SUPPLEMENTARY MATERIAL
Discovery and molecular characterization of a new cryptovirus dsRNA
genome from Japanese persimmon through conventional cloning and
high-throughput sequencing
M. Morelli1, M. Chiumenti1, A. De Stradis1, P. La Notte1, A. Minafra1
1
Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP), UOS Bari, Via Amendola 122/D, 70126 Bari,
Italy
Corresponding author
Dr. Massimiliano Morelli, Ph.D.
Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP), UOS Bari
Via Amendola 122/D, 70126 Bari, Italy
Tel. 0039.0805443065 Fax. 0039.0805443608
E-mail: [email protected]
Tab. S1 List of oligonucleotide primers used in this study, with indication of sequence and genome position (when available)
Primer
F090
R090
CryKaF
CryNeR
ADPcm
M13F
M13R
DOP
Sequence 5’-3’
AGTTTCGAGTCGTCTTCGGCGG
GTGTTGTTCACCGGTGTAGTAACT
ACGTCCACGACCGATTGTGC
ACGAAGACACGTAACACGCAGTGG
GAGAAGGATGCGGTT
TTGTAAAACGACGGCCAGT
CAGGAAACAGCTATGACC
OHCCGACTCGAGNNNNNNATGTGGOH
Genome nt position
dsRNA-1 467-488
dsRNA-1 290-313
dsRNA-2 285-304
dsRNA-2 405-428
RLM-RACE universal primer
Cloning universal primer
Cloning universal primer
DOP-PCR universal primer
Tab. S2 Summary of statistical data retrieved from Illumina HiScanSQ deep-sequencing of SSPI accession and subsequent bioinformatics analysis
General statistics
Sequencing data size
5,190 Gb
Raw reads
33,632,035
Unique reads
1,994,231
PeCV reads
586,451
Assembled contigs
1,829
Average read number
Mean coverage index
PeCV contigs 54,320
Other contigs
376
PeCV contigs 2225.3
Other contigs
Assembled reads
22.9
Reads Count
Fwd.
Persimmon cryptic virus dsRNA-1
(PeCV, GenBank HE805113)
Persimmon cryptic virus dsRNA-2
(PeCV, GenBank HE805114)
Rev.
421,937
213,739
208,198
164,514
78,288
86,226
Reference
Length (nt)
% Total
% Coverage
Mean
Coverage
reads/pos.
1,577
21.0
100.0
13,489
1,491
8.0
100.0
5,563
Tab. S3 Persimmon accessions used in this study for RT-PCR detection of PeCV, with indication of their provenance and host
Accession
SSPI
BOS1
BOS2
SAL
PAL
GIA1
GIA2
ORI1
ORI2
SEM1
SEM2
SEM3
SEM4
SEM5
SEM6
Provenance
Santo Spirito (Bari, Italy)
Gioia del Colle (Bari, Italy)
Gioia del Colle (Bari, Italy)
Terlizzi (Bari, Italy)
Locorotondo (Bary, Italy)
Santeramo in Colle (Bari, Italy)
Santeramo in Colle (Bari, Italy)
Oria (Brindisi, Italy)
Oria (Brindisi, Italy)
Sammichele di Bari (Bari, Italy)
Sammichele di Bari (Bari, Italy)
Sammichele di Bari (Bari, Italy)
Sammichele di Bari (Bari, Italy)
Sammichele di Bari (Bari, Italy)
Sammichele di Bari (Bari, Italy)
Private orchard
Private orchard
Private orchard
Private orchard
Private orchard
Private orchard
Private orchard
Private orchard
Private orchard
Nursery seedling
Nursery seedling
Nursery seedling
Nursery seedling
Nursery seedling
Nursery seedling
RT-PCR
+
+
+
+
+
+
+
+
+
+
+
+
Fig. S1 Illumina HiScanSQ deep-sequencing analysis of PeCV reads obtained from SSPI accession,
showing graphical distribution of read matches at each nucleotide position of genomic dsRNA-1 and dsRNA2. Rectangular boxes below the graphs represents coding ORFs
Fig. S2 Secondary structure 2D view of PeCV dsRNA-1 (A) and dsRNA-2 (B) 5’-UTR plus-strand sequences, with the highlighting of putative hairpin-loop
structures and indication of related free energy contribution
Fig. S3 Phylogenetic tree constructed on capsid protein (CP) of PeCV and members of the family Partitiviridae. Tree was generated using neighbor-joining-based
bootstrap analysis with 1,000 replicates. Numbers on branches indicate bootstrap values above 600. Bar represents 0.2 amino-acid substitutions per site. AoV-1,
ABV30675, Aspergillus ochraceous virus 1; BCV-1, ACA81389, Beet cryptic virus 1; BFV-1, YP_001686789, Botryotinia fuckeliana partitivirus 1; CaCV,
ACL93278, Carrot cryptic virus; CCV, AET80948, Cannabis cryptic virus; CiLCV, AGH12859, Citrullus lanatus cryptic virus; CCCV-2, AGJ83769, Crimson clover
cryptic virus 2; CrV-1, AAU26069, Ceratocystis resinifera virus 1; DCV-2, AGJ83771, Dill cryptic virus 2; DdV-1, NP_620301, Discula destructiva virus 1; FChCV,
AAZ06131, Fragaria chiloensis cryptic virus; FCV, CBW77436, Fig cryptic virus; FpV-1, AAC98734, Fusarium poae virus 1; GaRV, AAM12240, Gremmeniella
abietina RNA virus MS1; HTCV-2, YP_007889825, Hop trefoil cryptic virus 2; MCV-1, GU145316, Mulberry cryptic virus 1; PCV-1, AEJ07890, Pepper cryptic
virus 1; PCV-2, AEJ07892; Pepper cryptic virus 2; PeCV, HE805113, Persimmon cryptic virus; PmV-1, ABW82141, Primula malacoides virus 1; PoV-1,
ACX43952, Pleurotus ostreatus virus 1; PsV-S, YP_052856, Penicillium stoloniferum virus S; RCCV-2, YP_007889823, Red clover cryptic virus 2; RCV-1,
YP_001686786, Rose cryptic virus 1; RnV-1, YP_392480, Rosellinia necatrix partitivirus 1; RsCV-1, YP_656506, Raphanus sativus cryptic virus 1; RsV-717,
AF133290, Rhizoctonia solani virus 717; VCV, ABN71241, Vicia cryptic virus; WCCV-1, AAU14888, White clover cryptic virus 1