Development of chloroplast genome library for DNA tracking and conservation of endangered Aquilaria species native to Malaysia
Aquilaria Lam., species [Aquilaria beccariana Tiegh., (gaharu buaya), Aquilaria crassna Pierre ex Lecomte, (gaharu), Aquilaria hirta Ridl., (chandan bulu), Aquilaria malaccensis Lam., (ching karas), Aquilaria microcarpa Baill., (gaharu putih), Aquilaria rostrata Ridl., (candan gunung), Aquilaria...
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/104753/1/FPAS%202022%2017%20IR.pdf http://psasir.upm.edu.my/id/eprint/104753/ |
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| Summary: | Aquilaria Lam., species [Aquilaria beccariana Tiegh., (gaharu buaya), Aquilaria
crassna Pierre ex Lecomte, (gaharu), Aquilaria hirta Ridl., (chandan bulu),
Aquilaria malaccensis Lam., (ching karas), Aquilaria microcarpa Baill., (gaharu
putih), Aquilaria rostrata Ridl., (candan gunung), Aquilaria sinensis (gaharu) and
Aquilaria subintegra Ding Hou, (gaharu)] is a naturally distributed in the
Indomalesian region and are protected against over-exploitation. Aquilaria is
highly prized for its unique scented resin, agarwood, which is often the subject
of unlawful trade activities. Survival of the tree is heavily threatened by
destructive harvesting and agarwood poaching, leading to its protection under
the Convention on International Trade in Endangered Species of Wild Fauna and
Flora (CITES). Ambiguous species delimitation and limited genetic information
within Aquilaria are among the impediments to conservation efforts. In this study,
a comparative analysis was performed on eight Aquilaria species complete
chloroplast (cp) genomes, of which seven were newly sequenced using Illumina
HiSeq X Ten platform followed by de novo assembly. All Aquilaria species
utilised in this study were collected during fieldwork and the Aquilaria germplasm
of Forest Research Institute of Malaysia (FRIM), and grown in the greenhouse
of the Faculty of Forestry and Environment, Universiti Putra Malaysia. All
Aquilaria species have been authenticated by experts. To conduct comparative
cp genome analysis, the sequences of all eight Aquilaria cp genomes were
aligned using MAFFT v7 and then imported into DnaSP v5.10.1 to determine
nucleotide diversity in the total genome, LSC, SSC, and IR regions. The
boundaries between the IR and SC regions were manually examined to
determine the differences in length variation between Aquilaria cp genomes.
Aquilaria cp genomes possess a typical quadripartite structure including gene
order and genomic structure. The length of each of the cp genome is about 174
kbp and encoded between 89 and 92 proteins, 38 tRNAs, and 8 rRNAs, with 27
duplicated in the IR (inverted repeat) region. Besides, 832 repeats (forward,
reverse, palindrome and complement repeats) and nine highly variable regions (HVR) were also identified. The phylogenetic analysis performed using
Maximum likelihood (ML) and Bayesian inference (BI) suggests that the topology
structure of Aquilaria cp genomes were well presented with strong support
values based on the cp genomes data set and matches their geographic
distribution pattern. Five of the nine HVR (matK-rps16, ndhF-rpl32, psbJ-petA,
trnD, and trnT-trnL) were selected based on a cut off value of >0.01. These
regions were further analyzed using the neighbor-joining (NJ) method to assess
their ability at discriminating the eight species. Coupled with in silico primer
design, two potential barcoding regions, psbJ-petA and trnT-trnL, were identified.
Their strengths in species delimitation were evaluated individually and in
combination, via DNA barcoding analysis. The results showed that the combined
dataset, psbJ-petA+trnT-trnL, effectively resolved members of the genus
Aquilaria by clustering all species into their respective clades. In addition, this
study show great potential for agarwood identification that the newly proposed
DNA barcode was capable at identifying the species of origin of six commercial
agarwood samples that were included as unknown samples. Such achievement
offers a new technical advancement, useful in the combat against illicit agarwood
trades and in assisting the conservation of these valuable species in natural
populations. The identification of simple sequence repeats (SSR) across
complete cp genome sequences from eight Aquilaria species has never been
reported before. Perhaps because developing these genetic are regarded as
laborious and expensive. To overcome this issue, the current work also aims to
find SSRs in Aquilaria cp genomes using silico methods. For identification of the
simple sequence repeats (SSRs), MISA PERL script which had a repeat length
of 12 for mononucleotides (mono-), 6 for dinucleotides (di-), 4 for trinucleotides
(tri-), 3 for tetranucleotides (tetra-), pentanucleotides (penta-), and
hexanucleotides (hexa-), respectively, along with frequency were utilized. From
a total of 312 SSRs that were discovered, merely 50 (16%) were found localized
within the coding region while the majority (84%) were within the intergenic
regions, with an average of one SSR per 4.5 kb. The mean length of the SSRs
were 11.63 bp. Mono- repeats were the predominant motifs (29.2%), followed by
tetra- (28.8%), di- (20.5%), tri- (19.9%), and penta- (1.6%). Whereas the most
recurring motifs were A/T (97.8%) for mono-, AT/AT (87.5%) for di-, AAT/ATT
(48.4%) for tri-, and AAAT/ATTT (45.6%) for tetra-. GO analysis using the
REVIGO software identified four molecular functions, six biological processes
and three cellular components. In conclusion, findings of this SSR offer a
scientific foundation for future phylogenetics, evolutionary genetics, diversity
studies and breeding programs on Aquilaria species. |
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