UNIVERSITI PUTRA MALAYSIA ASSESSMENT OF DNA … · Sebagai kesimpulan, kajian ini menunjukkan...
Transcript of UNIVERSITI PUTRA MALAYSIA ASSESSMENT OF DNA … · Sebagai kesimpulan, kajian ini menunjukkan...
UNIVERSITI PUTRA MALAYSIA
ASSESSMENT OF DNA BARCODING REGIONS FOR IDENTIFICATION OF MALAYSIAN BANANA CULTIVARS
ABDUL RAHMAN B. ZULKIFLLI
FS 2013 22
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ASSESSMENT OF DNA BARCODING REGIONS FOR IDENTIFICATION OF MALAYSIAN BANANA CULTIVARS
By
ABDUL RAHMAN B. ZULKIFLLI
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science
January 2012
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ASSESSMENT OF DNA BARCODING REGIONS FOR IDENTIFICATION OF MALAYSIAN BANANA CULTIVARS
By
ABDUL RAHMAN B. ZULKIFLLI
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science
January 2013
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science
ASSESSMENT OF DNA BARCODING REGIONS FOR IDENTIFICATION OF MALAYSIAN BANANA CULTIVARS
By
ABDUL RAHMAN B. ZULKIFLLI
January 2013
Chair: Associate Professor Siti Khalijah Daud, PhD
Faculty: Science
To date, there is no exclusive barcode for bananas and plantains being
documented, hence, this study was undertaken to find out the most suitable
barcodes for banana cultivars identification. The aim of this study was to
determine the most appropriate DNA regions to be assigned as the DNA
barcoding for banana cultivars identification. In this study, genes from
chloroplast genome were chosen as the barcode candidates, namely matK,
rpoB, rpoC1, rbcL, trnH-psbA primers and their combinations. In addition,
ITS2 gene from nuclear genome was also selected.
Six banana cultivars, namely Pisang Jari Buaya, Pisang Raja Udang, Pisang
Tanduk, Pisang Rastali, Pisang Nangka and Pisang Nipah were sampled
from UPM banana germplasm collection, located in Kompleks Ladang
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Bersepadu, Taman Pertanian Universiti, Universiti Putra Malaysia. Four
criteria have been used to assess the barcoding properties of the chosen
barcode regions, i.e. the ability to be screened and amplified from the whole
genome, their genetic divergence characteristic, the ability to form
monophyletic clade, and the DNA barcoding gap.
By using the suggested primer pairs, Polymerase Chain Reaction (PCR)
technique was employed to screen and amplify the desired regions. The
genetic divergence characteristic, inter and intraspecific genetic distances
were assessed from the genetic distance matrices based on Kimura 2
Parameter model using MEGA5 software. The monophyletic clade
assessment was done following phylogenetic technique. The phylogenetic
trees involved were inferred using Neighbour Joining (NJ), Maximum
Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI)
methods. As for the DNA barcoding gap, the data from previous genetic
distance matrices were used and the graphs were created using Microsoft
Excel 2007 software.
The assessment revealed that the used primers generally performed well in
PCR amplification with the average PCR efficiency of 89%. However, ITS2
showed that it was the easiest marker to work with. On the other hand, trnH-
psbA has the highest interspecific divergence and the lowest intraspecific
divergence (0.0261 and 0.000 respectively). For phylogenetic analysis, ITS2
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managed to resolve monophyletic clades for all cultivars, which were
strongly supported by bootstrap and posterior probability values.
Nevertheless, none of the barcode candidates exhibited clear “barcoding
gap”.
In conclusion, this study revealed that chloroplast genome was not suitable
for identification of hybrid plants, such as banana cultivars, because it only
can elucidate a single line of genetic inheritance since chloroplast mode of
inheritance only either through paternal or maternal line. Nuclear gene, ITS2,
was shown to be the best candidate for highly hybrid plants such as banana
cultivars. Thus, this study suggested that ITS2 region to be used for DNA
barcoding of Malaysian banana cultivars. For future study, the number of
cultivars should be increased and Single Nucleotide Polymorphism (SNP)
marker can be established based on the barcoding region. By employing
High Resolution Melting temperature (HRM) technique, faster, rapid and
high throughput identification can be possible.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENILAIAN BARKOD DNA UNTUK PENGENALPASTIAN
KULTIVAR PISANG MALAYSIA
Oleh
ABDUL RAHMAN B. ZULKIFLLI
Januari 2013
Pengerusi: Profesor Madya Siti Khalijah Daud, PhD
Fakulti: Sains
Sehingga kini, masih tiada barkod yang ekslusif untuk pisang dan plantain
didokumentasikan. Oleh itu, kajian ini dijalankan untuk menentukan barkod
yang sesuai bagi pengenalpastian kultivar pisang. Tujuan kajian ini adalah
untuk menentukan kawasan barkod yang paling sesuai sebagai pengkodan
DNA bagi pengenalpastian kultivar pisang. Dalam kajian ini, gen dari
genom kloroplas dipilih sebagai calon barkod, iaitu primer matK, rpoB, rpoC1,
rbcL dan trnh-psbA, serta gabungan antara mereka. Sebagai tambahan, gen
ITS2 dari genom nuklear juga telah dipilih.
Enam kultivar pisang, iaitu Pisang Jari Buaya, Pisang Raja Udang, Pisang
Tanduk, Pisang Rastali, Pisang Nangka dan Pisang Nipah telah disampel
dari koleksi germplasma pisang UPM yang terletak di Ladang Bersepadu,
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Taman Pertanian Universiti, Universiti Putra Malaysia. Empat kriteria telah
digunakan untuk menilai sifat-sifat calon barkod yang telah dipilih, iaitu
kebolehan untuk disaring dan digandakan dari keseluruhan genom, ciri-ciri
pencapahan genetik, kebolehan untuk membentuk klad monofiletik dan
jurang barkod DNA.
Dengan menggunakan pasangan primer yang telah dicadangkan, teknik
Polymerase Chain Reaction (PCR) digunakan untuk menyaring dan
menggandakan kawasan yang dikehendaki. Ciri pencapahan genetik, jarak
genetik interspesifik dan intraspesifik dinilai daripada matrik jarak genetik
yang berdasarkan kepada model Kimura 2 Parameter menggunakan perisian
MEGA5. Penilaian klad monofiletik dilakukan dengan menggunakan teknik
filogenetik. Pokok filogenetik ini dibina berdasarkan kaedah Neighbour
Joining (NJ), Maximum Parsimony (MP), Maximum Likelihood (ML) dan juga
Bayesian Inference (BI). Untuk analisis jurang barkod DNA pula, data dari
matriks jarak genetik yang sama digunakan dan graf jurang barkod
dihasilkan dengan menggunakan perisian Microsof Excel 2007.
Penilaian ini menunjukkan pasangan primer yang digunakan pada amnya
adalah baik dalam penggandaan PCR dengan purata kecekapan PCR
sebanyak 89%. Walau bagaimanapun, ITS2 menunjukkan yang ia adalah
penanda barkod yang mudah untuk dikendalikan. Sebaliknya, trnH-psbA
mempunyai pencapahan interspesifik paling tinggi (0.0261) dan pencapahan
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intraspesifik yang paling rendah (0.000). Untuk analisis filogenetik pula, ITS2
mampu untuk merungkai klad monofiletik bagi semua kultivar pisang yang
terlibat yang mana ia disokong kuat oleh nilai bootstrap dan nilai
kebarangkalian posterior. Namun begitu, tidak ada satu pun calon barkod
yang berjaya mempamerkan jurang barkod yang jelas.
Sebagai kesimpulan, kajian ini menunjukkan bahawa genom kloroplas tidak
sesuai bagi pengenalpastian tumbuhan hibrid, seperti kultivar pisang,
kerana ia hanya mampu menjelaskan satu arah pewarisan genetik sahaja. Ini
adalah kerana mod pewarisan kloroplas diwariskan hanya melalui bapa atau
ibu sahaja. Gen nukleus, ITS2, didapati sangat baik digunakan bagi
pengenalpastian tumbuhan yang sangat hibrid seperti kultivar pisang. Oleh
itu, kajian ini mencadangkan supaya kawasan ITS2 digunakan sebagai
penanda barkod DNA untuk kultivar pisang Malaysia. Untuk kajian akan
datang pula, bilangan kultivar pisang perlu ditambah dan penanda Single
Nucleotide Polymorphism (SNP) pula boleh digunakan berdasarkan kawasan
pengkodan ini. Dengan menggunakan teknik High Resolution Melting
temperature (HRM) pula, pengenalpastian yang cepat, pantas dan secara
besar-besaran dapat dilakukan.
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ACKNOWLEDGEMENTS
My sincere gratitude goes to,
My supervisory committee, Assoc. Prof. Siti Khalijah Daud and Dr. Christina
Yong Seok Yien for their dedication, guidance and support.
My laboratory mates, Nadiatul Hafiza, Hasnita, Azlina, Fitri, Meng Han,
Syamim and Faezeh for their friendship and support.
The undergraduate students, Ayu, Adib, Aishah, Shin, Alin and Li Moi for
helping me during the course of this study. I owed them some parts of this
thesis.
The Departments‟ lecturer and staffs for helping me out during my stay at
the department and all peoples that I met along my academic life for the
support and experience.
Last but the most important one is to my family. I love you all.
Thank you.
ABDUL RAHMAN ZULKIFLLI
JANUARY 2013
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I certify that an Examination Committee has met on 11th January 2013 to conduct the final examination of Abdul Rahman b. Zulkiflli on his Master of Science thesis entitled “Assessment of DNA barcoding regions for identification of Malaysian banana cultivars” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Masters of Science.
Members of the Examination Committee were as follows: Muskhazli b. Mustafa, PhD
Associate Professor Faculty of Science Universiti Putra Malaysia 43400 UPM Serdang (Chairman) Hishamuddin b. Omar, PhD
Faculty of Science Universiti Putra Malaysia 43400 UPM Serdang (Internal Examiner) Maheran bt. Abd Aziz, PhD
Associate Professor Faculty of Agriculture Universiti Putra Malaysia 43400 UPM Serdang (Internal Examiner) Badrul Munir Md. Zain, PhD
Associate Professor Fakulti Sains dan Teknologi Universiti Kebangsaan Malaysia 43600 Bangi, Selangor (External Examiner) __________________________ SEOW HENG FONG, PhD
Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Masters of Science. The members of the Supervisory Committee were as follows:
Siti Khalijah Daud, PhD
Associate Professor Faculty of Science Universiti Putra Malaysia (Chairperson)
Christina Yong Seok Yien, PhD
Senior Lecturer Faculty of Science Universiti Putra Malaysia (Member)
___________________________ BUJANG BIN KIM HUAT, PhD
Professor and Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. _______________________________________
ABDUL RAHMAN B. ZULKIFLLI
Date:
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS vii
APPROVAL ix
DECLARATION xi
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xvii
CHAPTER
1 INTRODUCTION 1 2 LITERATURE REVIEW 8 2.1 The genus Musa 8 2.1.1 Distribution of genus Musa 9 2.1.2 Morphology and Botanical Description of
Banana 10
2.1.3 Development of banana cultivars 15 2.1.4 Cultivated banana varieties in Malaysia
a) Pisang Rastali b) Pisang Nangka c) Pisang Tanduk d) Pisang Nipah
18 19 20 21 23
2.1.5 Characteristics of banana plants 29 2.2 DNA barcoding 30 2.2.1 Plant DNA barcoding 33 2.2.2 matK gene 38 2.2.3 rpoB gene 40 2.2.4 rpoC gene 41 2.2.5 rbcL gene 42
2.2.6 trnH-psbA region 42 2.3 DNA barcoding assessment: Concepts and Theories 45 2.3.1 Genetic distance 46 2.3.2 Genetic divergence 51 2.3.3 Monophyly of the groups 52 2.3.4 DNA barcoding gap 55 3 MATERIALS AND METHODS 56 3.1 Sampling 56
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3.2 DNA extraction 56 3.3 DNA quality confirmation 58 3.4 Region screening and amplification 59 3.4.1 The primers 59 3.4.2 The mastermix and thermal cycling 61 3.5 DNA purification and sequencing 65 3.6 Sequence analysis and assessment of DNA barcodes 66 4 RESULTS 68
4.1 Barcode candidate screening and amplification 68 4.2 Single region analysis 70 4.2.1 Genetic divergence characterization between
barcoding region 70
4.2.2 Analysis of ability to resolve monophyletic clades for each barcoding region
72
a) matK 72 b) rpoB 80 c) rpoC1 88 d) rbcL 95 e) trnH-psbA 102 4.2.3 Assessment of DNA barcoding gap 109 4.3 Multi region analysis 111 4.3.1 Genetic divergence characterization 112 4.3.2 Analysis of the ability to resolve monophyletic
clades 113
4.3.3 Analysis of DNA barcoding gap 115 4.4 Analysis of nuclear gene, ITS2 133 4.4.1 Genetic divergence characterization 134 4.4.2 Analysis of ability to resolve monophyletic clades 135 4.4.3 DNA barcoding gap analysis 142 5 DISCUSSION 143
6
SUMMARY, CONCLUSION AND RECOMMENDATION FOR FUTURE RESEARCH
149
REFERENCES 152 BIODATA OF STUDENT