EVALUATION ON GENETIC DIVERSITY AND IDENTIFICATION MOLECULAR MARKER FOR VIETNAMESE NATIVE DENDROBIUM SPECIES

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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 1 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, 2 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 3 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 4 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 5 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. 7 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. 9 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 10 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 11 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 18
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