TRANSACTIONS ON SCIENCE AND TECHNOLOGY (28S rRNA, 23S rRNA, ITS1, 5.8S rRNA and ITS2), mitochondrial cytochrome C oxidase ( oxidase C cytochrome mitochondrial ITS2), and rRNA 5.8S ITS1, rRNA, 23S rRNA, acid (28S ribonucleic ribosomal the targeting reaction chain polymerase of analysis comparative based (Donaldson AFLP 1998), (Patwary as RAPD such markers molecular DNA using by species seaweed of differentiation and characterization physical other genetic biodiversity of seaweeds is still in its infancy. and var size intraspecificofassessmentknown the because the poorly still theirbiodiversityaddition,is In Malaysia. of structure, classification and branch identification the coloration, on are which morphologies, based observations phenotypic seaweed varieties but decades, recent in significantly waters cul as reported been have varieties 15 than more where carrageenophytes, Agardh J. s cultivated commonly the where INTRODUCTION © Transactions on Science and Technology Received Full Article V Eucheuma ISSR Transac Kappaphycus; KEYWORDS: of development for applications other various and technology breeding seaweed industries. in used be can analysis ISSR from obtained tools molecular as applied be can these and samples seaweed 30 seaweed varieties have thesame profiles. band Interestingly, 22identical variety results polymorphic. were 94.3% which of amplified, successfully were bp 4,600 to 180 of bands 1494 of total of total A method. characterize to forthinter using fingerprinting genetic the set on based varieties study present The disease. to resistance and rates faste content, carrageenan higher as such characteristics desired exhibit species same the of varieties seaweed Malaysia ABSTRACT arieties rvos tde rpre ta svrl molecul several that reported studies Previous Seaweedstheoneof are economically important thefor crops carrageenan industry Malaysia, in tions on Science and Technology
30 August 2017
showed a clear differentiation amo differentiation clear a showed - (Tan
Agricultural biotechnology .
Identification of the different varieties of varieties different the of Identification - Icesn dmns ae e t te ai ices i te amn o these of farming the in increase rapid the to led have demands Increasing . PCR Kappaphycus
e Eucheuma seaweeds. t al. t Online eight ISSR primers were used to produce distinct and reproducible patterns of polymorphic bands. A A bands. polymorphic of patterns reproducible and distinct produce to used were primers ISSR eight t al. et Grace Joy Wei Lie Chin Wei Lie Joy Grace
. , 2013). ,
The rudimentarymethodof identificationcurrently
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28 28 November 2017 19) M13 1993), , F rom Sabah, Malaysia
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Eucheuma 2017 , No. et et al. production of these economically important seaweeds has seaweeds important economically these of production 19) ad SP (Zuccarello SSCP and 1998), , ly influenced by environmental factors. This has led to confusion in confusion to led has This factors. environmental by influenced ly
Biotechnology Research 3
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eaweeds are belonging to genera to belonging are eaweeds 201x. Wilson Thau Lym Yong Lym Wilson Thau 420 - igrrnig (Coyer fingerprinting ng the cultivar varieties of varieties cultivar the ng
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Transactions on Science and Technology. 425 ewes r cmol cliae fr carrag for cultivated commonly are seaweeds farmer , 201
inter # Kappaphycus 7 Corresponding author. E
s - simple sequence r sequence simple Institute, - # simple sequence repeat sequence simple had , Mohd Za , Mohd eis of ieties
Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, MALAYSIA. encounter
and
for the identification of seaweed varieties. The data The varieties. seaweed of identification the for r prahs ae en ple in applied been have approaches ar et al. et - Kappaphycus Kappaphycus Mail: [email protected] Eucheuma Kappaphycus rifi Mohamad, Salahaudin Maili, Salahaudin Mohamad, rifi epeat t al. et ed 94, irstlie (Wattier microsatellites 1994), ,
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Kenneth Francis Rodrigues Rodrigues Francis Kenneth 3 – ), challenges 19) Mr rcnl, sequence recently, More 1999). , - – polymerase chain reaction (ISSR reaction chain polymerase Kappaphycus 420 specific ISSR were bands obtained from Kappaphycus seaweeds is important because certain because important is seaweeds polymerase chain reaction (ISSR reaction chain polymerase
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The dendogram The ewes in seaweeds and
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TRANSACTIONS ON SCIENCE AND TECHNOLOGY xenl opooia ciei dfeetaig oa vreis wie h wl sampl wild the while varieties; local differentiating individually described. their on criteria based named morphological and classified were external samples Cultivated diving. scuba and snorkeling via (Seban Sempornaarea the aroundlocations various from collected Seaweed samples METHODOLOGY of authentication the for varieties at genom method molecular valuable a is technique ISSR the demonstrate will data These 2012). (Sarwat, biology evolutionary and mapping, fingerprinting, genomic ISSR the polymorphic study highly to of markers use the involves which method quick and effectiveness, cost simple, of varieties different among of level variation the differentiate Eucheuma cannot and level, species to up differentiate al et thebetween and ., 2012; de 2012; ., inter study, this In ttl f 0 amd n wild and farmed 30 of total A cox2 -
3 seaweed species.
spacer), and plastid plastid and spacer), Kappaphycus - Barros Barretto Barros
ic DNAic level.
Table 1.
- ipe eune eet IS) ehd a ue to used was method (ISSR) repeat sequence simple seaweeds(Zuccarello
Chin
Details of collected seaweed samples thisin study Kappaphycus et et al. et al., et
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7 201 ISSN ISSN 2289 .
ee ae en tlzd o vlae h gntc relationship genetic the evaluate to utilized been have gene Kappaphycus Transactions on Science and Technology. 2; Tan 2;
- and and 8786. et al., al., et eei dvriy pyoey gn tgig genome tagging, gene phylogeny, diversity, genetic et al. et http://transectscience.org/ Eucheuma
and , 2006; Conklin2006; , 2013). Eventually these approaches could only could approaches these Eventually 2013). Eucheuma ewes Ti tcnqe s reliable, a is technique This seaweeds. Kappaphycus
4 ( 3 -
3 ewe smls Tbe ) were 1) (Table samples seaweed gkat and Selakan Island) in Sabahin Island) Selakan and gkat ) , et al. et 420
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425 , 2009; Zhao & He, 2011; Tan2011; He, Zhao & 2009; ,
and
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Eucheuma Kappaphycus
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and 421
TRANSACTIONS ON SCIENCE AND TECHNOLOGY prxmtl 10 o 4,600 polymorphismthat ratesea to 180 here shown approximately (not primers ISSR 8 alvarezii H= Heterozygosity, PIC = Polymorphic information content BRIAGG4 seaweed varieties. 3. Table RESULT DISCUSSIONAND arithmetic mean (UPGMA). with method group pair unweighted by dendrogram a develop to performed was analysis cluster d genetic Nei’s the comparison, Data analysis QIAxcel DNA High Resolution Kit. (O’Doherty cycler 2007). thermal a on Sherwood, min 10 for 72°C at extension final lastly and 72°C, at extension denaturing s 20 of cycles d initial with performed was PCR The polymerase. MgCl mM 1.5 buffer, PCR amplification (PCR) cons µl 20 of volume a in out carriedwere amplification reaction chain polymerase DNA isolatio BRIAAC4 BRICCT4 ISSR 12 ISSR 10 ISSR Primer ISSR 5 ISSR 2 ISSR 1 ISSR oa DA a etatd sn te rtcl f Yang of protocol the using extracted was DNA Total eei dvriy n gntc eainhp ewe 3 saed ape o te seis ( species 3 the of samples seaweed 30 between relationship genetic and diversity Genetic bands. of (0) presence and (1) absence on based primer each for performed was Scoring
,
K. striatus K.
SR igrrns aa o te molecu the for data fingerprints ISSR ISSRPrimer BRIAGG4 BRIAAC4 BRIAAC6 BRICTT4 alleles ISSR 12 ISSR 10 ISSR No. ofNo. n andPCR amplification
6 5 5 5 ISSR 5 ISSR 2 ISSR 1 ISSR 3 3 1 1 ------
12 11 13 12 5 8 9 8
adn pten wr aaye wt te Ixe Avne Sse using System Advanced QIAxcel the with analyzed were patterns Banding and
430 280 370 180 300 230 30 600 size (bp) size
Product Product 0 E. denticulatum E. ------
1900 2100 4300 3100 4600 1800 3000 2900 at 95°C, 40 s annealing at 40 at annealing s 40 95°C, at ₂ GGG AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC GGG , 0.2 mM dNTPs of each nucleotide, 10 pmol primer, and 2.5 U U 2.5 and primer, pmol 10 nucleotide, each of dNTPs mM 0.2 , ch testedTableISSR3. primer ch is shownin produced Chin
Table 2.
et et al. CTC TCT CTC TCT CTC TGC CTC TCT CTC TCT CTC TCT CTC istance matrix (Nei, 1972) was generated among the 30 varieties and varieties 30 the among generated was 1972) (Nei, matrix istance GGG AAC AAC AAC AAC AAC AAC AAC GGG AGG AGG AGGAGG CCC p ee ucsfly observed. successfully were bp of scored bands ofscored CAC ACA CAC ACA GG ACA CAC ACA CAC GGG CTT CTT CTT CTT CTT CTT CTT GGG Total number number Total ,
201
GAG GAG GAG CC GAG GAGGAG CAC CAC CAC GC CAC CAC CAC , la ad erdcbe N bns agn i sz from size in ranging bands DNA reproducible and clear ),
Sequence (5’ (5’ Sequence 7 ISSN ISSN 2289 .
Transactions on Science and Technology. ISSR primers tested thisin study 240 212 238 148 166 138 272 ) 80 were analyzed. According to the ISSR the to Accordinganalyzed. were CTC TCT CTC TAC CTC TCT CTC
- 8786. http://transectscience.org/ – a caatrzto of characterization lar
polymorphic bands polymorphic 3’) Total number of number Total
ntrto a 9° fr min 3 for 95°C at enaturation sn e using
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isted of the genomic DNA (50 DNA genomic the of isted
224 206 224 138 164 122 268 50°C according to primer used and 1 min 1 and used primer to according 50°C 74
t al et gt SR rmr (al 2) (Table primers ISSR ight
4 ( 3 (03 ad sd meitl for immediately used and (2013) . - 3 ) Tm (˚C) Tm , 420
oa nme o bns and bands of number Total 41.0 50.5 46.7 46.2 44.4 53.7 44.5 53.0 44.7
-
Polymorphism 425
Kappaphycus
93% 97% 94% 93% 99% 88% 98% 93% - O’Doherty and and O’Doherty PCR results using results PCR
Table 3 showedTable3 that This study This Reference Sherwood Sherwood (2007)
olwd y 30 by followed
and -
60 ng/μl), 1 x 1 ng/μl), 60
0.38 0.43 0.35 0.35 0.36 0.29 0.48 0.39 H
Eucheuma Taq
.
DNA
PCR 0.31 0.34 0.29 0.29 0.30 0.25 0.36 0.31 PIC For the 422 K. &
TRANSACTIONS ON SCIENCE AND TECHNOLOGY amplified DNA fragments DNA amplified the among varieties the differentiate to unable Eucheuma were primers ISSR the all contrast, In study. this alvarezii with with together grouped with which agiven branch appeared 1000in bootstrap replications. offrequencies percentagethe are nodes at givenprimers).Thenumbers all of(combinationseaweed 1. Figure and tree of varieties 30 the between relationship prime eight from of varieties between similarity of degree tool for studying population geneti and 0.36 ISSRtechnologyvariation,The issensitive of geneticto lowlevelsproviding usefula very molecular to (Palai 0.25 method detection the with from varies shown been rangedhas polymorphism of level used the markers high ISSRshow usually markers ISSR the 0.48. to 0.29 of from ranged heterozygosity values PIC (88%). to BRIAAC4 compared as varietiesprimer (99%) polymorphism seaweed of 30 degree highest the the from exhibited ISSR1 produced Primer fragmentspolymorphic. of (94.3%) majority vast The analysis. cluster UPGMA for used were which fragments, DNA 1,494 with loci ISSR 192 of total a were there lhuh the Although PM cutr nlss sn te tnadzd eei dsacs ee sd o iulz the visualize to used were distances genetic standardized the using analysis cluster UPGMA as identified initially were which KAS12_30 and KAS12_29 Sample K. alvarezii was the sequence of , it is interesting to note that there were genetic variationsgenetic were there that note to interesting is it , divided into 3 clades, which are which clades, 3 into divided
Coalescent tree showed the relationship between varieties of varieties between relationship the showed tree Coalescent seaweed, producing the same fingerprinting profiles. All eight ISSR primers revealed the revealed primers ISSR eight All profiles. fingerprinting same the producing seaweed,
(77% BS value). rs were combined together to construct a coalescent tree and observed the overall the observed and tree coalescent a construct to together combined were rs . denticulatum E. K. striatus K. striatus K. that were unique to ten varieties from varieties ten to unique were that Chin et et al.
deposited thein NCBI GenBank databases (Acc , . The sequence of both samples also demonstrated 85% similarity 85% demonstrated also samples both of sequence The .
201 a cutrd oehr n h sm gop with group same the in together clustered was cs (Zietkiewicz 7 ISSN ISSN 2289 .
Transactions on Science and Technology. Kappaphycus E. denticulatum E. Kappaphycus - 8786. http://transectscience.org/ et al.,et
and
and and
1994; Raina (100% BS value), BS (100% Eucheuma Eucheuma
4 ( 3 - 3 in Kappaphycus )
, 420 et al.,
seaweeds (Figure seaweeds Kappaphycus seaweeds. All the binary data binary the All seaweeds.
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425
Kappaphycus
2001).
K. polymorphism although polymorphism
Kappaphycus striatus .
and and no.
species detected indetected species : JN645178). Eucheuma . striatus K. (59% BS value), BS (59%
& and
1
), where ),
Rout, 2011). Rout,
p. were spp. Eucheuma
which
or or were
t he 423 K.
TRANSACTIONS ON SCIENCE AND TECHNOLOGY Management Deliveryand Unit (PEMANDU) under the Prime Minister Department of Malaysia. ACKNOWLEDGEMENTS relationships among inter ofunderstanding the facilitate and spe which locus, ISSRunique and valuable authentication the diagnostic of tool molecular for provided study a fromobtained this ISSRprofiles of varieties CONCLUSION markers and applied in marker assisted selection (MAS). may bewhich linked or to agroup atrait oftraits with agronomic importance can be used asspecific seaweeds. identificationof discriminationand the for methodmolecular valuable a be seaweed varieties atgenomic DNA level. auth the for method molecular valuable a is technique ISSR the demonstrated data These profiles. genotyping ISSR unique five about produced which ISSR12 and ISSR5 markers especially varieties, seaweed of authentication the for tools molecular represent (Table 12 Alien and 13 buaya Alien 14, buaya Alien green, Alien Tangan, brown, Alien flower, Yellow flower,Green Cacing, Brown, are Table 4 Our studydemonstratedOur has t hs eerh a spotd y rsac gat GR/P/) rm h Performance the from (GPRL/SPS/1) grant research a by supported was research This the Although Eucheuma . List ofISSR specific locus for identification of BRIAGG4 BRIAAC4 BRICCT4 K. alvarezii K. ISSR 12 ISSR 10 ISSR Primer ISSR 5 ISSR 2 ISSR 1 ISSR seaweeds. This will lead to the development of varietydevelopmentof the to lead will seaweeds.This
Kappaphycus
Kappaphycus
and and Size (bp) Size 2781 2260 1935 1 1704 2014 1239 676 646 483 357 560 520 464 433 185 500 818 300 238 495 410 338
K. striatus K. Chin
et et al.
and
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7 and intra and ISSN ISSN 2289
. Eucheuma
still showed a genetic variation except for except variation genetic a showed still K sp. Kappaphycus sp. Kappaphycus sp. Kappaphycus sp. Kappaphycus sp. Kappaphycus Transactions on Science and Technology. Eucheuma E. denticulatum E. 4 appaphycus sp. appaphycus . hs IS pies mlfig pcfc N fragme DNA specific amplifying primers ISSR These ). K.alvarezii K.alvarezii K.alvarez K.alvarezii K.alvarezii K.alvarezii K.alvarezii K.alvarezii K.alvarezii K.striatus K.striatus K.striatus K.striatus K.striatus K.striatus
Species cifically identified the varietal differences of differences varietal the identified cifically - 8786. - species gene flow, genetic diversity and evolut and diversity genetic flow, gene species http://transectscience.org/
seaweeds. ewes r smlr t h peoyi level phenotypic the at similar are seaweeds ii
Kappaphycus
Alien buaya 14 buaya Alien Alien buaya 14 buaya Alien 13 buaya Alien Yellow flower Yellow Green flower Green flower Green Alien brown Alien Alien green Alien green entication of entication Alien 12 Alien 12 Alien 12 Alien 12 Alien 12 Alien 12 Variety Tangan Tangan Cacing Brown Brown Brown B
rown
4 primersdevelopedthis forcanstudy ( 3 - 3 ) ,
and 420
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Collection ID Collection KAABA14 KAABA14 KAABA13 KAAG KAAG KAS12 KAS12 KAS12 KAS12 KAS12 KAS12 KACG KABN KATN KABN KABN KABN KATN KAAB KSGF KSGF KSYF E. denticulatum E. seaweed varieties
and
ISSR markerISSR
Kappaphycus
Eucheuma ionary . The .
the ,
nts 424 s
TRANSACTIONS ON SCIENCE AND TECHNOLOGY [17] [16] [16] [15] [14] [13] [12] [11] [10] [9] [8] [7] [6] [5] [4] [3] [2] [1] REFE
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