Tóm tắt Luận án 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 1 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 2 molecular levels of the Vienamese native Dendrobium species, which is basis for selection of elite germplasm, and new breeding; - The results of the research are significant in identifying a number of varieties/species of Vietnamese native Dendrobium species, as a basis for conservation, economic value and register on GenBank. 3.2. Practical Implications - Collection and preservation genetic resources of Vietnamese native Dendrobium species; - The results will be contributing on the conservation, selection and breeding new varieties to improving the Vietnamese orchid products. 4. Research subjects and scope of the project study 4.1. Research subjects - Thirty two Vietnamese native Dendrobium species were collected from different provinces. 4.2. Location and time study: - The experiment was carried out at the Department of Genetic Engineering, Institute of Genetics Agriculture, Pham Van Dong road, Tu Liem district, Hanoi city. - Research period: From 2010 to 2014. 5. The contributions of the thesis Thesis was the first studied methodically systematic assessment of genetic diversity by morphological indicators combined with molecular markers, identification of Vietnamese native Dendroium species based on the ITS sequences. The results of the thesis have significance in the classification, the conservation service, and selection and breed new varieties; these will be contributing to improve Vietnam orchids. CHAPTER 1: OVERVIEW 1.1. Glance of Dendrobium 1.1.1. Classification systems In the botanical classification systems, the gennus Dendrobium are belong the spread (Orchidales), subclass (Liliidae), monocots (Liliopsida), delimitation of higher plants (Cosmobionia), and the plant kingdom (Plantae) (Tran Hop, 1998; Hoang Thi Be, 2004; Leitch et al., 2009; Evans et al., 2012). Classification systems of Dendrobium species in Vietnam have often based Seidenfaden system (1985). This system is clear, straightforward, high reliability, and consistent with the representatives of Dendrobium in Vietnam (Tran Hop, 1998; Duong Duc Huyen, 2007). 1.1.2. Morphological characteristics 1.1.2.1. Stems The stems of Dendrobium species are segmented, cylindrical shaped spindles, clavate, ovoid, with lengths ranging from 2-3cm to 120cm or sometimes more, common size is 20-50cm (Tran Hop, 1998). The stems can 3 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, 4 including the description of the characteristics and structure external morphology, namely stems, leaves, flowers. 1.3.3.2. Methods based on biochemical The isozyme is defined as various forms of an enzyme (protein) has the same function or close together in the same individuals (Salazar, 2003). 1. 3. 3. 3. Evaluation genetic diversity based on molecular marker methods Evaluation genetic diversity based on molecular marker methods such as: Restriction Fragment Length Polymorphism (RFLP); Amplified Fragment Length Polymorphism (AFLP); Random Amplified Polymorphic DNA (RAPD); Microsatellite or Simple Sequence Repeates (SSR); Inter-simple sequence repeats (ISSRs) (Singh et al., 2012; Shangguo et al., 2013; Swati Das et al., 2014). 1.3.3.4. Classification methods of plant on gene sequences * ITS region (internal transcribed spacers) is a non-functional RNA segment, located between the RNA of the ribosome structure is usually translated code. Structure ITS region including ITS1 - 5.8S - ITS2. Another advantage of the ITS include two separate loci (ITS1 and ITS2) and they are connected together via locus 5.8S. 5.8S locus is insulated region, in fact there is enough phylogenetic signal discrimination and the sector level (Richardson et al., 2001; Sharma et al, 2012). * The gene rbcL (ribulose-bisphosphate carboxylase) is used more to build phylogenetic trees. However, for genetic relationships at the species under analysis on this gene encounter many limitations (Kress and Erickson, 2007). * The matK gene (gene coding for maturaseK): The study used gene sequencing matK to construct phylogenetic trees as showed genetic diversity matK than other genes in chloroplasts and therefore genetically matK become an important indicator gene to help sort plants (Asahina et al., 2010; Sharma et al., 2012). 1. 4. Current status in study of orchids in the world and in Vietnam 1.4.1. Evaluation genetic diversity and identification Dendrobium species in the world 1.4.1.1. Research on morphological indicators Theres were many research on Denedrobium species such as Pellegrino et al., 2005; Krishnan et al., 2011). 1.4.1.2. . Research on molecular markers * RFLP markers Recently, Surin et al., 2014 for using RFLP technique to identify 25 Dendrobium varieties from Thailand. 23 of the 25 species have been identified, only two species such as D. crumenatumand D. formosum were unidentified. * RAPD markers There have been studies using indicators to analyze RAPD polymorphism in orchids, the study focused on several categories like spreads Dia lan 5 (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. 6 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. 2.3.3.2. Purification PCR products and sequenes 2. 3.3.3. Aligment ITS sequencing in GenBank database 2. 3.3.5. Phylogenetic trees 2.4. Data processing software - Research datas were analyzed, processed on the Excel version 5.0 software, software PCORD, software NTSYS, MEGA: version 5.2.1, to construct phylogenetic trees. 7 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 422 st , 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 8 Table 3.1. List of Vietnamese native Dendrobium species Symbols Vietnamese names Latin names Origin (province) Symbols Vietnamese names Latin names Origin (province) D1 Hoang Thao Long nhan Lai Chau D. fimbriatum Lai Chau D17 Hoang Thao Kim Diep D. capillipes Lam Dong D2 Hoang Thao Chuoi ngoc Dien Bien D. findlayanum Dien Bien D18 Hoang Thao Chuoi Ngoc D. findlayanum Kontum D3 Hoang Thao Thai Binh D. moschatum Thai Binh D19 Hoang Thao Kieu Trang Dong Nai D. farmeri Dong Nai D4 Hoang Thao Phi Diep tim D. anosmum Hoa Binh D20 Hoang Thao Tieu Hoang lap D. chrysotoxum Kontum D5 Hoang Thao Tram tim D. parishii Son La D21 Hoang Thao Dai Hoang lap D. chrysotoxum Lam Dong D6 Hoang Thao Tram trang D.parishii.var alba Dien Bien D22 Hoang Thao Bach hac langbiang D. wattii Lam Dong D7 Hoang Thao Ngoc Van Vang D. chrysanthum Yen Bai D23 Hoang Thao Hac vi D. aphylum Da Lat Hoang Thao Phi Diep vang D. chrysanthum Dien Bien D24 Hoang Thao Vay rong la nho D.lindleyi Quang Tri D9 Hoang Thao Long tu Bac D. primulinum Son La D25 Hoang Thao Vay rong la trung D. lindleyi Lam D