Doctoral thesis summary: Evaluation on genetic diversity and identification molecular marker for Vietnamese native Dendrobium species

Research objectives: Thesis was the first studied methodically systematic assessment of genetic diversity by morphological indicators combined with molecular markers, identification of Vietnamese native Dendroium species based on the ITS sequences. The results of the thesis have significance in the classification, the conservation service, and selection and breed new varieties; these will be contributing to improve Vietnam orchids.

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RULAR

DEVELOPMENT

VIETNAM ACEDEMY OF AGRICULTURAL SCIENCES

TRAN DUY DUONG

EVALUATION ON GENETIC DIVERSITY AND IDENTIFICATION MOLECULAR MARKER FOR

VIETNAMESE NATIVE DENDROBIUM SPECIES

Discipline: Genetics and Crop Selection

Number Code: 62.62.01.11

SUMMARYOF DOCTOR THESIS

HA NOI - 2015

The thesis was raging at:

INSTITUTE OF AGRICULTURAL GENETICS

VIETNAM ACEDEMY OF AGRICULTURAL SCIENCES

Scientific supervisors:

1 Association Prof Dr La Tuan Nghia

2 Dr Nguyen Thi Thanh Thuy

Search the thesis in:

Vietnam Academy of Agricultural Sciences Library

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INTRODUCTIONS

1 Main of objective

The genus Dendrobium one of the largest genera in the Orchidaceae,

includes more than 1148 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all

major groups of this genus, and 2nd orchids after Bulbophyllum orchids (Bulbophyllum) (Leitch et al., 2009) Southeast Asia can be considered as the homeland of Dendrobium with more than hundreds of species, particularly in

Vietnam has more than 100 species (Tran Hop, 1998; Nguyen Xuan Linh, 2002; Averyanov, 2004; Duong Duc Huyen, 2007), and they are widely distributed across the regions of the country

In the conservation and sustainable use of plant genetic resources, assessing the gene bank is extremely important step not only for the identification of similar/different species, but also to understand the relationship genetic system between varieties/species to conserve genetic diversity The strong developments of methods and techniques in the field of molecular biology have created an effective tool and quickly are applied in studies of biodiversity conservation

The advantage of the molecular technique is capable to identify the genetic diversity, provide the basis for assessing the conservation value of species and populations Therefore, the creation of a database of DNA fingerprinting of the varieties/species, gene banks registered in the world, national sovereignty of plant genetic resources of our country as well as the determine the rights to plant varieties and implementation of intellectual property rights in the name of plant varieties, endemic to Vietnam in general

and in particular Dendrobium species are very important issues, urgency, carry

scientific and high practicality, not only for immediate benefits but also long term goal oriented conservation in effectively exploiting genetic resources program serves the country's key economic

Starting from a theoretical basis and practical, we conducted research

Vietmaese native Dendrobium species ”

2 Main of objective

- Evaluation on genetic diversity in morphological level combined with molecular markers to determine genetic relationships among varieties/species of

Vietnamese native Dendrobium species for classification, selection and

breeding new varieties

- Using the ITS marker to identify genetic resources of Vietnamese native

Dendrobium species for the conservation, database, and DNA barcode

3 Meaning scientific and practical significance of the subject

3.1 Meaning scientific

- Providing scientific datas on genetic diversity in the morphology and

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molecular levels of the Vienamese native Dendrobium species, which is basis

for selection of elite germplasm, and new breeding;

- The results of the research are significant in identifying a number of

varieties/species of Vietnamese native Dendrobium species, as a basis for

conservation, economic value and register on GenBank

3.2 Practical Implications

- Collection and preservation genetic resources of Vietnamese native

Dendrobium species;

- The results will be contributing on the conservation, selection and

breeding new varieties to improving the Vietnamese orchid products

4 Research subjects and scope of the project study

4.1 Research subjects

- Thirty two Vietnamese native Dendrobium species were collected from

different provinces

4.2 Location and time study:

- The experiment was carried out at the Department of Genetic Engineering, Institute of Genetics Agriculture, Pham Van Dong road, Tu Liem district, Hanoi city

- Research period: From 2010 to 2014

5 The contributions of the thesis

Thesis was the first studied methodically systematic assessment of genetic diversity by morphological indicators combined with molecular

markers, identification of Vietnamese native Dendroium species based on the

ITS sequences The results of the thesis have significance in the classification, the conservation service, and selection and breed new varieties; these will be

contributing to improve Vietnam orchids

CHAPTER 1: OVERVIEW

1.1 Glance of Dendrobium

1.1.1 Classification systems

In the botanical classification systems, the gennus Dendrobium are belong

the spread (Orchidales), subclass (Liliidae), monocots (Liliopsida), delimitation

of higher plants (Cosmobionia), and the plant kingdom (Plantae) (Tran Hop, 1998; Hoang Thi Be, 2004; Leitch et al., 2009; Evans et al., 2012)

Classification systems of Dendrobium species in Vietnam have often

based Seidenfaden system (1985) This system is clear, straightforward, high

reliability, and consistent with the representatives of Dendrobium in Vietnam

(Tran Hop, 1998; Duong Duc Huyen, 2007)

1.1.2 Morphological characteristics

1.1.2.1 Stems

The stems of Dendrobium species are segmented, cylindrical shaped

spindles, clavate, ovoid, with lengths ranging from 2-3cm to 120cm or sometimes more, common size is 20-50cm (Tran Hop, 1998) The stems can

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piece, sometimes up inside either thick or fat to form beads

1.1.2.2 Root

The roots of the Dendrobium species are usually slender, biogas roots,

cylindrical, green and brown when converted into old age, they often embrace could price or slouched down

1.1.2.3 Leaves

Leaves placed in two staggered rows, but only hug relatives’ sheath, rarely without sheath Leaves are usually hard, skin types, shadows, seldom lean and tender, often smooth surface Leaves often shaped marks, ellipses, sometimes shaped swords, rarely leaves oblong or long lozenge (Averyanov, 2004).)

1.1.2.4 Flower groups

Flower groups have usually many flowers blossom, sometimes little flower or flowers solitary Flower groups have often pendulous flowers hanging down long, many species have beautiful flower (Tran Hop, 1998)

In Vietnam, Dendrobium species have discovered more than 100 species

and these were distributed mainly in mountain areas throughout North, Central, South and on some coastal islands Vietnam (Tran Hop, 1998; Averyanov, 2004)

1.2 Evaluation of Dendrobium species

Besides on ornamentals, some several of Dendrobium will also to make medicines (Do Huy Bich et al., 2004; Cai et al., 2012; Feng et al., 2013)

1.3 Overview of research methods, evaluation on genetic diversity and

identification molecular maker in Dendrobium species

1.3.1 The concept of genetic diversity

Genetic diversity is the diversity of the composition of genes between individuals of the same species and between different species, is the variety of genes can be inherited in a population or between populations

1.3.2 The significance of the study of genetic diversity

Biodiversity is essential for the survival of the species, natural communities and very important for humans

1.3.3 The method of evaluation on genetic diversity and indentification in plants

1.3.3.1 Methods based on morphological

Evaluation genetic diversity on morphologicals are traditional method,

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including the description of the characteristics and structure external morphology, namely stems, leaves, flowers

1.3.3.2 Methods based on biochemical

The isozyme is defined as various forms of an enzyme (protein) has the same function or close together in the same individuals (Salazar, 2003)

1 3 3 3 Evaluation genetic diversity based on molecular marker methods

Evaluation genetic diversity based on molecular marker methods such as: Restriction Fragment Length Polymorphism (RFLP); Amplified Fragment Length Polymorphism (AFLP); Random Amplified Polymorphic DNA (RAPD); Microsatellite or Simple Sequence Repeates (SSR); Inter-simple

sequence repeats (ISSRs) (Singh et al., 2012; Shangguo et al., 2013; Swati Das

et al., 2014)

1.3.3.4 Classification methods of plant on gene sequences

* ITS region (internal transcribed spacers) is a non-functional RNA

segment, located between the RNA of the ribosome structure is usually translated code Structure ITS region including ITS1 - 5.8S - ITS2 Another advantage of the ITS include two separate loci (ITS1 and ITS2) and they are connected together via locus 5.8S 5.8S locus is insulated region, in fact there is enough phylogenetic signal discrimination and the sector level (Richardson et

al., 2001; Sharma et al, 2012)

* The gene rbcL (ribulose-bisphosphate carboxylase) is used more to

build phylogenetic trees However, for genetic relationships at the species under analysis on this gene encounter many limitations (Kress and Erickson, 2007)

* The matK gene (gene coding for maturaseK): The study used gene

sequencing matK to construct phylogenetic trees as showed genetic diversity

matK than other genes in chloroplasts and therefore genetically matK become

an important indicator gene to help sort plants (Asahina et al., 2010; Sharma et al., 2012)

1 4 Current status in study of orchids in the world and in Vietnam

1.4.1 Evaluation genetic diversity and identification Dendrobium species in

the world

1.4.1.1 Research on morphological indicators

Theres were many research on Denedrobium species such as Pellegrino et al., 2005; Krishnan et al., 2011)

1.4.1.2 Research on molecular markers

* RFLP markers

Recently, Surin et al., 2014 for using RFLP technique to identify 25 Dendrobium varieties from Thailand 23 of the 25 species have been identified, only two species such as D crumenatumand D formosum were unidentified

* RAPD markers

There have been studies using indicators to analyze RAPD polymorphism

in orchids, the study focused on several categories like spreads Dia lan

varieties in the North of India Results showed that, total of 124 bands were

analyzed and have 25 individual bands between different of Dendrobium spcies were indentified

* SSR markers

There was less research in Orchids by using SSR marker For Dendrobium

only a few studies on the use of SSR directive to evaluate genetic diversity in recent years as the study by Fan et al., 2009, Cai and et al., 2012; Liu et al., 2014)

* ISSR markers

In 2009, Wang et al were used 17 ISSR marker to evaluate genetic diversity of

31 Dendrobium from China Results showed that, in 2368 total the band was amplified 278 ISSR locus have polymorphic with percent homology at 100%

* The research is based on DNA sequences

Building the world's first use of DNA barcoding to distinguish Dendrobium species with Chinese authors published in 2009 (Yao et al., 2009) In this study, the

region located in between psbA-constructions on chloroplast genome of 17 varieties

of Dendrobium Also on the subject on Dendrobium, Japanese authors (Asahina et al., 2010) to use genetic sequencing to identify matK and rbcL gene while studying

the phylogenetic group of Dendrobium in medicine

1.4.2 Situation study orchids in Vietnam

Since 1985, there have been a number of research institutions and universities such as the Institute of Ecology and Biological Resources, the Institute of Tropical Biology, University of Agricultural Sciences I, University of Forestry, Institute of Agricultural Genetics, and Institute of Fruits and Vegetables has focused on research, investigation, classification group kept some orchids in our country For Dendrobium

orchid, Nguyen Thi My Duyen et al., 2012 studied on morphological characteristics and found three species of D pulchellum, and D moschatum Gatton Sunray has

relationship proximity, similarity rate is 96.5% and respectively 95% In 2007, Le Tran Phuc Tran Khoa was conducted a study on "Evaluation on genetic diversity of

wild Dendrobium species” which were collected in Binh Phuoc and Bao Loc Town

(Lam Dong province) with RAPD technique." The researchers were evaluated the genetic diversity Hoang Thao spread very least accurate identification marker on the

object identifier based spread Hoang Thao sequenced genome regions ITS, matK, rbcL Therefore, the deployment and conduct of the subject on the object Hoang

Thao spreads have important implications for the conservation, preservation and

development of this orchid species Recently, Tran Hoang Dung et al., 2012 had

applied DNA technology to classify and identify Hoang Thao Tram rung

(Dendrobium parishii) and Phi Diep tim (Dendrobium anosmum) species in

Vietnam

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CHAPTER II MATERIALS, CONTENTS AND METHODS 2.1 Plant materials

32 samples of Vietnamese native Dendrobium (aged 2-3 years) species

were made by the Institute of Agricultural Genetics and Prof Dr Tran Duy Quy Information about plant materials, including locaties of colection in the different provinces and they were growth at the Institute Agricultural Genetics and 422, Pham Van Dong, Tu Liem district, Hanoi city

2.2.1.2 Evaluation on genetic diversity for Vietnamese native Dendrobium

species by using RAPD markers

2.2.2 Content 2: ITS sequences of ribosome to indentify Vietnamese native

Dendrobium species

2.3 Methods

2 3.1 Methods based on morphological indicators

- The Dendrobium species were descripted by Pellegrino, 2005 such as

characteristics and structure external morphology, namely stems, leaves, flowers

2.3.2 Evaluation on genetic diversity for Vietnamese native Dendrobium

species by using RAPD markers

2.3.2.1 Extraction of DNA

2.3.2.2 The quantative and quality of DNA check

2.3.2.3 Check the DNA by agarose gel

2.3.2.4 DNA testing by spectrophotometer

2.3.2.5 PCR-RAPD reations

RAPD and ITS primers were provided by Opero and Sigma company

2.3.3 ITS sequences

2 3.3.1 Amplification on ITS region by PCR products

ITS region was amplified using primers ITS1 and ITS4

2.3.3.2 Purification PCR products and sequenes

2 3.3.3 Aligment ITS sequencing in GenBank database

2 3.3.5 Phylogenetic trees

2.4 Data processing software

- Research datas were analyzed, processed on the Excel version 5.0 software, software PCORD, software NTSYS, MEGA: version 5.2.1, to construct phylogenetic trees

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2 5 Location and duration

- Location and time study: Department of Genetic Engineering - Institute of Agricultural Genetics

- Duration: From 2010 to 2014

CHAPTER III: RESULTS AND DISCUSSIONS

3.1 Evaluation on genetic diversity for Vietnamese native Dendrobium

species

32 of Vietnamese native Dendrobium species were collected in the North,

Central and South There lived mainly in mountainous conditions, epiphyte on large trees in the forest, and preserved in the garden at 422st, Pham Van Dong Road, Tu Liem district, Hanoi city (Table 3.1) Among the study, some species were endemic pattern of the Northern provinces of Vietnam as Hoang Thao Chuoi Ngoc (D2), which was located in Dien Bien province, and Hoang Thao Tam Dao (D15) is endemic in Tam Dao -Vinh Phuc province These were

endangered Dendrobium species, was added to our catalog of Vietnam Red

Book, should be preserved, and nurtured The flowers such as D4, D7, D8, D12, D13, D14, D17, D19, D20, D21, D22, D23, D24 and D25 have a wide distributed range from North to South The remaining flower species were found only in some areas of some Central provinces or South were including Hoang Thao Vani and Hoang Thao Dai Bach hac species, which was only found in Kontum province

3.1.1 Evaluation on genetic diversity for Vietnamese native Dendrobium

species by using morphology marker

The stems of Dendrobium species were representatives segmented, with a

variety of shapes such as spindle formation, clavate, square, rosaries, rounded, and the most common are cylindrical The stems has many different forms such

as stem fragments clinging, fat bloated, long stems, short stems bloated, but mostly a long stems, slouched down Particularly, some species only thicken the line, and there is almost no slang, making rosaries shaped stems (D2) or gradual thickening is independent in each chicken thigh slang in forms serial (D28) This

is the hallmark that identifies Hoang Thao Dien Bien species (D2) which were located in Dien Bien province

Leaves were staggered in two rows, sheath hugs in the stems Leaves were distributed along the stems but at much represents the 2-6 leafs concentrate in the stems peak when the top stems has only flowers without leaves Leaves often exist when were flowering The number of leaves changing from a lot (D15- average 35.9 leaf/plant; D23- 31.7 average leaf /plant) to low (D22 average 6.46 leaf/plant) Leaves may be hard or soft, usually smooth

surfaces Most of the leaves of the 32 Dendrobium species were marks

tongue-shaped leaves, leaf oval, pointed leaves peaks, sharp deviation or imprisonment

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Table 3.1 List of Vietnamese native Dendrobium species

Origin (province)

D1 Hoang Thao Long nhan

D2 Hoang Thao Chuoi ngoc

D3 Hoang Thao Thai Binh D moschatum Thai Binh D19 Hoang Thao Kieu Trang

D4 Hoang Thao Phi Diep tim D anosmum Hoa Binh D20 Hoang Thao Tieu Hoang lap D chrysotoxum Kontum

D5 Hoang Thao Tram tim D parishii Son La D21 Hoang Thao Dai Hoang lap D chrysotoxum Lam Dong

D6 Hoang Thao Tram trang D.parishii.var alba Dien Bien D22 Hoang Thao Bach hac langbiang D wattii Lam Dong

D7 Hoang Thao Ngoc Van Vang D chrysanthum Yen Bai D23 Hoang Thao Hac vi D aphylum Da Lat

Hoang Thao Phi Diep

D9 Hoang Thao Long tu Bac D primulinum Son La D25 Hoang Thao Vay rong la trung D lindleyi Lam Dong

Lam Dong

D13 Hoang Thao Kieu vang D thyrsiflorum Tuyen Quang D29 Hoang Thao long tu da D primulinum Tay Nguyen D14 Hoang Thao Kieu trang D farmeri Tuyen Quang D30 Hoang Thao Moi to D delacourii Guill Tay Nguyen

D15 Hoang Thao Tam Dao D daoense Tam Dao D31 Hoang Thao Nhat diem hong D draconis Rchb.f Lam Dong

D16 Hoang Thao Hoang lap

Tay Bac

D

chrysotoxum Lao Cai D32 Hoang Thao Dai bac hac D christyanum Kontum Note: D Dendrobium

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Flowers could have many flowers, or have fewer flowers Flowers have often pendulous hanging down length, or alternately (Figure 3.4) Most of Dendrobium orchid species were different markedly among themselves on the size, number, arrangement and color, shape sepals, petals, and lip States usually not too big, diameter were ranging from 1.35 to 5.8 cm Lip sample have many different colors such as dark yellow or pale yellow, white, yellow or orange spots in the middle, white or black throat (D3) or purple (D4), yellow throat black (D1), light purple yellow throat etc Frequent lip shape is round, nearly round shape checkered, funnel-shaped, heart-shaped, oval, or ovoid shaped chin (D32) From the results of

morphological data such as stems, leaves, flowers of 32 Dendrobium species, we

used to software Pcord 4.0 to construct phylogenetic trees between 32 Vietnamese

native Dendrobium species (Figure 3.6)

Figure 3.4 Some types of flowers in Dendrobium species

According to the phylogenetic trees, 32 Vietnamese native Dendrobium

species were divided into five groups with percent homology at 37% homology

Group I had eight samples including D1, D3, D4, D5, D6, D10, D11, and

D15 species with the percent of homology between different subgroups was from 55

% to 100 % homology and divided in two subgroups:

Subgroup I.1 had 6 samples including D1, D3, D4, D5, D6 and D11, which

D4, D5 and D6 species were in same group In this group, the percent of homology between different subgroups was from 98 % to 100 % homology, and D5 (Hoang Thao Tram tim) and D6 (Hoang Thao Tram trang) species had 100 % percent homology

Subgroup I.2 had two samples incuding D10 (Hoang Thao Truc) and D15 (Hoang Thao Tam Dao) species

Group II had 15 samples including D2, D7, D9, D12, D13, D14, D17, D18,

D19, D22, D23, D26, D27, D29, and D30 species with the percent of homology between different subgroups was from 37 % to 100 % homology At 43% pecent of homology, 15 samples were and divided in two different subgroups:

These species had called namely as Hoang Thao Kieu in the North or called Thuy Tien of Southerners The same floral pattern on morphological characteristics such as stems, leaves, flower shape very similar, differing only in color In these of species were characterized by the large flowering, often covered lip shag, have convex However the big difference here is the spindle-shaped body, focusing on the top leaves, inflorescences often many flowers (Clements, 2003; Duong Duc Huyen,

2007)

Group III had 4 samples including D8, D28, D31 and D32 species In this

group, D31 and D32 species were difficult to indentify morphological characteristics such as stems, leaves, roots, flowers are relatively similar Two different samples of this variety characteristics lip shape, form shapes like D31 have labels, like D32 sample also shaped chin

Group IV had 3 samples including D16, D20 and D21 In this group, D20 and

D21 species were distributed in Dong Nai and Lam Dong provinces In terms of flower morphology they have similar color patterns more like stems; leaves and roots are the same, differing only in size leaves

Group V had 2 samples including Hoang Thao Vay rong la nho (D24) and

Hoang Thao Vay rong la trung (D25) species These species were distributed throughout North, Central and South have the same morphological characteristics and differ only in the size of the leaves It can be noticed that the two varieties are a different species and only in the distribution to the growth and development we have

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little difference especially leaves

The classification Dendrobium species so far is one of the problems in their

complex diversity of Orchidaceae by morphology, wide range of distribution, large number of species, and due to variation of morphological overlap between the species So far, the vast majority of taxonomists are divided into groups spread Hoang Thao (section) Some authors choose to divide into subgenra of Dendrobium

(subgenra) (Dressler, 1993; Kumar et al., 2011; Schuiteman, 2011) Therefore, in

this study, the use of indicators to assess patterns of genetic diversity can only distinguish relatively accurate 32 Vietnames native Dendroium species s However,

in order to better define the genetic relationship between the breeds studied samples

in the classification of Vietnamese native Dendrobium species, we need to

incorporate the results of the analysis of genetic diversity in the morphological and molecular level using PCR-based technique RAPD marker

3.1.2 Evaluation on genetic diversity for Vietnamese native Dendrobium

orchids by using molecular marker

Using 20 total random pimer RAPD markers, we were able to amplify 408 in

2858 bands with different size of 640 reations 20 random primers have polymorphic, clear, and without any primer application form Smallest size of DNA band was arranged 230 bp in Hoang Thao Chuoi Ngoc Dien Bien (D2) sample and largest sized bands from approximately 4.5 kb were observed in Hoang Thao Thai Binh (D2) sample with OPA2 primer

OPN9 primer was mutipled most 254 bands in total 20 random primers Some typical RAPD primers were bellowed (Figure 3.11) In total 20 RAPD primers used

in the study, there were 26 individual bands or bands with appearance OPN7 had six different bands These primes such as OPN1, OPN2, OPN3, OPN6, OPN8, OPN11, OPN12, OPN13, OPN16, OPN19, OPN20, OPA3, and OPA6 had one or two individual bands or bands with appearance Base on results, we found that it could rely on the individual bands appear in each primer to each seed sample to accurately

identify some native Dendrobium species in Viet Nam