Transcript CO1 Barcoding of Penicillium, Aspergillus, Fusarium and

Agriculture and
Agri-Food Canada
Agriculture et
Agroalimentaire Canada
Cox1 Barcoding of Penicillium
and Fusarium
Keith A. Seifert , Scott Gilmore and Karol Rivera
Biodiversity, Agriculture & Agri-Food Canada, Ottawa, Ontario, Canada.
Canada
Gene 1 tree
T 474 Outgroup 1 24432 T
T 478 Outgroup 1 45782
Taxon
Taxon 3
1
complex
T 483 Outgroup 1 45672
T 475 Outgroup 1 45872
Taxon
2
Gene 2
T 485 Outgroup 2 46272
100
T 486 Outgroup
2 43993
Taxon
3
T 488 Outgroup 2 50873
Cox1
Gene 1
T 487 Outgroup 2 18472
T 354 Taxon 3 13567
100
100
T 352 Taxon 3 47984
T L Taxon 3 22130
98
T 357 Taxon 3 23438
99
T 066 Taxon 2 44978
T 068 Taxon 2 46695 T
100
1 change
Taxon 2
T 071 Taxon 2
T 342 Taxon 1 101032
100
Taxon 3
T 347 Taxon 1 46595
T 348 Taxon 1 46495
Taxon 1
Taxon 8
Taxon 1
Taxon 6
Taxon 2
Taxon 4
Taxon 3
Taxon 2
Taxon 1
Taxon 4
Taxon 5
Taxon 6
Taxon 5
Taxon 3
Taxon 7
beta-tubulin
Gene 2 tree
Cox1
Taxon 7
Taxon 8
Gene 1 tree
Comparison of Three Potential
Barcoding Loci
Mean bp
Infraspecific
range
Interspecific
x
range
Species
resolution
Or
PCR success rate
Introns
Gaps
Gene 1
Gene 2
Gene 3
545
0.06
0-0.6
5.6
0-15.4
67.1
446
0.83
0-4.9
14.4
0.5-67.2
81.2
555
0.51
0-5.3
5.1
0-26.8
24.5
92
95%
0
0
100
75%
3
258
33
95%
0
150
Comparison of Three Potential
Barcoding Loci in Penicillium subgenus
Penicillium
Mean bp
Infraspecific
range
Interspecific
x
range
Species
resolution
Or
PCR success rate
Introns
Gaps
Cox1
BenA
ITS
545
0.06
0-0.6
5.6
0-15.4
67.1
446
0.83
0-4.9
14.4
0.5-67.2
81.2
555
0.51
0-5.3
5.1
0-26.8
24.5
92
95%
0
0
100
75%
3
258
33
95%
0
150
R
R
F
F
75
59
59
62
99
0.02
Taxon 3 genome 1
Taxon 2 DAOM235753
Taxon 4 DAOM 235795
Taxon 5 genome **
Taxon 2 DAOM 235752 b1
Taxon 2 DAOM 235752 b5
Taxon 1 DAOM 235624
Taxon 1 DAOM 235800
Taxon 3 genome 2
Taxon 6 BBA 67786
Taxon 7 DAOM 235678 **
55 Taxon 8 BBA 69792 **
Taxon 9 BBA 63606 **
Taxon 10 BBA 68459 **
Taxon 2 DAOM 235758
Taxon 1 DAOM 235624 i4b **
Taxon 11 BBA 64400
Taxon 12 BBA 64535 **
66 Taxon 13 DAOM 235643 **
Taxon 14 DAOM 235647 i4b **
Taxon 1 DAOM 235621 i4b **
80
Taxon 15 BBA 64798 **
Taxon 16 BBA 64465
Taxon 17 BBA 62149 **
Taxon 18 BBA 63784 i4b **
Taxon 18 BBA 63784
Taxon 19 BBA 71782 **
Taxon 1 DAOM 235621
Taxon 20 DAOM 235731 **
Taxon 21 DAOM 235651 **
99
Taxon 2 DAOM 235753 b5
Taxon 1 DAOM 235621 seq2
Taxon 14 DAOM 235647
Outgroup 1 DAOM 2266670
Outgroup 2 DAOM 226847
Outgroup 3 DAOM 232048
Outgroup 4 DAOM 229273 **
Outgroup 5 DAOM 226707
Outgroup 6 DAOM 237735
Outgroup 7 LRC1370
Outgroup 7 DAOM 2335220
Outgroup 8 KAS 2832
3 species from 3
taxonomic sections
of genus. Identical
barcodes.
F
R
2 species from 2
taxonomic sections
of genus. Identical
barcodes.
R
R
F
F
75
59
59
62
99
0.02
F. oxysporum genome 1
F. circinatum DAOM235753
F. sacchari DAOM 235795
F. verticillioides genome **
F. circinatum DAOM 235752 b1
3 species from 3
F. circinatum DAOM 235752 b5
taxonomic sections
F. graminearum DAOM 235624
of Fusarium.
F. graminearum DAOM 235800
Identical barcodes.
F. oxysporum genome 2
F. equiseti BBA 67786
F. babinda DAOM 235678 **
55 F. pallidoroseum BBA 69792 **
F
F. diversisporum BBA 63606 **
F. incarnatum BBA 68459 **
F. circinatum DAOM 235758
F. graminearum DAOM 235624 i4b **
F. coeruleum BBA 64400
R
F. flocciferum BBA 64535 **
66 F. graminum DAOM 235643 **
F. dimerum DAOM 235647 i4b **
F. graminearum DAOM 235621 i4b **
80
F. lateritium var. majus BBA 64798 **
F. torulosum BBA 64465
F. acuminatum BBA 62149 **
Fusarium sp. nov. BBA 63784 i4b **
Fusarium sp. nov. BBA 63784
F. avenaceum BBA 71782 **
F. graminearum DAOM 235621
2 species from 2
F. polyphialidicum DAOM 235731 **
taxonomic sections
F. solani DAOM 235651 **
of Fusarium.
99
F. circinatum DAOM 235753 b5
Identical barcodes.
F. graminearum DAOM 235621 seq2
F. dimerum DAOM 235647
Acremonium chrysogenum DAOM 2266670
Monocillium mucidum DAOM 226847
Hypocrea jecorina DAOM 232048
Lanatonectria flocculenta DAOM 229273 **
Emericellopsis minima DAOM 226707
Neighbour joining analysis of Cox1 exon
Metarhizium anisopliae DAOM 237735
region between Fusarium intron 4b and end of
Beauveria bassiana LRC1370
Beauveria bassiana DAOM 2335220
barcode (439bp).
Cylindrocarpon sp. KAS 2832
Hypocreales – Primer Design
primer name
Sequence 5' to 3'
AHyLe-Fa
TCAGGATTATTAGGTACAGCATTT
TTAAGTGGACCTGGTGTTCAAT
TTAAGTGGCCCAGGAGTACAAT
CTTAGTGGGCCAGGAGTTCAATA
ACCTCAGGGTGTCCGAAGAAT
ACCTCAGGRTGYCCGAAGAAT
TCAGGRTTAYTAGGWACAGCATTT
TTAAAAGTATCGAAAAATCAAAAAGGTGT
ATCTATCTCTTATTTCTTGGCTCATTGGTT
CCTTTAAAACTAGTACCGCAGAC
AHyLe-F3
AHyMe-F
AHyFu-F
AHyFu-R
Peziz-R
Peziz-F
FI3-F
FI3-R
FI4b-F
FI3-F
AHyFu-F
1
2
3
Barcode forward primer designed from Lecanicillium muscarium NC_004514
Barcode forward primer designed from Metarhizium anisopilae NC_003388
Barcode forward primer designed from Fusarium oxysporum AY874423
Barcode reverse primer designed from Fusarium oxysporum AY874423
Degenerate primer in position of Fu-R. Designed to work across Pezizomycotina
Degenerate primer in position of Le-Fa. Designed to work across Pezizomycotina
Intron 3 forward primer, for Fusarium
Intron 3 reverse primer, for Fusarium
Intron 4b forward primer to end of barcode, for Fusarium
FI4b-F
4
FI3-R
Description
Barcode forward primer designed from Lecanicillium muscarium NC_004514
5
6
7
8
9
10
11
AHyFu-R
Acknowledgements
• This work was funded by grants to the
Canadian Barcode of Life Network by
– The Natural Sciences and Engineering
Research Council of Canada (NSERC)
– Genome Canada