Chlorella vulgaris

FUSANT USING 18SrDNA GENE

Hermin Pancasakti Kusumaningruma*, Muhammad Zainurib

aGenetics Laboratory, Biology Department, Faculty of

Mathematics and Natural Sciences, Diponegoro University, Jl. Prof.

Soedarto, SH Tembalang, Semarang – 50275, Indonesia bMarine laboratory. Faculty of Faculty of Fisheries and Marine

Science, Diponegoro University, Jl. Prof. Soedarto, SH Tembalang,

Semarang – 50275, Indonesia

Article history

Received

5 November 2015

Received in revised form

11 February 2016

Accepted

15 February 2016

*Corresponding author

herminsakti@gmail.com

Graphical abstract

Abstract

Protoplast fusion was found to be an efficient method in improving carotenoid production

from fusant of carotenogenic microalgae D. salina and C. vulgaris. Molecular

characterization is needed for identifying the dominant parental genome in the fusant

using ribosomal DNA sequences. The research was carried out by analyzing the gene

encodes for 18S rDNA of fusant and determining relationship of fusant with D. salina and C.

vulgaris species from GenBank. Quantitative analysis showed that C. vulgaris was not

remarkably dominant in fusant with 84 % similarity compare to D. salina with 82 % similarity.

The result indicated that the fusant gained both character from their parents.

Keywords: Chlorellla, Dunaliella, protoplast fusion, 18SrDNA

© 2016 Penerbit UTM Press. All rights reserved

1.0 INTRODUCTION

Recent decades has shown remarkable developing of

the biotechnology of microalgae. Valuable product

for food and other applications will extend into

broader area. Production of genetically improved

strain by hybridization and somatic fusion on algae

have been reported in algae. Protoplast fusion was

done on intraspesific species green microalgae

Chlamydomonas reinhardii (P. A. Dang, 1888) [1]. The

protoplast fusion between two different phyla which

sexually incompatible also has been conducted

between the red alga Porphyridium cruentum (S.F.

Gray) Nägeli, 1849) with D. bardawil (Ben-Amotz &

Avron, 1982) or D. salina (Dunal) Teodoresco 1905 [2].

D. salina is the source of β-carotene and glycerol [3, 4].

Chlorella is widely used as a healthy food and feed

supplement, as well as in the pharmaceutical and

cosmetics industries [5]. Improvement of valuable

metabolites from Chlorella and Dunaliella microalgae

was done using biotechnological methods that

allowed somatic hybridization by protoplast fusion [6].

This technique is required because crosses between

two strains will not occur naturally and a diploid is

desired. This intergeneric fusion also enables nuclear

and cytoplasmic genomes to be combined, fully or

partially, at the intergeneric levels. Carotenogenic

microalgae which live in different environmental

salinity, namely Chlorella and Dunaliella have

produced stable hybrids possessing combined

characteristics of the parents [6]. Their application as

natural supplement for aquaculture animal was

potential for synthetic feed substitution since it

contains proteins, carbohydrates, lipids and vitamins,

carotenoid as antioxidants, and trace elements [6–9].

Supplementation feeding on Penaeus monodon

postlarvae with microalgae exhibited significant effect

on growth, weight, survival related to microbial

diseases resistance and immune response in high and

low salinity [10–12]. In the present study, fusant

62 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

between D. salina and C. vulgaris were obtained. The

purpose of this study was conducting molecular

characterization on fusant using 18SrDNA to asses

genetic exchange among parental strains during

fusant formation. The methodologies were conducted

by isolation and amplification of 18SrDNA region of

fusant, followed by analysis on variable and conserved

sequences that were contained in the 18SrDNAs of the

organisms. The comparison of rDNA sequences

between parental and progeny is a potencial tool for

deducing dominancy and combination of each

parental to improve their advantages in fusant. The

present study expands on the use of DNA technology

for the genotype within the fusant comparing to

parental.

2.0 EXPERIMENTAL

2.1 D. salina and C. vulgaris

D. salina and C. vulgaris microalgae were obtained

from Brackishwater Aquaculture Development Centre

(BBPBAP) on Jepara, Indonesia. They were held in

seawater tanks, recirculated and aerated, with the

temperature set at 25 °C to 28 °C and salinity at 30 ‰

to 32 ‰. The tanks were cleaned daily. The

microalgae were cultivated using sea water enriched

with Walne media. A twocomponent gel, it is easy to

modify the molecular structure of either of the two

components.

2.2 Microalgae Media

Walne media for microalgae growth and cultivation

consist of H3BO3 3.36 g L1, NaNO3 10 g L1, FeCl3 0.15 g

L1, MnCl2.4H2O 0.36 g L1, Na2EDTA 45 mg L1, NaH2PO4

20 g L1, trace metal solution 1 mL L1, and distilled

water. Trace metal solution was consist of H3BO3 2.86 g

·L1; MnCl2.4H2O 1.81 g L1; ZnSO4.7H2O 0.222 g L1;

NaMoO4.5H2O 0.39 g L1; CuSO4.5H2O 0.079 g L1;

Co(NO3)2.6H2O 0.0494 g L1; pH 6.8. The ingredients

were dissolved in 200 mL of distilled water. The solution

was boiled for 10 min while adjusting the pH to 7.6 with

HCl or NaOH, filtered and bring to 1 L. Sterilization was

done by autoclaving at 15 lb in2 (103 kPa and 120 oC).

The medium was using by adding 0.1 mL solution to

each 10 mL of seawater [13, 14].

2.3 DNA Extraction

The DNA was extracted from 3 L media of the fusant in

logarithmic phase of growth. The preparation of a

DNA genomic of fusants was carried out by

modification of CTAB methods [15, 16]. DNA was kept

on -20 °C or used directly for PCR.

2.4 Amplification of 18SrDNA Microalgae

The 18SrDNA fragment was amplified using specific

primers. Sequence of forward primer was 5’

GTAGTCATATGCTTGTCT3’, reverse primer was 5’

GCTGGCACCASACTTGCCCT3’ [17]. PCR was carried

out in mixture containing 50 ng of genomic DNA,a 2.5

L PCR buffer (KAPA), a 10 mM concentration of

deoxynucleoside triphosphate mix (KAPA), 2.5 pmol of

each primer, and 0.625 U (1 U=1/60 micro katal) of Taq

Extra Hotstart DNA Polymerase and ddH2O until

volume 25 L. PCR conditions were performed

according to the PCR protocol using the

manufacturer’s instructions and protocols. To amplify

the 18SrDNA, amplification reactions were performed

on a T-Gradient thermocycler (Biometra GmbH,

Gottingen, Germany). Aliquots (1 L) of the reaction

mixtures were analyzed by 0.8 % horizontal agarose gel

electrophoresis to confirm the presence of product.

The PCR products were purified using the Gel PCR

Clean-Up System (Applied Biosystems, Foster, CA).

Sequencing reactions were performed using a Dye

Deoxy Terminator Cycle Sequencing Ready Reaction

Kit (Applied Biosystems, Foster City, CA), and

sequencing fragments were analyzed on a ABI Prism

377 DNA Sequencer.

2.5 Sequencing and Phylogenetic Analysis

The 18S rDNA partial fragment gene sequences from

the fusant were searched against GenBank using

BLAST as illustrated in Table 1. The nucleotides were

aligned using the program ClustalX, respectively.

Sequences containing fewer than 400 nucleotides or in

excess of 1 000 nucleotides were removed, and

sequences not belonging to D. salina and C. vulgaris

microalgal species were discarded from this study. A

phylogenetic tree was constructed using the

neighborjoining (NJ) algorithm [18] using Kimura’s

twoparameter model of sequence evolution, as

implemented in the MEGA5 program package [19].

The bootstrap consensus tree inferred from 1 000

replicates [20] is taken to represent the evolutionary

history of the taxa analyzed. Branches corresponding

to partitions reproduced in less than 50 % bootstrap

replicates are collapsed. The percentage of replicate

trees in which the associated taxa clustered together

in the bootstrap test are shown next to the branches.

The evolutionary distances were computed using the

Jukes-Cantor method [21] and are in the units of the

number of base substitutions per site. Codon positions

included were 1st + 2nd + 3rd . All positions containing

gaps and missing data were eliminated.

63 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

Table 1 Dunaliella and Chlorella species rDNA sequence from Genbank used in this study

Species Accs number Species Accs number

C. vulgaris AB080308.1 D. salina strain KU13 KF825552.1

C. vulgaris isolate UMT-M1 KJ561358.1 D. salina strain KU07 KF825551.1

C. vulgaris isolate KS-MA2 KJ561357.1 D. salina strain KU11 KF825550.1

C. vulgaris KF574391.1 D. salina strain KU07 KF825551.1

C. vulgaris cc CCAP 211/79 FR865683.1 D. salina isolate B32 HQ735296.1

C. vulgaris strain KTP2 KF746940.1 D. salina isolate I3 HQ735295.1

C. vulgaris strain nm27 JQ256478.1| D. salina strain B34 JF900404.1

C. vulgaris isolate YL-2 KP341004.1 D. salina strain B24 JF831044.1

C. vulgaris strain A1-65 KF661335.1 D. salina EF195157.1

C. vulgaris strain CCAP211/21A KJ756823.1 D. salina strain CCAP 19/12 KJ756842.1

C. vulgaris strain CCAP211/75 KJ756813.1 D. salina str BuriRam KU01 JN052202.1

C. vulgaris strain LC9 KF569735.1 D. salina strain I2 JF831045.1

C. vulgaris strain LC8 KF569734.1 D. salina strain CCAP 19/18 EF473745.1

C. vulgaris strain LF5 KF569724.1 D. salina strain SAG 19-3 EF473739.1

C. vulgaris strain AG-35_ZF1 AB699112.1 D. salina strain Dsge EF473731.1

C. vulgaris cc CCAP211/11P FR865658.1 D. salina strain KMMCC 1428 JQ315781.1

C. vulgaris strain CCAP211/110 FN298918.1 D. salina M84320.1

C. vulgaris strain CCAP211/109 FN298917.1 D. salina strain CCC HQ843776.1

C. vulgaris strain CCAP211/82 FM205855.1 D. salina strain JR102 EU589200.1

C. vulgaris strain CCAP211/81 FM205854.1 D. salina EU239363.1

C. vulgaris strain CCAP211/80 AM231734.1 D. salina AF506698.1

C. vulgaris strain CCAP 211/11F AY591515.1 D. salina M84320.1

C. vulgaris strain CCAP 211/63 FR865681.1 D. salina strain CCC HQ843776.1

C. vulgaris strain SAG 211-11b FM205832.1 D. salina strain JR102 EU589200.1

C. vulgaris strain KMMCC FC-15 HQ702287.1 D. salina EU239363.1

C. vulgaris strain KMMCC FC-12 HQ702286.1 D. salina AF506698.1

C. vulgaris strain KMMCC FC-12 HQ702286.1 D. salina M84320.1

C. vulgaris strain KMMCC C-111 GQ122346.1 D. salina strain CCC HQ843776.1

C. vulgaris strain KMMCCFC-16 HQ702294.1 D. salina strain JR102 EU589200.1

C. vulgaris strain KMMCCC-117 HQ702318.1 D. salina strain B33 JF831042.1

C. vulgaris strain KMMCCC-119 HQ702309.1 D. salina strain UTEX LB 200 DQ009779.1

C. vulgaris strain KMMCCFC-42 HQ702285.1 Dunaliella sp. BBPBAP KC875350

C. vulgaris strain KMMCCEC-5 HQ702321.1 D. salina isolate NIOT-28(ANCOST-17) KC470060.1

C. vulgaris strain KMMCCFC-33 HQ702295.1 D. salina strain CCAP 19/30 DQ447648.1

C. vulgaris cc KMMCC FC-1 GQ122369.1 C. vulgaris cc KMMCC C-27 GQ122334.1

C. vulgaris strain NIES-1269 AB488579.1 C. vulgaris var vulgaris strNIES-642 AB488577.1

C. vulgaris strain: PS-2670 AB488582.1 C. vulgaris var vulgaris strNIES-227 AB488575.1

Chlorella sp. WO10-1 FJ946886.1 C. vulgaris var vulgaris str NIES-641 AB488576.1

C. vulgaris strain KMMCCEC-10 HQ702292.1 C. vulgaris var vulgaris str NIES-686 AB488578.1

C. vulgaris strain KMMCCEC-3 HQ702293.1 C.vulgaris isolate D2 JX185298.1

C. vulgaris strain KMMCCFC-16 HQ702294.1 C. vulgaris strain IAM C-27 AJ242757.1

C. vulgaris strain KMMCCC-119 HQ702309.1 C. vulgaris strain KMMCCC-111 GQ122346.1

C. vulgaris strain KMMCCFC-42 HQ702285.1 C. vulgaris cc KMMCC C-88 GQ122340.1 Note: cc = culture collection; v= varian; accs = accession

3.0 RESULT AND DISCUSSION

3.1 Amplification of 18SrDNA fusant

The products of the 18SrDNA fusant amplification are

shown in Figure 1. Several annealing temperature were

applied due to anticipation of genetic diversity inside

fusant genome. Almost all temperature annealing

showed positive bands with 55.2 °C exhibited the best

result. Those data confirmed that primer design was

suitable to conserved region of fusant.

Figure 1 Amplification of 18SrDNA fusant with several

annealing temperature using gradient PCR. Lane 1:

molecular weight marker. Lanes 2 to 10: amplification with

annealing temperature 45 °C; 46.6 °C; 48.4 °C; 50.9 °C; 52.9

°C; 55.2 °C; 57.2 °C; 59.9 °C; 61.7 °C; and 63 °C, respectively

M 1 2 3 4 5 6 7 8 9 10 M

64 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

Furthermore, the primer also can be used to amplify

fusant and also obtained similar band from parental

D. salina confirmed that primers are highly specific

to D. salina. On the other hand, by applying primer on

parental C. vulgaris single band was obtained but the

sequences were not as good as D. salina and fusant.

This result indicated that the primer was not in the

conserved region of C. vulgaris in amplifying specific

band. Implication of this result also showed the

divergence of sequences in C. vulgaris comparing

with D. salina. However, this result has not interfered

with the homology analysis of fusant sequences since

C. vulgaris showed conserved region among them.

Fusant 19 ATTGTACTCATTCCGATT-GCAGAACCCGAAGGGCTCCGCATCGTTATTTATTGTCACTACCTCCCTG 85

|||||||||||||| ||| ||| | | || || | | || ||||||||||||||||||||||| |

D.salina 469 ATTGTACTCATTCCAATTACCAG-A-CAAAAATGCCCGGTATTGTTATTTATTGTCACTACCTCCCCG 402

Fusant 86 TGTTAGGATTGGGTAATTTACGCGCCTGCTGCCTTCCTTAGATGTGGTAGCCGTTTCTCAGGCTCCCT 153

|||| |||||||||||||| ||||||||||||||||||| ||||||||||||||||||||||||||||

D.salina 401 TGTTGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCT 334

Fusant 154 CTCCGGAATCGAACCCTAATTCTCCGTTACCCGTTAACGCCACGGTAGGCCAATACCCTACCGTCGAA 221

|||||||||||||||||||| |||||| |||||||| | ||| |||||||| || |||||| |||||

D.salina 333 CTCCGGAATCGAACCCTAATCCTCCGTCACCCGTTACCACCATGGTAGGCCTCTATCCTACCATCGAA 266

Fusant 222 AGCTGATAGGGCAGAAACTTGAATGAACCATCGT-GCCG-AA-GCA---CGATTCGCTTAGTTATTAT 283

|| |||||||||||||| ||||||||| ||||| || || || ||||||| |||||| |||

D.salina 265 AGTTGATAGGGCAGAAATTTGAATGAAACATCGCCGGCATAAAGCCGTGCGATTCGTGAAGTTATCAT 198

Fusant 284 GACTCACCA-G-G--G---ATT---G-CTGG--TTGTATCTAATAAATACACCTCTTGC-GAGGTTGG 337

|| || ||| | | | | | |||| || |||||||||||||| | ||| | || || ||

D.salina 197 GATTCGCCAAGAGTCGGGCAAGCCCGGCTGGCCTTTTATCTAATAAATACGTCCCTTCCAGAAGTCGG 130

Fusant 338 -----ACGCATGTATTAGCTCTAGAATTACTACGGTTATCCAAGTAGTAGGGGACTATCAAATAAACT 400

||||| |||||||||||||||||||||||||||||| |||| ||| || ||||||||||||

D.salina 131 GATTTACGCACGTATTAGCTCTAGAATTACTACGGTTATCCGAGTAA-AGGT-ACCATCAAATAAACT 66

Fusant 401 ATAACTGATATAATGAGCCATTCGCAGTTTCACCGTATAA-AGGCTTATACTTAG-ACATGCA 461

||||||||| ||||||||||||||||||||||| |||||| | |||||||||| |||||||

D.salina 65 ATAACTGATTTAATGAGCCATTCGCAGTTTCACAGTATAAGCAGTTTATACTTAGGACATGCA 3

Figure 2 Similarity among fusant and D. Salina

Fusant 1 CATGTCT-AGTAT-AGC--CTTTATACGGTG-AACTGCGAATGGCTCATTATATCAGTTATAGTTTAT 63

||||||| ||||| | | |||||||| ||| ||||||||||||||||||| ||||||||||||||||

C.vulgaris 53 CATGTCTAAGTATAAACTGCTTTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTAT 120

Fusant 64 TTGATAGTCCCCTACTACTTGGATAACCGTAGTAATTCTAGAGCTAATACATGCGT-----CCAACCT 131

||||| || ||||||||| ||||| ||||||||| |||||||||||||| ||||| || || |

C.vulgaris 121 TTGATGGT-ACCTACTACTCGGATACCCGTAGTAAATCTAGAGCTAATACGTGCGTAAATCCCGACTT 187

Fusant 132 C-GCAAGAGGTGTATTTATTAGATACAA--CC-A----GCAAT--CC--CT---GGTGAGTCATAATA 185

| | ||| | |||||||||||||| || || | || | || || ||||| |||| |||

C.vulgaris 172 CTGGAAGGGACGTATTTATTAGATAAAAGGCCGACCGGGCTCTGCCCGACTCGCGGTGAATCATGATA 255

Fusant 186 ACTAAGCGAATCG--T-G-CTT-CGGC-ACGATGGTTCATTCAAGTTTCTGCCCTATCAGCTTTCGAC 241

||| ||||||| | | ||| || | ||||| ||||||||| |||||||||||||| |||||||

C.vulgaris 256 ACTTCACGAATCGCATGGCCTTGCGCCGGCGATGTTTCATTCAAATTTCTGCCCTATCAACTTTCGAT 323

Fusant 242 GGTAGGGTATTGGCCTACCGTGGCGTTAACGGGTAACGGAGAATTAGGGTTCGATTCCGGAGAGGGAG 309

|||||| || |||||||| ||| | |||||||| |||||| ||||||||||||||||||||||||||

C.vulgaris 324 GGTAGGATAGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAG 391

Fusant 270 CCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAGGCGCGTAAATTACCCAATCCTAACACAGGGA 377

|||||||||||||||||||||| ||||||||||||||||||| ||||||||||||||| |||||||||

C.vulgaris 352 CCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAGGGA 459

Fusant 330 GGTAGTGACAATAAATAACGATGCGGAGCCCTT-CGGGTTCTGC-AATCGGAATGAGTACAATTTAAA 443

||||||||||||||||||| || | | ||| || | ||| ||| ||| |||||||||||||| ||||

C.vulgaris 412 GGTAGTGACAATAAATAACAATACTGGGCCTTTTCAGGT-CTGGTAATTGGAATGAGTACAATCTAAA 525

Fusant 390 CCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAG 487

|||||||||||||||| |||||||||||||||||||||||||||

C.vulgaris 472 CCCCTTAACGAGGATCAATTGGAGGGCAAGTCTGGTGCCAGCAG 570

Figure 3 Similarity among fusant and C. vulgaris

65 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

3.2 Sequence Analysis of Fusant

Sequence analysis of 18S rDNA fragmen nucleic acid

in GenBank GenBank and European Bioinformatics

shows close relationship between fusant and D. salina

strain BBPBAP and C. vulgaris. Analysis of homology

between fusan and D. salina showed 82 % homology

(Figure 2). Analysis of homology between fusan and C.

vulgaris showed 84 % homology (Figure 3). Identities

founded 320 in 435 with gaps 6 %.

Homology analysis inside the 18SrDNA sequences of

fusant and parental identified 77 % similarity in the

region as illustrated in Figure 4. Both D. salina and C.

vulgaris exhibited conservation region between

sequences and the similarity among them was 94 % in

the 18SrDNA region which suggested that this

sequence was well conserved between species

of Dunaliella and Chlorella. Analysis and comparison

of sequences on fusant and parental detected 77 %

similarity with 33 SNPs and 24 deletion in the 18SrDNA

region (Figure 4). A number of base were inherited

between two parents into the fusant exhibiting by

several substitution. Combination of bases from two

parents into the fusant made differences from both

parent but C. vulgaris tend to inherited more dominant

comparing with D. salina.

C.vulgaris : C-TAAGTATAAACTGCTTTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGAT : 69

D.salina : CCTAAGTATAAACTGCTT-ATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGAT : 70

Fusant : CATGTCTAGTATAGCCTTTATACGGTGAA-CTGCGAATGGCTCATTATATCAGTTATAGTTTATTTGAT : 70

C TaagTAtaAactgCTTtATACtGTGAAaCTGCGAATGGCTCATTAaATCAGTTATAGTTTATTTGAT 70

80 * 100 * 120 *

C.vulgaris : GGT-ACCTACTACTCGGATACCCGTAGTAAATCTAGAGCTAATACGTGCGTAAATCCCGACTTCTGGAA :139

D.salina : GGTACCTT--TACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCGTAAATCCCGACTTCTGGAA :137

Fusant : AGTCCCCTACTACTTGGATAACCGTAGTAATTCTAGAGCTAATACATGCGT-----CCAACCTC-GCAA :135

gGT cCcTacTACTcGGATAaCCGTAGTAAtTCTAGAGCTAATACgTGCGTaaatcCCgACtTCtGgAA 69

140 * 160 * 180 * 200

C.vulgaris : GGGACGTATTTATTAGATAAAAGGCCGACCGGGCTCTGCCCGACTCGCGGTGAATCATGATAACTTCAC :208

D.salina : GGGACGTATTTATTAGATAAAAGGCCAGCCGGGCT-TGCCCGACTCTTGGCGAATCATGATAACTTCAC :209

Fusant : GAGGTGTATTTATTAGATA------CAACCAG--------CAATCCCTGGTGAGTCATAATAACTAAGC :196

GgGacGTATTTATTAGATAaaaggcCaaCCgGgct tgccCgActC tGGtGAaTCATgATAACTtcaC 68

* 220 * 240 * 260 *

C.vulgaris : GAATCGCATGGCCTTGCGCCGGCGATGTTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGAT :278

D.salina : GAATCGCACGGCTTTATGCCGGCGATGTTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGAT :279

Fusant : GAATCGTG----CTTCGGC--ACGATGGTTCATTCAAGTTTCTGCCCTATCAGCTTTCGACGGTAGGGT :260

GAATCGca ggccTT GCcggCGATGtTTCATTCAAaTTTCTGCCCTATCAaCTTTCGAtGGTAGGaT 67

280 * 300 * 320 * 340

C.vulgaris : AGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAA :348

D.salina : AGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAA :349

Fusant : ATTGGCCTACCGTGGCGTTAACGGGTAACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAA :330

AgaGGCCTACCaTGGtGgTAACGGGTgACGGAGgATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAA 70

* 360 * 380 * 400 *

C.vulgaris : CGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACA :418

D.salina : CGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCAACACGGGGAGGTAGTGACA :419

Fusant : CGGCTACCACATCTAAGGAAGGCAGCAGGCGCGTAAATTACCCAATCCTAACACAGGGAGGTAGTGACA :400

CGGCTACCACATCcAAGGAAGGCAGCAGGCGCGcAAATTACCCAATCCtaACACaGGGAGGTAGTGACA 70

420 * 440 * 460 * 480

C.vulgaris : ATAAATAACAATTACTGGGCCTTTTCAGGTCTGGTAATTGGAATGAGTACAATCTAAACCCCTTAACGA :488

D.salina : ATAAATAACAAT-ACCGGGCATTTTT--GTCTGGTAATTGGAATGAGTACAATCTAAATCCCTTAACGA :487

Fusant : ATAAATAACGAT-GCGGAGCCCTT-CGGGTTCTGCAATCGGAATGAGTACAATTTAAACCCCTTAACGA :468

ATAAATAACaAT aC GgGCctTTtc gGTctgGtAATtGGAATGAGTACAATcTAAAcCCCTTAAC 65

* 500 * 520

C.vulgaris : GGATCAATTGGAGGGCAAGTCTGGTGCCAGCAG :521

D.salina : GTCTCCTTTGGAAGGCAAGTCTGGTGCCAGCAG :520

Fusant : GGATCCATTGGAGGGCAAGTCTGGTGCCAGCAG :501

GgaTCcaTTGGAgGGCAAGTCTGGTGCCAGCAG

Figure 4 Homology region among fusant, D. salina and C. vulgaris

66 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

Figure 5 Phylogenetic evolutionary tree displaying the evolutionary relationship of fusant within a lineage shared by the C. vulgaris

species with Dunaliella sp. BBPBAP as an outgroup (str = strain)

Selection on related similarity of fusant with other D.

salina and C. vulgaris species with the

genus Dunaliella, exhibited the 100 and 102 most

related species. Alignment result from selected species

showed that all the species had quite high

percentage of similarity throughout the sequences (81

% to 85 %), presenting such differences inside the

sequences. Analysis by multiple alignment methods

revealed a close relationship of fusant with member of

D. salina and C. vulgaris as illustrated from consensus

tree using phylogenetic evolutionary analisis in Figure 5

and Figure 6. The reliability of the tree topology was

estimated by bootstrapping. The 80 % bootstrap

proportion consensus Neighbor Joining tree for

18SrDNA sequences is shown in Figure 5 and Figure 6.

Homology analysis with C. vulgaris species retrieved

from GenBank is illustrated in Figure 5. which shows

close similarities between a green algae isolates with

those of C. vulgaris strain KMMCC FC-41 and C.

vulgaris strain nm27. According to the phylogenetic

tree, the studied fusant appeared as individual entity

separated from the rest cluster. It was clearly shown

Duna liella sp. BBPBAP Fusant

C. vulgaris str nm27

C. vulgaris CCAP 211/79 C. vulgaris AB080308

C. vulgaris KMMCCFC-1

C. vulgaris strKMMCCFC-15

C. vulgaris str KMMCCFC-42

C. vulgaris strKMMCCFC-12

C. vulgaris str KTP2 C.vulgaris isolate C. vulgaris str CCAP 211/63 C. vulgaris strA1-65

C. vulgaris strCCAP

C. vulgaris strCCAP 211/80

C. vulgaris strLF5

C. vulgaris ccCCAP 211/11P C. vulgaris str

C. vulgaris str: AG-35_ZF1

C. vulgaris strCCAP 211/109

C. vulgaris str IAMC-27

C. vulgaris str: NIES-2172 C. vulgaris var. vulgaris str: NIES-641

C. vulgaris var. vulgaris str: NIES-227 C. vulgaris str: NIES-2170

C. vulgaris var. vulgaris str :NIES-642

C. vulgaris str: NIES-1269

C. vulgaris str: PS-2670

C. vulgaris var. vulgaris strNIES:-

C. vulgaris strKMMCCEC-16

C. vulgaris strKMMCCEC-3

C. vulgaris strKMMCCEC-10

C. vulgaris strKMMCCFC-333

C. vulgaris strSAG 211-11b

C. vulgaris strCCAP 211/110

C. vulgaris strCCAP 211/75

C. vulgaris str

C. vulgaris strCCAP211/8 1 C. vulgaris strCCAP 211/8 2

C. vulgaris strY2

3

671

C vulgaris . strOW01 C .vulgaris strUMTM1

C.vulgaris strKSMA2

C . vulgaris KF574391 C. vulgaris ccKMMCCC-27 C. vulgaris strKMMCCC-111

C. vulgaris ccKMMCCC-88

C. vulgaris strKMMCCC-117

959

669

417

513

C. vulgaris ccKMMCC:FC-

C. vulgaris strKMMCC FC-41

566

1000

0.01

67 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

that fusant, instead of having almost equal similarities

with all member of other C. vulgaris, it also had close

relationship with Dunaliella sp. BBPAP as the other

parental.

Figure 6 Phylogenetic evolutionary tree displaying the evolutionary relationship of fusant within a lineage shared by the D. Salina

species. C. vulgaris is used as an outgroup sequence

Homology analysis with D. salina species in Figure

6. showed closest similarities between a green algae

isolates with those of D. salina KU 13. However,

phylogenetic evolutionary tree showed the position

of fusant outside of the cluster of parental. These

results suggested that fusant gained different

character with parental species. In this sense, C.

vulgaris seemed to be more related to D. salina,

which was consistent with homology analysis result.

Almost all of the fusant obtained from protoplast

fusion process suggested higher total carotenoid

production after the process compared with the

parental strain (data not shown).

Although the two profiles of fusant and parental

shared similarity, but the position of fusant in different

cluster with parental indicating that there was indeed

a difference in the cell of the two algal strains. On

the basis of the gained results we could conclude

that the dominant genotype in fusants between D.

salina and C. vulgaris appertain to C. vulgaris. The

difference in the bases profiles is further evidence

that there is indeed a change at the genomic level.

The result also showed that 18SrDNA gene can be

used to calculate dominant genotype in fusant

resulted from protopast fusion process.

4.0 CONCLUSION

Molecular analysis showed that C. vulgaris was more

dominant in fusant comparing with D. salina based

on homology analysis of 18SrDNA sequences. The

result also indicated that the fusant gained both

character from their parents due to conserved

sequence of 18SrDNA in parental and progenitor. The

research showed possibilities in potential acquisition

of genomic combination of both parents.

Acknowledgement

This research was funded by Directorate Research

and Public Services (Ditlitabmas), General of Higher

Education (Ditjen Dikti), Ministry of Research,

Technology and Higher Education, Indonesia year

2015 according to number 023.04.02.1.67345/2015–

Chlorella vulgaris CCAP21111F

D. salina strain

D. salina strain

D. salina strain B34

D. salina str MBTD-CMFRI-S135

D. salina isolate B32

D. salina strain I2

D. salina isolate I3

D. salina strain B33

D. salina strain B25

D. salina strain N5

D. salina strain

D. salina isolate NIOT-28(ANCOST-17)

D. salina strain B24

917

D. salina strain KMMCC

600

D. salina strain KMMCC 1511

428

D. salina strain KMMCC 1064

D. salina str MBTD-CMFRI-S089

D. salina cc DCCBC:CCAP19/20

D. salina strain CS265

D. salina

D. salina strain UTEX LB 200

D. salina strain CCAP

D. salina strain Dsge

D. salina str BuriRam KU01

D. salina strain SAG 19-3

D. salina strain CCAP

D. salina

D. salina strain JR101

D. salina strain JR102

D. salina AF506698

D. salina strain UTEX LB 1644

D. salina strain CCAP

Dunaliella sp. BBPBAP

742

Dunaliella sp. BBPBAP

898

0.01

68 Hermin Pancasakti Kusumaningrum & Muhammad Zainuri / Jurnal Teknologi (Sciences & Engineering) 78:4–2 (2016) 61–68

DIPA 03 Maret 2015 which was gratefully

acknowledged.

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