Taxonomic identification of pureendophytic strains

Huong Minh Nguyen PhDINSTITUTE OF BIOTECHNOLOGY

Vietnam Academy of Science and Technology

Natural Product Research with Endophytic Fungi WorkshopSeptember 23, 2014

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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 moresensitive and affordable

Development of new sequencing technology creates large genomedatabases for diverse and accurate comparison

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DNA Isolation (1)

Grind 1g ofmycelium in

liquidnitrogen

Add 750l of lysisbuffer to 1g of

mycelium ground inliquid nitrogen

Lysis buffer: 50 mM Tris-HCl, 50 mMEDTA, 3% SDS, 1% 2-mercaptoethanol(add just before use)

Vortexmixture,

incubate at65C for 1

hour

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DNA Isolation (2)

Centrifuge at4000 rpm for5min at RT,

transfer aqueousphage to a new

tube

Add 700l of SEVAG,vortex mixture then

centrifuge for 12000 rpmin 10min, transfer upperphase to a new tube

SEVAG:chloroform:isoamylalcohol, 24:1

Add 20 l of3M NaOACand 1 vol ofisopropanol,mix well

Shouldobserve DNAprecipitation

Separatecellular debrisfrom DNA

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DNA Isolation (3)

Centrifuge at12000 rpm for3min at RT,discard

supernatant

Add 300l of EB topellet with 1l of100mg/ml RNAse,incubate at 65C for

15min

This step is to cleanupcontaminated RNA

Add 250l of 7.5MAlOAc, centrifugeat 12000 rpm for5min then transfertop layer to a new

tube

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DNA Isolation (4)

Pellet DNA byadding 1 vol ofisopropanol then

centrifuge at 12000rpm for 2min at RT

Wash pellet with 1 volof 70% EtOH twice,collect pellet by

centrifuge at 12000rpm for 1min

Resuspend pellet in50l 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

Contaminated RNA

Treatment withRNAse

X S

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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 small-

subunit ribosomal DNA (rDNA), 28S large-subunit rDNA or internaltranscribed spacer (ITS) regions

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PCR Amplification and Sequencing (2) PCR reaction component for amplification of fungal ITS region :

Component Volume/ 50l reactionDDW To 50l10x Pfu Buffer 510mM dNTPs mix 2.520 pmol primer mix 1DNA template 0.5 1Pfu (2 - 4U/l) 0.5

94C2min

94C30sec 52 - 57C

30sec

72C30s 1m

72C5min 4C

X 35

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PCR Amplification and Sequencing (3) 0.8 1.5% agarose gel is

used to check for the successof PCR amplification

Successful amplified productsare cleaned-up using PCRPurification kit (ThermoScientific) and an appropriateamount of cleaned-upsamples are sent forsequencing

Sacch

aromy

ces sp

.

ITS3-4 ITS1-4

Sacch

aromy

ces sp

.

Phell

inuss

p.

Phell

inuss

p.

Phell

inuss

p.

ITS3-4

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PCR Amplification and Sequencing (4)Previous results: Sequencing of Xanthomonas sp. 16S rRNA amplified targetusing 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 strainswith 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 BiotechnologyInformation (NCBI) Genome database listed 57 complete fungal genomescompared with >2700 bacterial genomes

Genomic sequence of sexual (teleomorph) and asexual (anamorph) forms ofa fungus are often annotated as different taxa in many databases, causingcomplication in identification

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THANK YOU