Kai-Shu Ling, Rugang Li, and W. Patrick Wechter USDA-ARS, Charleston, SC

Alvaro G. Hernandez

University of Illinois at Urbana-Champaign, IL

Zhangjun Fei Boyce Thompson Institute, USDA-ARS, Cornell

University, Ithaca, NY

Using Small RNA Technology to Efficiently Indentify Tomato Viruses and Viroids in Mixed-Infected Field Samples

Hydroponic Greenhouse Tomato Production

Bumble bees

Intercropping De-leafing

Grafted plants

Pepino Mosaic Virus Mexican Papita Viroid

Tomato Chlorotic Dwarf Pepino Mosaic Virus

Most Common Methods in Plant Virus Detection

RNA or DNA Coat protein

N. benthamiana S. melongena

S. lycopersicum

A Tomato Disease in ‘MX’ with Unknown Etiology

S. lycopersicum

Next Generation Sequencing for Plant Viruses

Host Virus RNA template NGS

technology

Reference

Tomato &

Gomphrena

globosa

PepMV, GMMV Total plant RNA Roche 454 Adams et al.,

2009

Grapevine 4 RNA viruses + 3

viroids

Total plant RNA &

dsRNA

Roche 454 Al Rwahnih

et al. 2009

Grapevine GLRaV-3 & 3

others

dsRNA Illumina Coetzee et al.

2010

Sweetpotato SPFMV, SPCSV

and 2 DNA viruses

siRNA Illumina Kreuze et al.,

2009

Tomato ,

Squash

TSWV, SLCV siRNA Illumina Hagen et al.,

2011

Grapevine HSVd, GYSVd siRNA Illumina Navarro et al.,

2009

Viral siRNA Biogenesis

AAA

RDR A A A

AAA

AAA

siRNA (21-24 nt)

AAA

RISC

Gene Silencing/Viral RNA

Degradation

Slicing

AGO AGO AGO AGO AGO

DCL

AGO

AGO

Dicing

Guiding

DCL

Illumina Small RNA Library Construction

Small RNA Barcoded

adaptors

Adaptor ligation

RT-PCR

cDNA Library

+

Illumina sRNA reads

de novo assembly

Virus genome contigs

Distribution of sRNA reads on virus genome

Fill gaps and detect virus variants

Tomato

sRNAs

Small RNA Bioinformatics Pipeline

Number of Reads after Removing Adaptor and Barcode

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

CAHN8 EF09_58

EF09_60 MX

No. reads No. unique reads

sRNA Size Distribution

0

500000

1000000

1500000

2000000

2500000

3000000

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 36

CAHN8

EF09_58

EF09_60

MX

nt

Validation of siRNA Assembly by Sanger Sequencing

of RT-PCR Product (EF09-60)

US1

EU

Replicase CP TGB1-3

RT-PCR

Nucleotide Substitut ion per 100 residues

0

16.5

246810121416

336288977 Italy SIC1-09

336288983 Italy SIC2-09

336288989 Italy SAR01

66576027 Chile Ch2

336288971 Italy SAR09

148283741 Poland PK

328879877 poland P22

328879871 Poland P19

222431628 Poland Pa

208401171 England Tomato

46093918 US2

57282624 Spain LE-2000

57282630 Spain LE-2002

21703327 Spain Sp-13

237861554 Netherlands EU

237861560 Netherlands DB1

EF09-58-EU

EF09-60-EU

CAHN8-EU

57282636 Peru LP-2001

79676039 Peru SM.74

EF09-60-US1

EF09-58-US1

66576021 Chile Ch1

237861566 US1

46093912 US1

Phylogenetic Relationship Among PepMV Isolates

CH2

Genotype

US2

EU

LP

US1

P1 CI NIa-

VPg

NIa-

Pro P3

1 6 5 3 7 4 8 9 2 10 kb 0

CP HC-Pro NIb AAA

Identification of a Novel Potyvirus in MX

siRNA

Contig

Summary and Perspectives

Deep sequencing of small RNA will likely

revolutionize virus detection and disease diagnosis

for known or unknown viruses and viroids.

This technology can be used to differentiate

closely related strains in a mixed infection.

Validation of de novo genome assembly of siRNA

with conventional Sanger sequencing was

necessary.

Acknowledgments

USDA-ARS, Charleston

Andrea Gilliard

Emily Fillippeli

USDA-NIFA SCRI program,

Project Director : Dr. Sally Miller

of the Ohio State University