Path: TopS2-ThesesChemistry-FMIPA2016

STUDI FORENSIK ENAM GALUR DIATOM LAUT TROPIS BERDASARKAN MORFOLOGI SEL DAN GEN PENANDA rbcL-3P, LSU D2/D3, DAN 18S rDNA DAERAH V4

FORENSIC STUDY OF SIX TROPICAL MARINE DIATOM STRAINS BASED ON CELL MORPHOLOGY AND GENETIC MARKERS OF rbcL-3P, LSU D2/D3, AND 18S rDNA AREA V4

Master Theses from JBPTITBPP / 2017-11-14 14:31:33
Oleh : ELVA STIAWAN (NIM : 20514008), S2 - Chemistry-FMIPA
Dibuat : 2016, dengan 8 file

Keyword : diatom laut tropis, forensik botani, citra SEM, gen penanda, rbcL-3P, LSU D2/D3, 18S rDNA daerah V4

Sebagai negara maritim, Indonesia memiliki keanekaragaman hayati laut yang sangat besar, diantaranya adalah diatom laut tropis yang memiliki prospek pemanfaatan luas. Namun, diatom-diatom tersebut tidak terkoleksi dengan baik dan belum teridentifikasi secara teliti sehingga rentan terhadap pencurian oleh warga negara lain. Penyidikan diatom secara umum didasarkan atas karakteristik morfologi dari sel. Kelemahan penyidikan diatom berdasarkan pendekatan tersebut adalah akurasi yang rendah khususnya bila sel telah mengalami kerusakan. Cara penyidikan yang lebih mutakhir adalah dengan cara kode batang DNA (DNA barcoding). Sejauh ini, konsensus kode batang DNA yang khas untuk menyidik diatom belum disepakati. Oleh karena itu, upaya untuk menelusuri gen penanda yang spesifik untuk kelompok diatom perlu dilakukan. Riset ini bertujuan untuk menyidik enam galur diatom secara morfologi berdasarkan tampilan sel di bawah mikroskop dan secara genetik melalui gen-gen penanda rbcL-3P (daerah gen pengode subunit besar dari RuBisCo di ujung 3’, 853–866 pb), LSU D2/D3 (bagian D2 dan D3 dari gen pengode 28S RNA ribosom, 549–622 pb), dan 18S rDNA daerah V4 (daerah V4 dari gen pengode 18S RNA ribosom, 392–599 pb). Tahapan riset meliputi penyediaan kultur murni diatom; pengambilan foto mikroskop cahaya dan citra SEM dari sel diatom; amplifikasi gen-gen penanda melalui PCR secara langsung dari sel diatom; pengurutan nukleotida gen-gen penanda; dan analisis data morfologi dan genetik dari enam galur diatom yang diteliti. Diatom yang diteliti terdiri atas empat galur diatom yang ditapis dari perairan laut di Teluk Jakarta dan Kepulauan Seribu dan dua galur diatom koleksi Laboratorium Biokimia ITB. Penyidikan morfologi sel di bawah mikroskop cahaya hanya berhasil dilakukan hingga tingkat genus, kecuali untuk galur CUJ yang berhasil tersidik memiliki kemiripan dengan Chaetoceros debilis. Penyidikan berdasarkan tampilan morfologi cangkang silika sel pada citra SEM menunjukkan bahwa galur TMA dan TMA1 masing-masing tersidik sebagai Cyclotella striata dan galur NLA sebagai Navicula salinicola. Hasil citra SEM untuk galur CUJ, CBO, dan TMGS tidak cukup memadai untuk keperluan penyidikan. Hasil analisis pohon filogenetik dari tiga gen penanda menunjukkan bahwa galur TMA dan TMA1 memiliki kemiripan paling tinggi dengan Cyclotella striata (kemiripan gen rbcL-3P 100%; LSU D2/D3 98%; 18S rDNA daerah V4 100%) dan galur NLA memiliki kemiripan tertinggi dengan Navicula salinicola (kemiripan gen rbcL-3P 98%; LSU D2/D3 98%; dan 18S rDNA daerah V4 99%). Galur TMGS tersidik sebagai genus Chaetoceros berdasarkan hasil analisis pohon filogenetik dari tiga gen penanda. Namun, pohon-pohon filogenetik galur TMGS menunjukkan hasil yang tidak konsisten di tingkat spesies, yaitu tersidik sebagai Chaetoceros muellerii (kemiripan 98%), Chaetoceros gracilis (kemiripan 95%), dan Chaetoceros calcitrans (kemiripan 98%) secara berturut-turut berdasarkan gen rbcL-3P, LSU D2/D3, dan 18S rDNA daerah V4. Sementara itu, hanya dua pohon filogenetik yang menunjukkan galur CUJ dan CBO sebagai genus Chaetoceros, yaitu pohon filogenetik dari gen rbcL-3P dan 18S rDNA daerah V4. Galur CUJ tersidik sebagai Chaetoceros cf. debilis berdasarkan gen rbcL-3P (kemiripan 96%) dan Chaetoceros debilis berdasarkan gen 18S rDNA daerah V4 (kemiripan 93%), sedangkan galur CBO tersidik sebagai Chaetoceros dayaensis berdasarkan gen rbcL-3P (kemiripan 96%); Spumella sp. berdasarkan gen LSU D2/D3 (kemiripan 92%); dan Chaetoceros cf. wighamii berdasarkan gen 18S rDNA daerah V4 (kemiripan 85%). Hasil penyidikan galur TMA, TMA1, dan NLA melalui BOLDSYSTEM menunjukkan hasil temuan yang konsisten dengan hasil analisis filogenetik dari tiga penanda, yaitu galur TMA dan TMA1 memiliki kemiripan tertinggi dengan Cyclotella striata (kemiripan 99,71%) dan galur NLA memiliki kemiripan tertinggi dengan Navicula salinicola (kemiripan 98,4%). Sementara itu, hasil penyidikan melalui BOLDSYSTEM secara konsisten menunjukkan galur CUJ, CBO, dan TMGS sebagai genus Chaetoceros, dengan CUJ memiliki kemiripan tertinggi dengan Chaetoceros debilis (kemiripan 96,03%); galur CBO memiliki kemiripan tertinggi dengan Chaetoceros lorenzianus (kemiripan 95,77%); dan galur TMGS memiliki kemiripan tertinggi dengan Chaetoceros constictus (kemiripan 91,39%). Sementara itu, area-area khas pada gen penanda telah ditemukan di masing-masing galur diatom. Area-area khas tersebut perlu dikonfirmasi lebih lanjut untuk dapat dinyatakan sebagai penciri khusus bagi enam galur diatom asal Indonesia.

Deskripsi Alternatif :

As a maritime country Indonesia has huge of marine biodiversity, such as tropical marine diatoms which have enormous potential for application in different areas. However, the diatoms have not been well collected and accurately identified so that it is very susceptible to biopiracy. Commonly diatoms are identified based on their cells’ morphological characteristics. This approach has a weakness due to relatively low accuracy of identification especially for the broken cell. DNA barcoding is a recently species identification methods. Currently, a specific DNA barcode consensus to identify diatoms does not exist yet. Therefore an effort to improve existing method to develop diatoms’ specific genetic markers is required. The objective of this research is to identify six diatom strains through morphological approach based on cells’ characteristics and genetic approach based on genetic markers of rbcL-3P (3’ end region of large subunit of RuBisCo encoding gene, 853–866 bp), LSU D2/D3 (D2 and D3 regions of 28S ribosomal RNA encoding gene, 549–622 bp), and 18S rDNA area V4 (V4 area of 18S ribosomal RNA encoding gene, 392–599 bp). Main steps in this research involve supplying of single cultures; light microscope and SEM imaging of diatom cells; DNA amplification; sequencing of genetic markers; and morphological and analysis of genetic data. The observed diatoms consist of four strains screened from Jakarta Bay and sea areas around The Thousand Islands of Jakarta and two strain are collection of Laboratory of Biochemistry, ITB. Cell morphological identification under light microscope was only successful until genus level, except for CUJ strain that was successfully identified as Chaetoceros debilis. Morphological identification based on silica frustules of diatoms’ cell through SEM image was successfully identified for three strains, i.e. TMA and TMA1 strains were identified as Cyclotella striata, and NLA strain was identified as Navicula salinicola. However, SEM images of CUJ, CBO, and TMGS strains were insufficient for identification. Phylogenetic trees based on three genetic markers showed that TMA and TMA1 have the highest similarity with Cyclotella striata (100% similarity with rbcL-3P; 98% similarity with LSU D2/D3; 100% similarity with 18S rDNA area V4), while NLA strain have the highest similarity with Navicula salinicola (98% similarity with rbcL-3P; 98% similarity with LSU D2/D3; 99% similarity with 18S rDNA area V4). TMGS strain identified as Chaetoceros genus based on phylogenetic trees analysis, whereas identification results of TMGS strain was not consistent on species level, i.e. TMGS strain was identified as Chaetoceros muellerii (98% of similarity), Chaetoceros gracilis (95% of similarity), and Chaetoceros calcitrans (98% of similarity) based on rbcL-3P, LSU D2/D3, and 18S rDNA area V4 gene, respectively. CUJ strain was identified as Chaetoceros cf. debilis based on rbcL-3P (96% of similarity) and Chaetoceros debilis based on 18S rDNA area V4 (93% of similarity), while CBO strain was identified as Chaetoceros dayaensis based on rbcL-3P (96% of similarity), Spumella sp. based on LSU D2/D3 (92% of similarity) and Chaetoceros cf. wighamii based on 18S rDNA area V4 (85% of similarity). Species identification results based on BOLDSYSTEM showed a consistent finding related to phylogenetic analysis of three genetic markers, i.e. TMA and TMA1 strains have the highest similarity with Cyclotella striata (99,71% of similarity) and NLA strain has the highest similarity with Navicula salinicola (98,4% of similarity). On the other hand, the investigation based on BOLDSYSTEM showed that CUJ, CBO, and TMGS strains have the highest similarity with Chaetoceros genus, consistently, i.e. CUJ strain has the highest similarity with Chaetoceros debilis (96.03% of similarity); CBO strain had the highest similarity with Chaetoceros lorenzianus (95.77% of similarity); and TMGS strain has the highest similarity with Chaetoceros constictus (91,39% of similarity). Moreover, several unique areas had been found in each diatom strains. It is required to confirm those areas to establish a specific marker for six of Indonesian diatom strains.

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OrganisasiS2 - Chemistry-FMIPA
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