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

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

Reviewer 1:……………………………………………………...

Reviewer 2:………………………………………………………

Reviewer 3:………………………………………………………

The dissertation will be defended before the doctoral thesis Councilatthe Vietnam Academy of Agricultural Sciences

In :……times…….date…….month…..year…..

Search the thesis in:

Vietnam Academy of Agricultural Sciences Library

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 “Evaluation on genetic diversity and identification molecular marker for 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

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

- Thirty two Vietnamese native Dendrobium species were collected from

breeding new varieties to improving the Vietnamese orchid products. 4. Research subjects and scope of the project study 4.1. Research subjects 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

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). 1.1.2.5. Flowers

Flowers have united bisexual, symmetrical two sides with muitiple colour

and have scent. 1.1.2.6. Capsule

Capsule usually have clavated or spindle formation, containing many

particles are mixed with the hair piece. 1.1.3. Ecoregion distributions

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,

the characteristics and structure external

including the description of 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).

Theres were many research on Denedrobium species such as Pellegrino et

* 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 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

(Cymbidiums), Lan hai (Paphiopedilum), Ho Diep (Phalaenopsis), Dai Chau (Rhynchostylis gigantea) and Hoang Thao (Dendrobium) (Bateman, 2001; Choi et al., 2006; Goh et al., 2005; Li et al., 2007; Parab et al., 2008; Zha et al., 2009; Chattopadhyay et al., 2012; Paromik and Suman, 2014). In the Dendrobium spcies, recently Pritam Chattopadhyay et al., 2012 was used to analyze 5 RAPD primers to determine relationship between 5 Dendrobium 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.

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. Research contents 2.2.1. Content 1: Evaluation on genetic diversity for Vietnamese native Dendrobium species 2.2.1.1. Evaluation on genetic diversity for Vietnamese native Dendrobium species by using morphology methods 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.

- Research datas were analyzed, processed on the Excel version 5.0 software, to construct

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 software PCORD, software NTSYS, MEGA: version 5.2.1, phylogenetic trees.

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

Table 3.1. List of Vietnamese native Dendrobium species

Symbols Vietnamese names Latin names Symbols Vietnamese names Latin names Origin (province) Origin (province)

D. fimbriatum Lai Chau D17 Hoang Thao Kim Diep D. capillipes Lam Dong D1

D. findlayanum Dien Bien D18 Hoang Thao Chuoi Ngoc D. findlayanum Kontum D2 Hoang Thao Long nhan Lai Chau Hoang Thao Chuoi ngoc Dien Bien

Hoang Thao Thai Binh D. moschatum Thai Binh D19 D. farmeri Dong Nai D3

Hoang Thao Phi Diep tim D. anosmum Hoa Binh D20 D. chrysotoxum Kontum D4 Hoang Thao Kieu Trang Dong Nai Hoang Thao Tieu Hoang lap

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

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

Yen Bai D23 Hoang Thao Hac vi D. aphylum Da Lat D7

D.lindleyi Quang Tri Dien Bien D24 Hoang Thao Ngoc Van Vang Hoang Thao Phi Diep vang D. chrysanthum D. chrysanthum Lam Dong D. lindleyi Hoang Thao Long tu Bac D. primulinum Son La D25 D9

Lam Dong D. haveyanum Hoang Thao Truc D. hancockii Ninh Binh D26 D10

D. aduncum Kon Tum Hoang Thao Xoan D. tortile Ninh Binh D27 D11

Hoa Binh

Da Lat D. nobile Tay Nguyen D. primulinum D. delacourii Guill Tay Nguyen Hoang Thao Kieu tim Hoang Thao Kieu vang Hoang Thao Kieu trang D. amabile D28 D. thyrsiflorum Tuyen Quang D29 Tuyen Quang D30 D. farmeri D12 D13 D14

D. draconis Rchb.f Lam Dong Hoang Thao Tam Dao D. daoense Tam Dao D31 D15 Hoang Thao Vay rong la nho Hoang Thao Vay rong la trung Hoang Thao Thuy tien mo ga Hoang Thao Vani Hoang Thao Dui ga Hoang Thao long tu da Hoang Thao Moi to Hoang Thao Nhat diem hong

Lao Cai D32 Hoang Thao Dai bac hac D. christyanum Kontum D16 Hoang Thao Hoang lap Tay Bac D. chrysotoxum Note: D. Dendrobium

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:

III

Figure 3.6. Phylogenetic tree of 32 Vietnamese Dendrobium species based

on morphology markers

Subgroup II.1 had 8 samples including D2, D9, D17, D18, D23, D27, D29 and D30 species, with D2 and D18 samples pecent of homology was 100 % homology. Subgroup II.1: had icluding D7, D12, D13, D14, D17, D19, and D26 species with percent of homology at 68% homology and they divided in two different subgroups with D7 and D17 in group; and D12, D13, D14, D19, and D26 species were located in other group.

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 10

little difference especially leaves.

Most of the DNA bands were clear, quite neat, evenly, with no faults or

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 3.1.2.1. DNA extraction impurities after run electrophoresis on the 0.8% agarose gel. 3.1.2.2. Polymorphic analysis of Dendrobium varieties by using PCR- RAPD 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.

* Results of some typical RAPD primers

Figure 3.11. PCR-RAPD products of 32 Dendrobium samples with OPN11 primer

Lanes 1- 32: Dendrobium varieties (D1-D32); M : M: 1kb ladder

3.1.2.3. Genetic relationships between Vietnamese native Dendrobium species

Genetic relationships between Vietnamese native Dendrobium species were indicated on pecent of homology and phylogenetic trees. At 32% of pecent of homology, 32 species were divided into five main groups (Fig.13).

Group I had 13 samples including D1, D2, D3, D4, D5, D6, D10, D11, D15, D18, D9, and D29 species and these were divided into three different subgroups with pecent of homology from 37 to 100% homology.

Subgroup 1 (I.1): Included 6 samples such as D1 (Hoang Thao Long nhan Lai Chau), D3 (Hoang Thao Thai Binh), D11 (Hoang Thao Xoan), D15 (Hoang Thao Tam dao), D10 (Hoang Thao Truc) and D30 (Hoang Thao Moi to) species with percent of homology from 50 to 80% homology.

Subgroup 2 (I.2) included 5 samples: D4, D5, D6, D7 and D17 species. In this subgroups, 3 samples such as D4 (Hoang Thao Phi diep tim), D5 (Hoang Thao Tram rung) and D6 (Hoang Thao Tram trang) species have high pecent of homollogy from 80- 100 % homology in subgroups (Fig. 3.14). D5 and D6 samples were similarity percent of homology at 100%. Two species such as Hoang Thao tram were collected in two different areas of S on La and Dien Bien and they have morphological characteristics such as stems, leaves, flowers same type but with different flower colors. Our observation agreed with study of Tran Hoang Dung et al., 2012, when they studied in two Vietnamese native Dendrobium species as Hoang Thao tram (Dendrobium parishii), and Hoang Thao Phi diep (Dendrobium anosmum) (Tran Hoang Dung et al., 2012).

Subgroup 3(I.3) were included 2 samples: D10 (Hoang Thao Truc) and D15 (Hoang Thao Tam Dao) species with percent of homology at 53% homology. Two varieties were collected in Ninh Binh and Tam Dao provinces, and these were different entirely on stems, leaves and flowers.

Group II had 8 samples including D9, D12, D13, D14, D29, D19, D26, and D27 species with pecent of homology at 47% homology and these divided into two subgroups:

Figure 3.13. Phylogenetic trees on genetic relationship of 32 Vietnamese native Dendrobium species by molecular RAPD marker

Figure 3.14. D4 (Hoang Thao Phi diep tim), D5 (Hoang Thao Tram rung) and D6 (Hoang Thao Tram trang) species

Subgroup 1 (II. 1) included 4 samples: D9, D29, D27 and D26 species with percent of homology from 51-75 %. In this subgroups, D9 (Hoang Thao Long tu Bac) and D29 (Hoang Thao Long tu da) have highest percent of homology at 75% homology. Subgroup 2 (II. 2) included 4 samples: D12, D13, D14 and D19 with percent of homology from 65-80% homology. The characteristics of namely as Kieu species s were identity in leaves, and flowers grow in clusters. They have only difference on the color of flowers. Two varieties as D13 (Hoang Thao Kieu vang) and D14 (Hoang Thao Kieu trang) have the highest of percent of homology at 80%.

Group III included D23 (Hoang Thao Hac vi) and D28 (Hoang Thao Dui ga)

species with the percent of homology at 50% homology.

Group IV had 6 species including D16, D20, D21, D22, D31, and D32 species, and they were divided into two subgroups with percent of homology from 33-50% homology.

Subgroup 1(IV.1) included D31 and D32 species with percent of homology at

50%; Subgroup 2(IV.2) included D16, D20, D21 and D22 species with percent of homology from 50-75% homology. In these subgroups, D20 and D21 species have highest the percent of homology at 75% homology.

Group V had 3 species including D8, D24, and D25 with high percent of homology from 50-100% homology. In these subgroups, two varieties as D24 (Hoang Thao Vay rong la nho) and D25 (Hoang Thao Vay rong la trung) have same the percent of homology at 100% homology. 3.1.3. Evaluation on genetic diversity for Vietnamese native Dendrobium species by morphological characteristics and molecular markers RAPD

Evaluation genetic diversity by morphological characteristics and molecular markers RAPD, the results indicated that 32 Vietnamese native Dendrobium species were divided into 5 different groups. The Hoang Thao orchids several closely related genetically, and integrated into a group. Among the sameples, which has the same form with the same percent of homology such as D5 (Hoang Thao Tram tim) and D6 (Hoang Thao Tram trang); D24 (Hoang Thao Vay rong la nho) and and D25 (Hoang Thao vay rong la trung); or Hoang Thao Kieu varieties have high the percent of homology from 65-80% homology.

In conclusion, to being research and evaluation genetic diversity of germplasm Dendrobium orchids are very necessary work to create a database for conservation methods, develop sustainable use genetic resources, and also have strategic significance in genetic improvement programs. The combination based on morphological classification and classification methods of genetic polymorphisms in the DNA level could complement and support each other in order to classify the varieties/species of Dendrobium species, service of collecting, conservation and bred new varieties. However, RAPD markers had limited to determine species/ subspecies. Indentification between species and subspecies should incorporate sequencing chloroplast DNA as the ITS, or matK gene, gene rbcL (Richardson et al., 2001; Sharma et al, 2012, Liu et al., 2014). 3.2. Molecular maker to indentiy Dendrobium species based on ITS region sequences 3.2.1. Amplification of ITS region based on PCR Using ITS1 and ITS4 primer pairs, we suscessfuly amplified ITS region by PCR products. These results were high quality with only one band in size from 700- 800 bp (Fig.3. 17). There results were also consistented with the findings of other authors to amplify the ITS region on Dendrobium species (Chiang and Tsong, 2012; Tran Hoang Dung et al., 2012; Liu et al., 2014). The bands in size were e clear, correct size should be able to use sequencing.

Figure 3.17. Electrophoresis of amplification ITS segment on 32 Dendrobium species by PCR with ITS1 and ITS4 primer pairs Lanes: 1-32: Dendrobium sampleso; M: 100bp ladder

3.3.2.1. Analysis of 32 Dendrobium species by sequences

The resulst of sequencing showed that the 32 Dendrobium species were amplified and sequenced the ITS region include partial 18S region, entire regions ITS1, 5.8S, ITS2 and partial 28S area, total length of nucleotides obtained from 652 to 715 nucleotide with average 685.3 nucleotides. The percent of nucleotide as T (U) = 22.4%; C = 24.3%; A = 23.7%; and G = 29.6%. This result is quite pretty consistent with the provisions of Xu et al., 2005; Sigh et al., 2012; Liu et al., 2014. 3.3.2.2. Aligment on Vietnamese native Dendeobium and Dendrobium species in the world

To compare the difference of Vietnamese native Dendrobium species and Dendrobium species in the world, we were conducted the analysis of the sample of Vietnamese species and the world based on the analysis coming aligned columns. Here are some results compare the ITS sequences of some orchids form Vietnamese native Dendrobium species and the world based on GeneBank. * Aligment of D2 (Hoang Thao Chuoi ngoc Dien Bien) and D18 (Hoang Thao Chuoi ngoc) in GeneBank

Through the analysis aligns upcoming column (alignments), ITS sequences of D2, D18 Vietnamese species with two species of D.findlayanum | KF143462.1 |, | HQ114257.1 | in the world, these results showed that in some statistical tables nucleotides 4 ITS sequences of species with fluctuations D.findlayanum number of nucleotide sequences from 688- 694 nucleotides when it was compared with the sample sequence shows the two samples D.findlayanum D2 and D18 had 19 different nucleotides. When comparing the two samples D2 and D18 with two reference samples, the samples D2 and D18 have around 8 different sequence than two samples of the world as D. Findlayanum species (Figure 3.19). This result showed that D2 and D18 speciess did not differ much from the world's D.findlayanum species.

two sequences of acession number D.

Figure 3.19. Alignment of D2 and D18 Vietnamese native Dendrobium sequences with findlayanum |KF143462.1|,|HQ114257.1| species

Similarly, most of Vietnamese native Denrobium species were also analyzed by alignment with Dendrobium species of the world to determine the difference in the order as well as identify species/subspecies based on the ITS regions. However, the comparison with the GenBank database on aim for a comparable result with similar taxa with the reference sequence. BLAST results could not conclude exactly the species. For instance BLAST similarities coverage and high identity sequences (99%), could not reverse impairment species name. BLAST results were only available showed the most homologous sequences in the GenBank. Therefore, in order to determine the correct species name should make more of other analysis as compared with morphological data and determined the relationship through phylogenetic tree based on ITS region sequences. Therefore, we wre decided to phylogenetic tree of Dendrobium species to determine the exact name of the samples in this study. 3.2.3. Phylogentic trees base on ITS region sequences

Arcoding the phylogenetic trees, 32Vietnamese native Dendrobium species

trees were divided in 17 diferent subgroups:

Group I included 3 Vietnamese species such as D4 (D. anosmum), D5 (D.parishi.var alba) and D6 (D.parishi); and 6 sequences with acession number HM590378.1, HM054736.1, HM054735.1, AB5936930.1 species on D.parishi, and 2 sequences on D.asosmum species with acession number EU477499.1, JN388570.1. In early diverging positions, D6 species was identified as D. parishi morphological but located near acession number EU47749.1, which was identified as D. anosmum, whereas 4 species with acession number included HM590378.1, HM054736.1, HM054735.1, and AB5936930.1 as D. parishi were located together in subgroups. This could be confirmed that the acession number EU47749.1 was D. parishi species, not anosmum, from which we deduced that D6 species was similar of D.parishi species. In late position divergence, D4, D5 and JN388570.1 samples were located in same subgroups. In terms of morphology, D4 sample was indentified

as anosmum species and D5 was identified as D parishi.var alba. However, two species as D4 and D5 were located with acession number JN388570.1 subgroup on percent idnetity at 99%. Therefore, D5 sample should be determined as D. anosmum.var alba. Our observation agreed with study of Tran Hoang Dung et al., 2012, when they were analyzed two Vietnamese species as Hoang Thao Tram rung (D. parishi) and Hoang Thao Phi diep (D. anosmum) (Tran Hoang Dung et al., 2012).

Group II: included D9, D29 species and 3 acession numbers KF14399.1, AB593641.1, AB59352.1 as D.primulinum species were located in subgroup. D9 and D29 species were same divided with acession number AB59352.1in subgroups at 99% boostrap index. D9 and D29 species were distributed in both Northern and Central highlands. Morphologically, they have similar characteristics and identified as D.primulinum species. Therefore, D9 and D29 species were accurately identified as D.primulinum species.

Group III inclued D23 species which was identified as D. aphylum morphology and it was same located in subgroups with 4 acession number HM590384.1, HQ114247.1, HQ114248.1 and KF143430.1 at 100% boostrap index. Therefor, D23 species was identified as Hoang Thao Hac vi (D. aphylum).

Group IV: D11 and D28 species were located in same group with 6 species in the world. In this group, D11 (D.tortile) divided with 4 different acession number EU4775071 (D.nobile), EU477507.1 (D.fiedricksianum), KF143518.1 (D.sp.Jin XHs.n .9) and EU477511.1 (D.tortile) in subgroup with bootstrap index up to 98%. In terms of morphology, D11 was different morphology as stems, leaves, flowers compared to acession number EU477507.1 (D.fiedricksianum), KF143518.1 (D. Sp.Jin XHs.n.9). From the results of the comparison on Balst, these templates are also messy unfocused. Therefore, we could not recognize the D11 sample as D.tortile or not. However, the D28 sample was located with acession numbers as KC205193.1 and JN38579.1 at 85% bootstrap index. Therfore, this indicated that D28 sample was indentified as D. nobile.

Group V had three species including D27, D2 and D18 divided in two subgroups: Subgoup 1 included D27 (D.aduncum) species and divided into one group with 3 different species sequences as acession numbers JF713083.1, KF143428.1 and JN388580.1 at 99% bootstrap index. On other hand, the characteristic of the morphology on D27 was similar with the 3 Dendrobium species. Therefore, D27 sample should be indentifing as D. aduncum; Subgoup 2 was included D2 and D18 samples, which were colleted in two provinces. In terms of their form exactly the same on stems, leaves, and flowers differs only Long tu Bac (D2) in the middle of the flower lip does not have spots. When we comparing sequences of them, it have only the bootstrap of 100% with 3 acession numbers as KF143246.1, HQ114257.1, and JN388589.1 belong D.findlayamum species. Therefore, D2 and D18 samples were indentified as D. findlayamum species.

71 64 90

D. parishii voucher NCHU-D89331201-1012|HM590378.1| D. parishii|AB593630.1| D. parishii voucher SBB-0528 |HM054736.1| D. parishii voucher SBB-0527 HM054735.1|

79 D. anosmum |EU477499.1|

D. parishi - D6

D.parishi.var alba - D5

100

D. anosmum - D4 D. anosmum isolate D4 |JN388570.1| D. primulinum voucher Jin X-H 10793 |KF143499.1| D. primulinum |AB593641.1|

D. primulinum TBG 118293|AB593521.1| D. primulinum -D9

99 90

100

D. primulinum - D29 D. aphyllum voucher NCHU-D89331201-1018 HM590384.1| D. aphyllum voucher PS2523MT01 |HQ114247.1| D. aphyllum voucher PS2523MT02 |HQ114248.1| D.aphyllum -D23 D. aphyllum voucher Jin X-H 10798 |KF143430.1|

D. tortile - D11 D. nobile |EU477507.1| D. friedricksianum |EU477505.1| D. sp. Jin X-H s.n. 9 |KF143518.1|

D. tortile |EU477511.1|

D. nobile |KC205193.1|

D. nobile - D28

D. nobile |JN388579.1|

85 59

D. aduncum - D27

100

D. aduncum voucher SBB-0309 |JF713083.1|

D. aduncum voucher Jin X-H 9522 |KF143428.1| D. aduncum |JN388580.1|

100

D. findlayanum -D2 D. findlayanum - D18 D. findlayanum voucher Jin X-H 11875|KF143462.1| D. findlayanum voucher PS2531MT01 |HQ114257.1| D. findlayanum isolate D23 |JN388589.1|

D. chysanthum - D7

D. chrysanthum |JN388584.1| D. chrysanthum |FJ384738.1| D. chysanthum- D8

D. chrysanthum voucher Jin X-H 11430 |KF143443.1| D. chrysanthum voucher SBB-0617 |JF713093.1| D. chrysanthum voucher SBB-0504 |JF713091.1| D. brymerianum - D30

100

D. brymerianum voucher PS2510MT01|HQ114233.1| D. brymerianum voucher Jin X-H10782 |KF143432.1| D. brymerianum |JN388581.1| D. brymerianum |EU477500.1|

100

D. pulchellum voucher Jin X-H 11878 |KF143503.1| D. moschatum -D3 D. moschatum voucher Jin X-H 11886 |KF143492.1| D. daoense - D15 D. fimbriatum isolate D22 |JN388588.1| D. fimbriatum - D1 D. fimbriatum voucher PS2507MT01 |HQ114229.1| D. fimbriatum voucher Jin X-H s.n. 16 (KF143461.1)

D. chrysotoxum - D20

D. chrysotoxum - D21

100

99 74

D. chrysotoxum |EU477501.1| D. capillipes voucher NCHU-D89331201 |HM590379.1| D. chrysotoxum voucher PS2501MT03 |HQ114223.1| D. chrysotoxum -D16 D. chrysotoxum voucher PS2501MT01 |HQ114221.1| D. chrysotoxum isolate D19 |JN388585.1|

D. hancockii voucher Jin X-H 13492 |KF143467.1| D. hancockii isolate D25 |JN388591.1| D. hancockii voucher PS2533MT01 |HQ114259.1| D. hancockii |AB593575.1|

D. hancockii - D10

D. capillipes - D17 D. capillipes |AF362035.1|

100

D. capillipes voucher Jin X-H 10757 |KF143433.1| D. capillipes voucher PS2502MT01 |HQ114224.1| D. capillipes |JN388582.1|

D. amabile - D12

D. thyrsiflorum - D13

100

D. farmeri -D14 D. farmeri -D19 D. haveyanum -D26 D. thyrsiflorum |KC205200.1|

D. thyrsiflorum voucher Jin X-H 10755 |KF143519.1|

100

D. lindleyi - D24 D. lindleyi -D25 D. jenkinsii voucher Jin X-H 10709 |KF143478.1| D. jenkinsii voucher PS2525MT01 |HQ114251.1|

D. jenkinsii voucher Jin X-H s.n. 4 |KF143479.1| D. jenkinsii isolate D29 |JN388595.1|

99 D. draconis Rchb.f - D31

75 100

D. draconis |HM054628.1| D. draconis voucher SBB-0545 |JF713101.1| D. draconis |EU477503.1|

D. wattii - D22

73 99

D. wattii voucher Jin X-H 11817 |KF143525.1| D. longicornu voucher Kurzweil H Lwin S 2647 |KF143484.1|

D. longicornu voucher Jin X-H 11663 |KF143485.1|

74 D. christyanum -D32 D. christyanum voucher Jin X-H s.n. 3 |KF143442.1| D. christyanum voucher Jin X-H 11045 |KF143441.1| D. christyanum |GU339106.1| D. christyanum |EF629325.1|

0.02

Figure 3.25. Phylogenetic trees based on ITS sequences

Group VI included D7 and D8 samples, these spcies were indentified as D.chrysanthum and located with 6 different D.chrysanthum species in the world as controls. D8 sample was indentity at 100% when it was compared with two acession number of D.chrysanthum as JN388584.1 and FJ384738.1. Therefore, we concluded that D7 and D8 were indentified as D.chrysanthum species.

Group VII included D30 (Hoang Thao Moi to- D.brymerianum) species, and 4 species as D.brymerianum: KF143432.1, HQ114233.1, JN388581.1 and EU477500. 1 in subgroups at 80% bootstrap index. D30 species was group with 4 4 species as D.brymerianum but relatively large genetic distance with the pattern of the world.

Group VIII included D3 (Hoang Thao Thai Binh - D.moschatum) species which was indentified as D. moschatum (KF143492.1) at 100% with bootstrap index. However, in this group there was one form as D.puchellum (KF1435031.1) similar D.moschatum (KF143492.1). In terms of morphology, these were identiccal on stems, leaves and flowers. However sample (KF1435031.1) was identified as D.puchellum. This may explain that, there was defected when the sample (KF1435031.1) was identified Latin name. In terms of morphology, D3 was identified as D.moschatum, thereby confirming that the D3 sample was corrected as D.moschatum species after sequencing the ITS region.

Group IX included 2 Vietnamese species such as D1 (HT Long Long nhan Dien Bien), D15 (HT Tam Dao) and 3 species of world as D. fimbriatum classification. In this group, the sample D1 (D. fimbriatum) and D15 (D.daoense) were indentity from 86-100% bootstrap index with 3 species of D. fimbriatum with acession number HQ114229.1, JN388588.1, KF143461.1. However, morphological identification on D15 species was identified as D.daoense. Therefore, we confirmed that D15 species was collected as D. fimbriatum, which was not as D.daoense species.

Group X included 3 Vietnamese species as D16, D20, D21 and 4 species as D. chynsotoxum with acession numbers HQ114221.1, JN388585.1, HQ114223.1, EU477501.1 and 1 sample of species D.cappillies (HM590379.1) In the 5 samples of this world, HM590379.1 sample sequencing was wrong because if this form is mandatory D.cappillies species it must be located on the group XII, could not locate into this groupd as sequences belong D. chynsotoxum. The D16, D20 and D21 were divided in two subgroups consisting D16 (Hoang Thao Hoang Hoang lap Tay Bac) into one group which located with 4 species as D. chynsotoxum. Other subgroup included D20 (Hoang Thao tieu Hoang lap), D21 (Hoang Thao Dai Hoang lap) and acession numbers EU477501.1 with high bootstrap index (98%). In terms of form, the form Hoang Thao Hoang lap species were shape, leaf shape and color of flowers marking the same, they are only about the size of a little flower and distributed in

different regions. Thereby may notice that, 3 sample as D16, D20 and D21 species have the genetic separation, but they were same species D. chynsotoxum.

Group XI included 5 species as D10 (D. hancokii) and 4 species D. hancokii sequences form a group with the 76-88% bootstrap index. However, D10 sample was separated genetic distance is quite far with 4 D. hancokii species of the world.

Group XII

included D17 (D.capillipes) and 4 D.capillipes species: AF362035.1, KF143433.1, JN388582.1, HQ114224.1, and they divided into two branches with D17 sample and acession numbers AF362035.1 with bootstrap index (88%) and the 3 samples were located together form a branch. In forms, D17 (Hoang Thao Kim Diep) sample was recognized as D.capillipes, thereby confirming that the corrected form is D.capillipes D17 after sequencing on the ITS region.

Group XIII included 7 sample which had 5 Vietnamese samples such as D12 (D.amabile), D13 (D.thyrsiflorum), D14 (D.farmeri), D19 (D.farmeri), D26 (D.haveyanum) and two acession numbers KF143519.1, KC205200.1 of D.thyrsiflorum, and they divided into subgroups. Subgroup 1 had 5 samples and acession number KC205200.1 with bootstrap index at 100%. D12 and D13 samples were separated within the same group with 91% bootstrap index, while D14, D19 and D26 have the same one branch with KC205200.1 samples with 100% bootstrap index. In terms of form, Hoang Thao Kieu varieties as D12, D13, D14, D19 and Thuy Tien Hoang Thao mo ga (D26) are very similar in morphology, such as stems, leaves, they differ only in the color of flowers and region distribution. However, in the phylogenetic tree showed that the samples collected have very high bootstrap index from 96-100%, while the morphological identification Latin name is different. So it could be concluded that the samples collected when no precision on morphological identification or they have extremely close genetic relationship to use ITS not enough to identify their exact species. Only could determine D13 (HT Kieu Vang) were recognized as D.thyrsiflorum species.

Group XIV included D24 and D25 (D.lindleyi species) species and 4 samples collected sequences are KF143479.1, JN388595.1, KF143478.1 and HQ114251.1 (D.jenkinsii species) with bootstrap index from 94-100%. In terms of form, D24 and D25 varieties were easy to get two wrong with D.jenkinsii. D.lindleyi scales very often confused with snake scales or fins, when comparing the flower stems and branches, stems larger scales scaly, flower scales have long stem from 5-15 flowers and D.jenkinsii species were less flowers than D.lindleyi. Therefore, the results of the ITS region sequencing and comparative phylogenetic tree showed that D24 (Hoang Thao Vay rong la nho) and D25 (Hoang Thao Vay rong la trung) were D.jenkinsii species.

Group XV inculded Hoang Thao Nhat diem hong (D. draconis Rchb.f - D31) with 5 species in acession numbers as HM054628.1, EU477503.1, JF713101.1 (D. draconis) with bootstrap index 100%. Therefor, D31 was indicated as D. draconis species.

Group XVI included D22 (Hoang Thao Bach hac langbiang- D.wattii) and 3 other varieties such as KF143525.1, KF143481.1, KF143485.1. In this group, the D22 was into branch with Dendrobium voucher Jin XH 11 817 (KF143525.1) at 73% bootstrap index. But D. wattii group has very close with D.longicomu (KF143481.1, KF143485.1) with bootstrap index from 74-99%. In terms of morphology, D. wattii and D.longicomu have similar morphology so easy to confuse the distinction. Therefore, D22 Hoang Thao Bach hac langbiang confirmed exactly D.wattii species

Group XVII included Hoang Thao Dai bach hac (D. christyanum -D32) and 4 species with acession numbers F143442.1, KF143441, GU339106.1, and EF629325.1. These varieties were indentified D. christyanum species with bootstrap index at 99%. This suggests that, the D32 sample was indentifield as D. christyanum species .

The information through the ITS region sequencing showed that the majority identifying compatible with the study was of samples collected during morphological identification. 23/32 species were indentified by recognition molecule forms such as D1, D2, D3, D4, D5, D7, D8, D9, D10, D13, D16, D17, D18, D20, D21, D22, D23, D27, D28, D29, D30, D31, and D32 species (Table 3.14). However, during the study, the varieties were identified based on morphological form, then sequencing the ITS region. Comparision on sequencing between 32 Vietnamese native Dendrobium orchids and and sequecing of Dendrobium orchids varieties in the world, we have determined the exact same pattern 23/32 of Dendrobium varieties. In the remaining 9 Vietnamese native Dendrobium species, and 4 species were edited form Latin names such as (Hoang Thao tram Trang), D15 (Hoang Thao Tam Dao), D24 (Hoang Thao Vay rong la nho), and D25 (Hoang Thao Vay rong la trung); 5 samples has not been pinpointed as the same form D11 (Hoang Thao Xoan), D12 (Hoang Thao Kieu tim), D14 (Hoang Thao Kieu trang), D19 (Hoang Thao Kieu trang Dong Nai), and D26 (Hoang Thao Thuy tien mo ga). Explanations for this phenomenon has many different causes, such as collecting seed samples confusion about different Latin name; The process of preservation can breed confusion or data on GeneBank insufficient information with the same sample by the Latin name and dissimilar ITS region.

Table 3.14. Identification on Vietnamese native Dendrobium species based on ITS sequences

Vietnamese names Morphological Symbols Vietnamese names Morphological Sym bols Indentification based on ITS sequencces Indentification based on ITS sequencces

D1 D. fimbriatum D. fimbriatum D. capillipes D. capillipes D17

D2 D. findlayanum D. findlayanum D. findlayanum D. findlayanum D18

D3 D. moschatum D. moschatum D. farmeri Chưa xác định được D19

D4 D. anosmum D. anosmum D. chrysotoxum D. chrysotoxum D20 Hoang Thao Long nhan Lai Chau Hoang Thao Chuoi ngoc Dien Bien Hoang Thao Thai Binh Hoang Thao Phi Diep tim

D5 Hoang Thao Tram tim D.parishii D.parishii D. chrysotoxum D. chrysotoxum D21

D6 D.anosmum.var alba D22 D. wattii D. wattii D.parishi .var alba Hoang Thao Kim Diep Hoang Thao Chuoi Ngoc Hoang Thao Kieu Trang Dong Nai Hoang Thao Tieu Hoang lap Hoang Thao Dai Hoang lap Hoang Thao Bach hac langbiang

D7 D. chrysanthum D. chrysanthum Hoang Thao Hac vi D. aphylum D. aphylum D23

D. chrysanthum D. chrysanthum D. lindleyi D. jenkinsii D24

D9 D. primulinum D. primulinum D. lindleyi D. jenkinsii D25 Hoang Thao Tram trang Hoang Thao Ngoc Van Vang Hoang Thao Phi Diep vang Hoang Thao Long tu Bac

D10 Hoang Thao Truc D. hancockii D. hancockii D. haveyanum Not indentify D26

D. tortile D. aduncum

D11 Hoang Thao Xoan D12 Hoang Thao Kieu tim D. amabile Not indentify Not indentify D. aduncum D. nobile D27 D28

D13 D. thyrsiflorum D. thyrsiflorum D. primulinum D. primulinum D29 Hoang Thao Vay rong la nho Hoang Thao Vay rong la trung Hoang Thao Thuy tien mo ga Hoang Thao Vani Hoang Thao Dui ga D. nobile Hoang Thao long tu da

D14 D. farmeri Not indentify Hoang Thao Moi to D.brymerianum D.brymerianum D30 Hoang Thao Kieu vang Hoang Thao Kieu trang

D15 Hoang Thao Tam Dao D. daoense D. fimbriatum D. draconis Rchb.f D. draconis Rchb.f D31

D. christyanum D. christyanum D16 D. chrysotoxum D. chrysotoxum D32 Hoang Thao Nhat diem hong Hoang Thao Dai bac hac Hoang Thao Hoang lap Tay Bac

In summary, although the species is still limited but the ITS sequences again showed they promote strength is a universal DNA barcode in the delimitation of species and subspecies level for plant group flowering. The result showed that the majority of varieties were identified their name and species. Some varieties were differ in name by morphological and ITS sequence region. Through the analysis shows that the location of the animals used in the phylogenetic tree based on sequence analysis of the ITS help confirm correct scientific names of varieties. The success of this study were make important prerequisite for us to expand the sample size like (the same sample in the species), Dendrobium species, or more species in Orchideacea), or selection some molecular marker (16 rRNA genes, gene matK ...) to construct DNA barcodes for valuable orchids in Vietnam.

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS: 1. Vietnamese native Dendrobium species have diverse stems, leaves, and flowers. On the basis of the phylogenetic tree, 32 varieties were divided in 5 different groups with percent of homology from 37-100% homology. Some species such as D5 (Hoang Thao Tram tim) and D6 (Hoang Thao Tram trang); D2 (Hoang Thao Chuoi ngoc Dien Bien) and D18 (Hoang Thao Chuoi ngoc have a genetic similarity coefficient was 100% homology. 2. Genetic diversity of 32 Vietnamese Dendrobium native species by using RAPD markers showed genetic similarity coefficient ranged from 23 to 100% homology. Some varieties included D5 (Hoang Thao Tram tim) and D6 (Hoang Thao Tram trang); D24 (Hoang Thao Vay rong la nho) and D25 (Hoang Thao Vay rong la trung) have a genetic similarity coefficient high as 100% indentity. 3. Evaluation genetic diversity by morphological characteristics and molecular markers RAPD, the results indicated that 32 Vietnamese native Dendrobium scpecies were divided into 5 different groups. The same pattern Hoang Thao orchids several closely related genetically, and integrated into a group. Among the sameples, which has the same form with the same percent of homology such as D5 (Hoang Thao Tram tim) and D6 (Hoang Thao Tram trang); D24 (Hoang Thao Vay rong la nho) and and D25 (Hoang Thao vay rong la trung). 4. Base on RAPD primers, we have indentified samples by individual bands or bands with appearance. For example, D2- Hoang Thao Chuoi Ngoc Dien Bien sample had identified by OPN7 and OPN20 prmers; or D9- Hoang Thao Long tu Bac was indentified by OPN16 and OPN20 primers. 5. Comparision on sequencing between 32 Vietnamese native Dendrobium and Dendrobium species in the world by using ITS sequences, we have determined the exacty same pattern 23/32 of Dendrobium species. In the remaining 9 Vietnamese native Dendrobium varieties, 4 varieties were edited form Latin names such as (Hoang Thao tram Trang), D15 (Hoang Thao Tam Dao), D24 (Hoang Thao Vay

rong la nho), and D25 (Hoang Thao Vay rong la trung); 5 samples has not been pinpointed as the same form D11 (Hoang Thao Xoan), D12 (Hoang Thao Kieu tim), D14 (Hoang Thao Kieu trang), D19 (Hoang Thao Kieu trang Dong Nai), and D26 (Hoang Thao Thuy tien mo ga) species. RECOMMENDATIONS:

Investigate and collect more Dendrobium species. In addition, to choosing some marker (16 rRNA genes, gene matK, rbcL region, the constructions-psbA...) to identify several closely related of Dendrobium species.