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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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By


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

© COPYRIG

HT UPM


By


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

© COPYRIG

HT UPM

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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


By


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


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. _______________________________________


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

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