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 their were 2nd orchids after bulbophyllum
orchid (Bulbophyllum) (Leitch et al. , 2009). Southeast Asia can be considered
as the homeland of dendrobium with 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
diversity in the gene level, provide the basis for assessing the conservation
value of species and populations. Therefore, the creation of a database of DNA
(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 orchid 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 orchids 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 orchids, which is basis
for selection of elite germplasm for the selected service and new breeding;
- The results of the research are significant in identifying a number of
varieties/species of Vietnamese native Dendrobium orchids, as a basis for
conservation, economic value and register on GenBank.
3.2. Practical Implications
- Collect and preserve genetic resources of Vietnamese native
Dendrobium orchids;
- The results are contribute 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
The thesis is the first study 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, select and breed new varieties, contribute to improving
Vietnam orchids.
CHAPTER 1: OVERVIEW
1.1. Glance of Dendrobium
1.1.1. Classification systems
In the botanical classification systems, the gennus Dendrobium
(Dendrobium) are belong the spread (Orchidales), subclass (Liliidae), class
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 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 are segmented, cylindrical shaped spindles,
clavate, ovoid, with lengths ranging from 2-3cm to 120cm or sometimes more,
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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 orchids 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 mountainou 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 species of Dendrobium were also to make
a Nontraditional medicine (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 indicators
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Evaluation genetic diversity on morphologicals are traditional method,
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 hay 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 includes two separate loci (ITS1 and ITS2) and they are
connected together via locus 5.8S. 5.8S pretty 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. Situation 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 orchids such as Pellegrino et
al., 2005; Krishnan et al., 2011).
1.4.1.2. . Research on molecular markers
* Research is based on on RFLP marker
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
* Research is based on RAPD marker
There have been studies using indicators to analyze RAPD polymorphism
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in orchids, the study focused on several categories like spreads Search
(Cymbidiums), Paph (Paphiopedilum), Phalaenopsis (Phalaenopsis), Dai Chau
(Rhynchostylis gigantea) and Hoang Thao orchids (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 orchids, recently Pritam Chattopadhyay et al., 2012 was used to
analyze RAPD 5 prey relationship between genetic 5 Dendrobium orchid
species in the North of India. Results showed that, with a total of 124 bands
were analyzed DNA is found to have 25 individual bands between different of
Dendrobium.
* Research is based on SSR marker
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).
* The research is based on the ISSR marker
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 of
Chinese authors published in 2009 (Yao et al., 2009). In this study, the authors
decode region located in between psbA-constructions on chloroplast genome of 17
species 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 study based on morphological
characteristics and found three species of D. pulchellum, D. 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 "Assessment of
genetic diversity of wild orchid Dendrobium 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. Most recently, Tran Hoang Dung et al., 2012 had
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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. Materials
Inheritance source materials were made by the Institute of Agricultural
Genetics and Prof. Dr. Tran Duy Quy, 32 samples of Vietnamese native
Dendrobium (aged 2-3 years) were collected in the different provinces and
their 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
orchids by using RAPD marker
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 marker
2.3.2.1. Extraction of DNA
2.3.2.2. The quantative and quality of DNA
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. Purified PCR products and sequencing
2. 3.3.3. Comparison ITS sequencing with 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,
6
software PCORD, software NTSYS, MEGA: version 5.2.1, to construct
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 samples of Vietnamese native Dendrobium orchids 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
sample varieties, some varieties were endemic pattern of the Northern provinces
of Vietnam as Hoang Thao Chuoi Ngoc (D2), which is endemic in Dien Bien
province, 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 samples
flower varieties found only in some areas of some Central provinces or South
were including Hoang Thao Vani, Hoang Thao Dai Bach hac variety which was
only found in Kontum province.
3.1.1. Evaluation on genetic diversity for Vietnamese native Dendrobium
species by using morphology methods
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.
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Table 3.1. Samples of Vietnamese native Dendrobium species
Symbols
D1
D2
Vietnamese names
Hoang Thao Long nhan
Lai Chau
Hoang Thao Chuoi ngoc
Dien Bien
Latin names
Origin
(province)
Symbols
Vietnamese names
Latin names
Origin
(province)
D. fimbriatum
Lai Chau
D17
Hoang Thao Kim Diep
D. capillipes
Lam Dong
D. findlayanum
Dien Bien
D18
Hoang Thao Chuoi Ngoc
D. findlayanum
Kontum
D. farmeri
Dong Nai
D. chrysotoxum
Kontum
Hoang Thao Kieu Trang
Dong Nai
Hoang Thao Tieu Hoang
lap
D3
Hoang Thao Thai Binh
D. moschatum
Thai Binh
D19
D4
Hoang Thao Phi Diep tim
D. anosmum
Hoa Binh
D20
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
Hoang Thao Ngoc Van
Vang
Hoang Thao Phi Diep
vang
D.
chrysanthum
D.
chrysanthum
Yen Bai
D23
Hoang Thao Hac vi
D. aphylum
Da Lat
Dien Bien
D24
D.lindleyi
Quang Tri
D9
Hoang Thao Long tu Bac
D. primulinum
Son La
D25
D10
Hoang Thao Truc
D. hancockii
Ninh Binh
D26
D11
Hoang Thao Xoan
D. tortile
Ninh Binh
D27
D12
D13
D14
Hoang Thao Kieu tim
Hoang Thao Kieu vang
Hoang Thao Kieu trang
D. amabile
D. thyrsiflorum
D. farmeri
Hoa Binh
D28
Tuyen Quang D29
Tuyen Quang D30
D15
Hoang Thao Tam Dao
D. daoense
Tam Dao
D31
Lao Cai
D32
D7
Hoang Thao Hoang lap D.
Tay Bac
chrysotoxum
Note: D. Dendrobium
D16
8
Hoang Thao Vay rong la
nho
Hoang Thao Vay rong la
trung
Hoang Thao Thuy tien mo
ga
Hoang Thao Vani
D. lindleyi
D. haveyanum
Lam Dong
Lam Dong
D. aduncum
Kon Tum
Hoang Thao Dui ga
Hoang Thao long tu da
Hoang Thao Moi to
Hoang Thao Nhat diem
hong
D. nobile
D. primulinum
D. delacourii Guill
Da Lat
Tay Nguyen
Tay Nguyen
D. draconis Rchb.f
Lam Dong
Hoang Thao Dai bac hac
D. christyanum
Kontum
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 very low (D22 average 6.46 leaf/plant).
Leaves may be hard or soft, usually smooth surfaces. Most of the leaves of the 32
Dendrobium orchid were marks tongue-shaped leaves, leaf oval, pointed leaves
peaks, sharp deviation or imprisonment.
Flowers can have many flowers, or have fewer flowers. Flowers have often
pendulous hanging down length, or alternately (Figure 3.4). Most of samples
Dendrobium orchid varieties 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 samples Dendrobium
orchids, we used to software Pcord 4.0 to build phylogenetic relationships between
32 samples of Dendrobium orchids varieties (Figure 3.6).
Figure 3.4. Some types of flowers in Dendrobium species
To examine the relationship between Vietnamese native species at 37%
homology, 32 samples of Dendrobium varieties were divided into five groups
according to the phylogenetic tree:
Group I had eight samples including D1, D3, D4, D5, D6, D10, D11; andD15
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 varieties 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) had 100 % percent homology.
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Subgroup I.2 had two samples incuding D10 (Hoang Thao Truc) and D15
(Hoang Thao Tam Dao).
I
II
III
IV
V
Figure 3.6. Phylogenetic tree of 32 Vietnamese Dendrobium varieties base
on morphology marker
Group II had 15 samples including D2, D7, D9, D12, D13, D14, D17, D18,
D19, D22, D23, D26, D27, D29, D30 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:
Subgroup II.1 had 8 samples including D2, D9, D17, D18, D23, D27, D29 and
D30 with D2 and D18 samples pecent of homology was 100 % homology.
Subgroup II.1: had icluding D7, D12, D13, D14, D17, D19 and D26 with
percent of homology at 68% homology and they divided in two different subgroups
with D7 and D17 in group; and five sameples in cluding D12, D13, D14, D19, and
D26 in group.
These varieties 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. In this group, D31
and D32 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 varieties were distributed in Dong Nai and Lam Dong provinces. In terms of
flower morphology they have similar color patterns more like stems; leaves and
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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). These varieties 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 little
difference especially leaves.
The classification Dendrobium 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 Dendroium varieties. However, in order to better
define the genetic relationship between the breeds studied samples in the
classification of species of Vietnamese Dendrobium, 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
Most of the DNA bands were clear, quite neat, evenly, with no faults or
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 1 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 orchid species in Viet Nam.
* Results of some typical RAPD primers
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Figure 3.11. PCR-RAPD products of 32 Dendrobium samples with OPN11
primer
Lane 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. The pecent of homology at
32%, 32 varieties 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 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) with percent of homology
from 50 to 80% homology.
Subgroup 2 (I.2) were included 5 samples: D4, D5, D6, D7 and D17. In this
subgroups, 3 samples such as D4 (Hoang Thao Phi diep tim), D5 (Hoang Thao Tram
rung) and D6 (Hoang Thao Tram trang) 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%. These were two varieties such as Hoang Thao tram were collected
in two different areas of Son La and Dien Bien and they have morphological
characteristics such as stems, leaves, flowers same type but with different flower colors.
This result was similar to the author Tran Hoang Dung et al., 2012, when they studied in
two Vietnamese native Dendrobium orchid species as Hoang Thao tram (Dendrobium
parishii), and Hoang Thao Phi diep (Dendrobium anosmum) (Tran Hoang Dung et al.,
2012).
12
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) varieties
Subgroup 3(I.3) were included 2 samples: D10 (Hoang Thao Truc) and D15
(Hoang Thao Tam Dao) 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
with pecent of homology at 47% homology and these divided into two subgroups
Subgroup 1 (II. 1) included 4 samples: D9, D29, D27 and D26 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 varieties were
similar 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)
samples with the percent of homology at 50% homology.
Group IV had 6 samples including D16, D20, D21, D22, D31, D32, and they
were divided into two subgroups with percent of homology from 33-50% homology.
13
Subgroup 1(IV.1) included D31 and D32 samples with percent of homology at
50%; Subgroup 2(IV.2) included D16, D20, D21 and D22 with percent of homology
from 50-75% homology. In these subgroups, D20 and D21 samples have highest the
percent of homology at 75% homology.
Group V had 3 samples 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 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); 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 orchids, 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 700800 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 band in size were e clear,
correct size should be able to use sequencing.
14
Figure 3.6. Electrophoresis of amplification ITS segment on 32 Dendrobium
samples were amplified by PCR with ITS1 and ITS4 primer pairs
Lanes: 1-32: Dendrobium sampleso; M: 100bp ladder
3.3.2.1. Analysis of 32 Dendrobium samples 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 sequences 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 Dendrobium species and the world based on GeneBank.
* Aligments analysis 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 with the
chamber 19 different. 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 D. findlayanum (Figure 3.19). This result shows that D2
and D18 Vietnamese speciess did not differ much from the world's D.findlayanum
species.
15
Figure 3.19. Alignments of D2 and D18 Vietnamese native Dendrobium
sequensed with two D. findlayanum |KF143462.1|,|HQ114257.1| in the world
Similarly, most of Vietnamese native Denrobium species were also analyzed
by alignments 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 cold 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
contruct 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
Figure 3. 25 showed that, the phylogenetic trees were divided 32 Vietnamese
spces in 17 diferent groups:
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 sample was identified samples as D.
parishi morphological but located near acession number EU47749.1 similar pattern
was identified as D. anosmum, whereas 4 samples 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 were D. parishi, not anosmum, from which we deduced that D6 sample
was similar patterns of D.parishi species. In late position divergence, D4, D5 and
JN388570.1 samples were located in same subgroups. In terms of morphology, D4
16
sample was indentified as anosmum species and D5 sample was identified as D
parishi.var alba. However, two samples 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. This result was coincided with the
study by Tran Hoang Dung et al., 2012 when researchers 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: D9, D29 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 samples 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 is D.tortile or
not. However, the D28 sample was located with acession number as KC205193.1
and JN38579.1 at 85%. bootstrap index. So, D28 sample was indentified as D.
nobile
Nhóm V had three samples including D27, D2 and D18, there were divided
in two subgroups:
Subgoup 1 was included D27 (D.aduncum) divided into one group with 3
different species sequences as JF713083.1, KF143428.1 and JN388580.1 at 99%.
bootstrap index. On other hand, the characteristics of the morphology on D27 was
similar with the 3 Dendrobium sample. Therefore, d27 smaple should be asv
D.aduncum.
Subgoup 2 was included D2 and D18 samples. There were colleted in two
provinces. In terms of their form exactly the same on stems, leaves, flowers differs
only Long tu bac (D2) in the middle of the flower lip does not have spots. When
comparing their sequences, they have only the bootstrap of 100% compared to the
sequence of the 3 samples as KF143246.1, HQ114257.1, and JN388589.1 belong
D.findlayamum species. Therefore, D2 and D18 were indentified as D. findlayamum
species
17
79
70
68
76
56
64
70
71 D. parishii voucher NCHU-D89331201-1012|HM590378.1|
64 D. parishii|AB593630.1|
90 D. parishii voucher SBB-0528 |HM054736.1|
79D. parishii voucher SBB-0527 HM054735.1|
82
D. anosmum |EU477499.1|
99
D. parishi - D6
D.parishi.var alba - D5
D. anosmum - D4
99
99
D. anosmum isolate D4 |JN388570.1|
100 D. primulinum voucher Jin X-H 10793 |KF143499.1|
D. primulinum |AB593641.1|
D. primulinum TBG 118293|AB593521.1|
97
87
99 D. primulinum -D9
90 D. primulinum - D29
D. aphyllum voucher NCHU-D89331201-1018 HM590384.1|
D. aphyllum voucher PS2523MT01 |HQ114247.1|
D. aphyllum voucher PS2523MT02 |HQ114248.1|
100
D.aphyllum -D23
D. aphyllum voucher Jin X-H 10798 |KF143430.1|
D. tortile - D11
96
D. nobile |EU477507.1|
98
D. friedricksianum |EU477505.1|
D. sp. Jin X-H s.n. 9 |KF143518.1|
77
62 D. tortile |EU477511.1|
D. nobile |KC205193.1|
D. nobile - D28
85
59 D. nobile |JN388579.1|
D. aduncum - D27
75
100
D. aduncum voucher SBB-0309 |JF713083.1|
99
D. aduncum voucher Jin X-H 9522 |KF143428.1|
D. aduncum |JN388580.1|
D. findlayanum -D2
75
D. findlayanum - D18
D. findlayanum voucher Jin X-H 11875|KF143462.1|
100
D. findlayanum voucher PS2531MT01 |HQ114257.1|
D. findlayanum isolate D23 |JN388589.1|
D. chysanthum - D7
D. chrysanthum |JN388584.1|
D. chrysanthum |FJ384738.1|
77 D. chysanthum- D8
D. chrysanthum voucher Jin X-H 11430 |KF143443.1|
D. chrysanthum voucher SBB-0617 |JF713093.1|
99
D. chrysanthum voucher SBB-0504 |JF713091.1|
80
D. brymerianum - D30
D. brymerianum voucher PS2510MT01|HQ114233.1|
100 D. brymerianum voucher Jin X-H10782 |KF143432.1|
D. brymerianum |JN388581.1|
D. brymerianum |EU477500.1|
D. pulchellum voucher Jin X-H 11878 |KF143503.1|
D. moschatum -D3
100
D. moschatum voucher Jin X-H 11886 |KF143492.1|
D. daoense - D15
86 D. fimbriatum isolate D22 |JN388588.1|
100 D. fimbriatum - D1
D. fimbriatum voucher PS2507MT01 |HQ114229.1|
D. fimbriatum voucher Jin X-H s.n. 16 (KF143461.1)
D. chrysotoxum - D20
95
97
D. chrysotoxum - D21
D. chrysotoxum |EU477501.1|
D.
capillipes
voucher NCHU-D89331201 |HM590379.1|
100
D. chrysotoxum voucher PS2501MT03 |HQ114223.1|
99 D. chrysotoxum -D16
74 D. chrysotoxum voucher PS2501MT01 |HQ114221.1|
D. chrysotoxum isolate D19 |JN388585.1|
D. hancockii voucher Jin X-H 13492 |KF143467.1|
88 D. hancockii isolate D25 |JN388591.1|
D. hancockii voucher PS2533MT01 |HQ114259.1|
76
D. hancockii |AB593575.1|
D. hancockii - D10
88 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|
91 D. amabile - D12
D. thyrsiflorum - D13
100 D. farmeri -D14
D. farmeri -D19
D. haveyanum -D26
100
D. thyrsiflorum |KC205200.1|
D. thyrsiflorum voucher Jin X-H 10755 |KF143519.1|
D. lindleyi - D24
96 D. lindleyi -D25
D. jenkinsii voucher Jin X-H 10709 |KF143478.1|
100 D. jenkinsii voucher PS2525MT01 |HQ114251.1|
D. jenkinsii voucher Jin X-H s.n. 4 |KF143479.1|
99 D. jenkinsii isolate D29 |JN388595.1|
D. draconis Rchb.f - D31
75
D. draconis |HM054628.1|
100
D. draconis voucher SBB-0545 |JF713101.1|
D. draconis |EU477503.1|
73
D. wattii - D22
99 D. wattii voucher Jin X-H 11817 |KF143525.1|
99
D. longicornu voucher Kurzweil H Lwin S 2647 |KF143484.1|
74 D. longicornu voucher Jin X-H 11663 |KF143485.1|
D. christyanum -D32
90
D. christyanum voucher Jin X-H s.n. 3 |KF143442.1|
D. christyanum voucher Jin X-H 11045 |KF143441.1|
98
D. christyanum |GU339106.1|
D. christyanum |EF629325.1|
0.02
Figure 3.25. Phylogenetic trees based on ITS sequences
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