VIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN NHU HOA CONSTRUCTION OF DNA SEQUENCE DATABASE FOR RAPID IDENTIFICATION AND DIVERSITY EVALUATION OF DENDROBIUM SPECIES IN SOUTHERN VIETN
Trang 1
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
NGUYEN NHU HOA
CONSTRUCTION OF DNA SEQUENCE DATABASE FOR RAPID IDENTIFICATION AND DIVERSITY EVALUATION
OF DENDROBIUM SPECIES IN SOUTHERN VIETNAM
Major: Biotechnology
Code: 9 42 02 01
SUMMARY OF DOCTORAL THESIS
Ho Chi Minh City, 2021
Trang 2The study was completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
Supervisor:
1 Duong Hoa Xo, Assoc Prof PhD
2 Tran Kim Dinh, PhD
The thesis can be referred at:
1 National Library of Vietnam
2 Library of Vietnam Academy of Agricultural Sciences
3 Library of Institute of Agricultural Sciences for Southern Vietnam
Trang 3INTRODUCTION
1 Rationale of the study
Orchid (family Orchidaceae), thanks to its highly diversified and beautiful flowers, has long attracted great interest from large groups
of spectators In Vietnam, it consists of a rich and special taxon with
significant value in many aspects Dendrobium is the largest genus
amongst the orchid family and also the most favored and sought in Vietnam Many works have been done on orchid conservation, including the Orchid Collection of the Biotechnology Center Of Ho Chi Minh City with 400 orchid samples including 190 from the
Dendrobium genus Morphological identification of the orchids
requires complete samples with all the anatomical components –
especially the flowers whose information is vital – which is not always possible, however Amongst molecular technologies, DNA
barcoding is acknowledged as a useful tool for plant identification,
including orchids
The study aimed to construct a DNA barcode reference database
for Dendrobium identification Morphology identification was
performed on 40 flowered samples collected in southern Vietnam
On the molecular part, the ITS, rbcL, matK, and trnH-psbA regions
of 71 samples from 25 species and 13 hybrid samples were sequenced, and taxonomic diversity and phylogenetic relationship were determined by bioinformatics tool The results were applied for the paternity test of several hybrid samples
2 Aim of the study
Trang 4This study aimed to construct a DNA sequence database and DNA barcodes for the identification and diversity evaluation of
Dendrobium samples collected in southern Vietnam
3 Scientific and practical significance
3.1 Scientific significance
The study successfully determined the genetic diversity of
Dendrobium orchids in southern Vietnam based on morphological
traits and DNA barcodes The data were submitted to GenBank and
enriched the Dendrobium database which laid the basis for further
researches Potential markers for rapid identification of Dendrobium were proposed to conserve the rare and valuable genetic resources in Vietnam
3.2 Practical significance
This study proposed the application of DNA barcoding for more
accurate and rapid identification of Dendrobium orchids for genetic
conservation
4 Object and scope of the study
4.1 Object of study
The orchid species in the Dendrobium genus
4.2 Scope of the study
The study was performed on orchid samples of the Orchid Collection, Biotechnology Center of Ho Chi Minh City The samples were also the results of the previous study “Collection, import, selection and rapid multiplication of the orchid cultivars for
domestic consumption and export” of Dr Hoa-Xo Duong et al
(2011)
Trang 5This study evaluated and constructed a phylogenetic tree based
on 72 morphological traits of 40 Dendrobium samples This study also evaluated the sequences of the rbcL, matK, trnH-psbA, and ITS
of 84 samples (including 71 samples of 25 Dendrobium species in
southern Vietnam and 13 hybrid samples)
5 Novel contributions of the study
This study provided the data of the markers ITS, matK, rbcL, and trnH-psbA of native Vietnamese Dendrobium species, submitted to GenBank database, amongst which many rbcL and
trnH-psbA sequences had not or limitedly published on that
database
This study preliminarily evaluated the genetic diversity of
Dendrobium DNA markers in southern Vietnam and proposed the
potential markers for Dendrobium identification
6 Structure of the thesis
The thesis consists of 127 pages excluding Appendixes It includes Introduction (5 pages), Chapter 1: Literature review and theoretical framework (38 pages), Chapter 2: Materials and methods (22 pages), Chapter 3: Results and discussion (48 pages), Conclusion and Recommendation (2 pages), and References (12 pages), uses 26 Vietnamese and 96 English reference works The thesis includes 11 tables, 43 figures, 8 appendixes, and 4 published works
Trang 6CHAPTER 1 LITERATURE REVIEW AND THEORETICAL
FRAMEWORK
The study referred to and summarized various Vietnamese and English literary works related to (1) Introduction of the Orchidaceae
family, (2) Introduction of the Dendrobium genus, (3) Current status
of Dendrobium conservation and identification in Vietnam and worldwide, (4) Studies related to Dendrobium genetic diversity, and
(5) DNA Barcoding and their applications in species identification
1.1 Introduction of the Orchidaceae family
The Orchidaceae, commonly known as the orchid family, is the largest monocot family and the second largest flowering plant family only after Asteraceae (Takhtajan, 1987)
1.2 Introduction to Dendrobium genus
1.2.1 Classification
Kingdom Plantae
Phylum Angiospermae (Magnoliophyta)
Class Monocotylendones (Liliopsida)
Dendrobium is found only in the Eastern Hemisphere, from
Oceania, throughout South Pacific, the Philippines, India, several
spotted in Japan, and mostly occurred in South East Asia
Trang 71.2.3 Diversity and richness
Dendrobium genus consists of 1600 species with high diversity in
color, size, and morphology for adaptation to a great range of ecological habitats
1.2.4 Morphology
1.3 Dendrobium conservation and identification in Vietnam and
worldwide
Dendrobium conservation worldwide has been carried out in
various centers and projects In vitro tissue culture also attracts
significant attention Accurate sample identification is essential for all conservation and cultivation efforts Morphological traits have long been employed for such works, however, in many cases, it is difficult to rely on morphology only Advances in molecular genetics, nucleotide sequence technology, bioinformatics, and DNA barcoding enable rapid and accurate identification of known taxa and allow data retrieval from them
1.4 Genetic diversity researches on Dendrobium
1.4.1 Markers for genetic diversity evaluation of Dendrobium
Various markers have been used for genetic diversity evaluation
of populations and species such as morphological, cytological, biochemical, and DNA markers Molecular markers currently are considered the most effective evaluation tools as they are not affected by changes in environment and phenotype There are many types of molecular markers and various technique employed in this field, some of them have been used for orchid diversity assessment such as AFLP (Amplified Fragment Length Polymorphism), RAPD (Randomly Amplified Polymorphism DNA), RFLPs (Restriction
Trang 8Fragment Length Polymorphisms), SSRs (Microsatellite or Simple Sequence Repeats), ISSR (Inter-Simple Sequence Repeat), and the method utilizing DNA sequence database
1.4.2 Genetic diversity researches on Dendrobium
Researches on Dendrobium genetic diversity, thanks to highly
developed biotechnology, have been strongly promoted in Singapore, Taiwan, or Thailand, using various markers such as SSR, AFLP, RAPD, ISSR, and RFLP
1.5 DNA barcoding in taxa identification
DNA barcode is one or several nucleotide sequences gene sequences taken from a standardized portion of the genome used to identify species The technique was first applied to animals
1.5.1 Sequences for construction of DNA barcodes
Plant DNA in the nucleus, chloroplast, and mitochondria are all used for molecular identification The mitochondrial genome has the slowest evolution rate, followed by the chloroplastic and the nuclear genomes, respectively
Kress et al (2005) proposed two potential regions for DNA barcoding in flowering plants which are the ITS and the trnH-psbA
The Kew, Royal Botanic Gardens (UK) proposed five candidate
regions including matK, rpoC1, rpoB, accD, and và YCF5, together with the combination of multiple region (rpoC1 + rpoB + matK, or
rpoC1 + matK + trnH-psbA) for better efficiency The CBOL
proposed a combination of matK and rbcL for plant barcoding
(2009) The China Plant BOL Group proposed combining ITS with
matK + rbcL for plant barcoding (2011) The 4th International
Trang 9Barcode of Life Conference proposed using matK + rbcL +
psbA-trnH
Based on the mentioned works, this study chose the region of
ITS, rbcL, matK, and psbA – trnH for the evaluation of Dendrobium
genetic diversity
1.5.2 DNA barcoding studies on orchids and Dendrobium
As soon as the concept of DNA barcoding was proposed, various researches, in Vietnam and worldwide, have been performed on building a DNA database for native species including orchids Many
studies have been done on the regions of ITS, ITS1, ITS2,
psbA-trnH, matK, rbcL, rpoB, or rpoC, most of which indicated a lower
discrimination power in chloroplastic barcodes than in nuclear ones – amongst which the ITS region has the highest power A barcode using multiple sequences is essential for plant species in general and
Dendrobium species in specific
The ITS and psbA-trnH sequences were seen as potential markers for Dendrobium species, and the more conserved matK and rbcL
could be used as data for phylogenetic analysis and in combination with other sequences for DNA barcoding
Trang 10CHAPTER 2 MATERIAL AND METHODS
2.1 Materials, time, and location
The study was carried out on Dendrobium spp samples
The morphological description was performed on 40
Dendrobium samples from the Orchid Collection of the
Biotechnology Center of Ho Chi Minh City
Sequencing was performed on leaf DNA extracted from 84
Dendrobium samples, including 71 samples from 25 native species
for the DNA barcoding database, and 13 hybrid samples for parental identification
This study was carried out from August 2014 to August 2018, at the Biotechnology Center of Ho Chi Minh City
Objective 4: Using DNA data for paternity test of Dendrobium
hybrids cultivated by the Biotechnology Center of Ho Chi Minh City and from other sources
2.3 Methods
Trang 112.3.1 Phylogenetic tree construction based on morphological traits
Morphological traits were described for 40 flowered samples from the Biotechnology Center of Ho Chi Minh City
2.3.1.1 Dendrobium morphological description
Forty flowered samples were described uniformly for 72
morphological traits, using the DUS criteria for Cymbidium and
Phalaenopsis Flower anatomy was analyzed horizontally and
vertically Photographic illustrations of each sample were recorded
2.3.1.2 Dendrobium phylogenetic relationship based on morphology
The phylogenetic tree was built using NTSYS PC software
2.3.2 DNA barcode reference database for the samples
2.3.2.1 Sample collection and storage
Fresh leaf samples were collected from morphologically healthy plants and stored in numbered tubes at -20oC to -80oC
2.3.2.2 Genomic DNA extraction
Leaf DNA was extracted using the CTAB method or specialized kit (GeneJET Plant Genomic DNA Purification Mini Kit)
2.3.2.3 PCR and gel electrophoresis
The ITS, matK, rbcL, and trnH-psbA sequences were amplified
by PCR using designated primers PCR products were examined by gel electrophoresis on 0.8% agarose gel, at 120V, 65mA for 20 minutes
Table 1 Primers and thermal cycles for amplification of the ITS,
matK, rbcL, trnH-psbA regions
Regions Primers PCR cycles
Trang 12Regions Primers PCR cycles
Trang 13Raw sequences were adjusted by FinchTV to remove the ambiguities and check the peak reliability A BLAST search was performed on the NCBI database for the sequence similarity and origin, and sequence alignment was performed by SeaView 4.0 The ITS2 sequence was obtained from the ITS region (JN388570.1)
2.3.3 Evaluation of Dendrobium genetic diversity by DNA markers
Genetic diversity evaluation based on phylogenetic branching was performed on discrete and combined markers The phylogenetic tree was constructed by MEGA 7.0 software using the Maximum Likelihood algorithm based on the Kimura-2 parameter model with
1000 bootstrap replicates Paphiopedilum sequence was used as an
outgroup
2.3.4 Evaluation of the marker discrimination power
The most optimal markers were chosen based on species discrimination power using the phylogenetic tree together with DNA indels (insertion and deletion mutations) data, and the “Best Match / Best Close Match” techniques in which the genetic distances of the target sequences were compared
Trang 14CHAPTER 3 RESULTS AND DISCUSSION
3.1 Phylogenetic tree based on Dendrobium morphology
3.1.1 Morphological description of the Dendrobium samples
This study successfully described the morphology of 40
Dendrobium samples including 37 natives and 3 hybrids; each was
recorded for 72 standardized traits based on DUS criteria, with five photographic images of high resolution Morphological evaluation
managed to detect errors in the identification of the Dendrobium
chrysotoxum Lindl – which was previously mistaken for Dendrobium thyrsiflorum Lindl
3.1.2 Genetic relationship between 40 Dendrobium samples
The data of 72 recorded morphological traits partly revealed the
genetic relationship between the 40 sampled Dendrobium, however,
several close taxa with similar appearance could not be discriminated
In detail, the morphological assessment categorized the 40
samples into two groups Group I included D amabile và D
palpebrae Group II included 2 sub-groups The sub-group IIA (17
samples) had only one member with pendulous stem, while the
sub-group IIB (21 samples) had 10 members with the same trait D
anosmum (7,8,9,10), D aphyllum (11,12), and D parishii (28) with
close phylogenetic relationship and morphological similarity, and the
hybrid of D anosmum and D aphyllum, all were grouped together However the hybrid (D anosmum x D parishii) was put in a different cluster, together with D hercoglossum, D heterocarpum, and D venustum D primulinum and D crepidatum due to the
morphological similarity were together grouped in a separated cluster