Natural Product Research with Endophytic Fungi Workshop

September 23, 2014

Taxonomic identification of pure

endophytic strains

Huong Minh Nguyen PhD

INSTITUTE OF BIOTECHNOLOGY
Vietnam Academy of Science and Technology

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An overall workflow

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Advantages of taxonomic identification using DNA markers

DNA-based methods are culture- independent, quick and unbiased

Amplification efficiency of PCR-based method allows for the use of
minimum starting fungal materials, making this method much more
sensitive and affordable

Development of new sequencing technology creates large genome

databases for diverse and accurate comparison

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DNA Isolation (1)

Grind 1g of
mycelium in
liquid
nitrogen

Add 750l of lysis

buffer to 1g of
mycelium ground in
liquid nitrogen

Lysis buffer: 50 mM Tris-HCl, 50 mM

EDTA, 3% SDS, 1% 2-mercaptoethanol
(add just before use)

Vortex
mixture,
incubate at
65C for 1
hour

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DNA Isolation (2)

Centrifuge at
4000 rpm for
5min at RT,
transfer aqueous
phage to a new
tube

Add 700l of SEVAG,

vortex mixture then
centrifuge for 12000 rpm
in 10min, transfer upper
phase to a new tube

Add 20 l of
3M NaOAC
and 1 vol of
isopropanol,
mix well

Separate

SEVAG:

Should

cellular debris

chloroform:isoamyl

observe DNA

from DNA

alcohol, 24:1

precipitation

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DNA Isolation (3)

Centrifuge at
12000 rpm for
3min at RT,
discard
supernatant

Add 300l of EB to
pellet with 1l of
100mg/ml RNAse,
incubate at 65C for
15min

This step is to cleanup

contaminated RNA

Add 250l of 7.5M

AlOAc, centrifuge
at 12000 rpm for
5min then transfer
top layer to a new
tube

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DNA Isolation (4)

Pellet DNA by
adding 1 vol of
isopropanol then
centrifuge at 12000
rpm for 2min at RT

Wash pellet with 1 vol

of 70% EtOH twice,
collect pellet by
centrifuge at 12000
rpm for 1min

Resuspend pellet in
50l of TE buffer,
store at -20C until
use

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DNA Isolation (5)

Previous results: Isolation of Xanthomonas sp. and Saccharomyces sp.
genomic DNA
X

Contaminated RNA

Treatment with
RNAse

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PCR Amplification and Sequencing (1)

The choice of target fragments for amplification can affect the efficiency
of amplification and the accuracy of comparison

Common targets for fungal taxonomic identification include 18S smallsubunit ribosomal DNA (rDNA), 28S large-subunit rDNA or internal
transcribed spacer (ITS) regions

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PCR Amplification and Sequencing (2)

PCR reaction component for amplification of fungal ITS region :
Component

Volume/ 50l reaction

DDW

To 50l

10x Pfu Buffer

10mM dNTPs mix

2.5

20 pmol primer mix

DNA template

0.5 1

Pfu (2 - 4U/l)

0.5

94C

94C

2min

30sec

X 35
52 - 57C
30sec

72C

72C

30s 1m

5min

4C

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PCR Amplification and Sequencing (3)

Purification kit (Thermo

Scientific) and an appropriate
amount of cleaned-up
samples are sent for
sequencing

ITS3-4

Phellinus sp.

are cleaned-up using PCR

Saccharomyces sp.

Successful amplified products

Phellinus sp.

of PCR amplification

ITS1-4

Phellinus sp.

used to check for the success

ITS3-4
Saccharomyces sp.

0.8 1.5% agarose gel is

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PCR Amplification and Sequencing (4)

Previous results: Sequencing of Xanthomonas sp. 16S rRNA amplified target
using IBT Sequencing Service

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PCR Amplification and Sequencing (5)

Genomic sequence comparison: using databases such as NCBI Genome Database

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PCR Amplification and Sequencing (6)

Taxonomic identification: build phylogenetic tree for isolated fungal strains
with closely related species

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(Endophytic) Fungal Taxonomic Identification

Remaining Challenges and Future Prospects
Limited database: as of February 2014, the National Center for Biotechnology
Information (NCBI) Genome database listed 57 complete fungal genomes
compared with >2700 bacterial genomes

Genomic sequence of sexual (teleomorph) and asexual (anamorph) forms of

a fungus are often annotated as different taxa in many databases, causing
complication in identification

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THANK YOU