www.sciencemag.org/cgi/content/full/336/6086/1304/DC1

Supplementary Materials for

Global Honey Bee Viral Landscape Altered by a Parasitic Mite Stephen J. Martin,* Andrea C. Highfield, Laura Brettell, Ethel M Villalobos, Giles E.

Budge, Michelle Powell, Scott Nikaido, Declan C. Schroeder*

*To whom correspondence should be addressed. E-mail: s.j.martin@sheffield.ac.uk (S.J.M.); dsch@mba.ac.uk (D.C.S.)

Published 8 June 2012, Science 336, 1304 (2012)

DOI: 10.1126/science.1220941

This PDF file includes:

Materials and Methods Table S1 Figs. S1 to S7 References (28–37)

2

Materials and Methods

Specimen sampling and RNA extraction

During November 2009, ~100 adult honeybees were sampled from 280 managed and

13 feral colonies across the four main Hawaiian Islands. During November 2010 an

additional 38 colonies were sampled from Big Island (6 apiaries) and 11 colonies

from one apiary on Oahu. All samples were kept frozen and transported back to the

UK on dry ice and then stored at -80 °C. From each colony, 30 asymptomatic bees

(i.e., without deformed wings) were individually checked for the presence of mites,

before being pooled and ground to a fine homogenous powder in liquid nitrogen using

a pestle and mortar, which allows subsequent detection of virus at more than a 10%

prevalence in a colony, with 95% confidence. Samples were then stored at -80 °C.

With each batch of 20–30 samples, ‘dummy’ control tubes were left open on the

bench to test for airborne contamination. Total RNA was extracted from 30 mg of

ground material using an RNeasy mini kit (Qiagen), according to the manufacturer’s

instructions, except that samples were eluted in 40 µl RNase free water. Total RNA

samples were quantified using a Nanodrop 8000 (Thermo Scientific). One microgram

of isolated RNA was treated with DNase I (Promega), according to the

manufacturer’s instructions, followed by Nanodrop quantification to standardise the

amounts of total RNA to 25 ng/µl, before storage at -80 °C. Two independent RT-

PCR methods were used, as these are 100-fold more sensitive than conventional non-

nested PCR (29).

Virus quantification using probe-based (TaqMan®) chemistry

Real-time RT PCR was performed using duel labelled (TaqMan®) probes designed to

detect six honeybee viruses: DWV (two assays, including one designed to detect

Varroa destructor variant-1 (VaDV-1) strains (22), KBV, SPV (minor and major

forms), ABPV (two assays), and IAPV (table S1). All 293 honeybee samples

collected in 2009 were tested against all probes. Reactions were set up in either a 96-

well or 384-well reaction plate using PCR core-reagent kits (Applied Biosystems),

according to the manufacturer’s protocols, with the addition of approximately 25 ng

of RNA, 5 units of M-MuLV reverse transcriptase enzyme (Fermentas Life Sciences)

and 0.1 mM DTT. Reactions were run on the ABI 7900 HT Sequence Detection

3

System using generic cycling conditions of 48°C for 30 min, 95°C for 10 min

followed by 40 cycles of 95°C for 10 s and 60°C for 1 min.

Additionally, SYBR green chemistry was used to screen for ABPV (29), using

SYBR green PCR Mastermix (Applied Biosystems), 25 pmol of each primer and 5

units of M-MuLV reverse transcriptase enzyme (Fermentas Life Sciences).

Absolute quantification of DWV using SYBR green qPCR

All samples that tested positive for DWV using TaqMan probes were analyzed using

SYBR green chemistry, as follows. Total RNA was analyzed in triplicate for each

sample using the one-step SensiMix with SYBR green kit (Bioline). PCR reactions

contained 50 ng RNA, 1× SYBR one-step Sensimix, 2.5 mM MgCl2, 5 units of RNase

inhibitor, and 7.5 pmol of each primer: DWVQ-F1 and R1 for DWV, and Actin F1

and R1, for the reference gene actin (Supplemental Table 1). Reactions were run on a

Rotorgene 6000 Thermocycler (Corbett Research) with an initial reverse transcription

stage at 49 °C for 30 min and a denaturation step of 95 °C for 10 min, followed by 40

cycles of denaturation for 15 sec at 95 °C, annealing for 20 sec at 54 °C for DWV, and

58 °C for actin, and extension for 20 sec at 72 °C. The SYBR green signal was

measured on the green channel after each extension step. To

ensure that a single

product was amplified, and that no

contamination was present in the reverse

transcription negative controls or in the no-template controls, a final dissociation

curve was performed between 72 °C and 95 °C, at 0.5°C increments, each with a 10

sec hold and acquisition to the green channel. Sample copy numbers were determined

for each target using Corbett analysis software where the threshold cycle (CT) number

was determined for each sample run in triplicate, before the average was taken.

DWV copy number was quantified in the positive samples using cRNA

standards previously developed (7), which were run in parallel to the samples

analysed. DWV copy number was normalised to actin for each run and the copy

number per bee was calculated (7). To increase confidence in the detection of low

DWV viral levels, three DWV runs (two with TaqMan® probes and one with

SYBRgreen) were compared and a positive sample was determined if two of the three

tests were positive. Only one of the 293 control samples (elongation factor 1, [EF1])

failed, and none of the 16 dummy controls tested positive, indicating that there was

minimal chance of cross-sample contamination. Reaction efficiency was calculated as

<90% for the TaqMan® probes.

4

Statistical analysis

Comparison of viral load for the five different viruses was conducted using the ∆CT

values derived from the TaqMan® probe analysis. Subsequently, both relative (DWV

& KBV using TaqMan® and a known positive) and absolute (DWV using

SYBRgreen and cRNA standards) (7) viral loads were obtained to allow comparisons

with other studies. Average viral loads were calculated only using positive samples.

The same distinctive pattern for DWV distribution and load was obtained across all

datasets, irrespective of the PCR assay method (TaqMan® or SYBRgreen) or

quantification method (relative or absolute) used, because the change in viral load was

so large.

Analysis of DWV RdRp sequences

Four colonies, one from Oahu, Maui, Kauai, and Big Island were analysed for DWV

sequence diversity using high resolution melt (HRM) analysis and clone

libraries/sequencing of a region of the RdRp gene. This is a highly conserved region

vital for viral survival so any substitutions in this region would be significant and

could contribute to variations in viral epidemiology, since any variation in this gene

could suggest a more significant genetic shift in the rest of the viral genome. HRM

analysis was performed using the Quantace one-step probe kit (Bioline), using the

same PCR reactions as the SYBR green kit, with the exception that the SYBR green

was replaced with 5× EvaGreen (Cambridge Biosciences). PCR proceeded with the

same protocol used for SYBR green, until the melt stage where a HRM melt curve

was performed between 72 °C and 95 °C, with 0.1 °C increments each with a 5 sec

hold and acquisition to the HRM channel. For HRM analysis fragments no greater

that 250 bp should be used (CorProtocolTM

6000-1-July06). DWV PCR products were

verified by gel electrophoresis and products were purified from the agarose gels using

the QiaexII gel extraction kit (Qiagen). Purified fragments were cloned using the

Invitrogen TA cloning kit (Invitrogen), according to the manufacturer’s instructions.

Positive colonies were screened for inserts using M13 colony PCR and these PCR

products were used for sequencing. Approximately 40 clones from each of the

samples from the four islands in 2009 were sequenced using the BigDye Terminator

V3.1 cycle sequencing kit (Applied Biosystems). Reaction mixtures contained 0.875×

Sequencing dilution buffer, 0.05× Big Dye V3.1, 3.3 pmol M13 forward primer, and

5

approximately 35 ng DNA. Reactions proceeded with an initial denaturation step at

95 °C for 1 min, followed by 39 cycles of denaturation at 94 °C for 10 sec, annealing

at 50 °C for 5 sec,

and extension at 60 °C for 4 min. Ethanol/EDTA/sodium acetate

precipitation was then carried out to purify the sequencing PCR products and to

remove dye terminators, before direct sequencing using the ABI3730 Genetic

Analyzer. Electropherograms were edited using CodonCode Aligner v1.6.3 and

unclear traces were re-sequenced using the reverse primer, before the two sequences

were aligned and edited. BioEdit was used to align sequences and to create the

consensus.

For HRM runs, thresholds were determined individually depending on the

amount of product yielded by the PCR, which is determined by the fluorescent signal

detected above the background noise. This is because a sample with a lower viral

load results in less fluorescence being produced so the resultant melt curve also has a

lower fluorescence. Consequently, the threshold used to determine the melt peak

temperature was adjusted accordingly so that the signal for each positive sample was

above the background noise level.

Variant sequences were determined based on the melting temperatures derived

from the HRM data, where a unique OTU (operational taxonomic unit) was defined

according to data acquired from running HRM on individual clones of the DWV PCR

product (fig. S5). Identical sequences were found to vary in their melt temperature by

up to 0.1°C and this temperature range was used to define a variant melt temperature,

i.e., 78.5 °C ± 0.1°C would be equivalent to a variant. Rarefaction curves for the

HRM data for each island were calculated using Analytic Rarefaction version 1.3

(http://strata.uga.edu/software/).

Analysis of other DWV sequences

To investigate the possibility of the formation of DWV/VaVD-1 hybrids we

sequenced regions of the Lp, VP3, helicase and RdRp genes using established primers

(L protein (DWV-F1153/DWV-B1806) de Miranda (personal communication), VP3

primers (DWV-F1/DWV-B1) (30), Helicase primers (DWV-6285F/DWV-6693R)

(31), RdRp (DWV-F15/DWV-B23) (30). DWV was extracted and sequenced from

three bees and three Varroa mites from an infested Oahu apiary in 2009. The methods

used were as given above, except PCR products were not cloned before sequencing.

The similarity of the six Oahu isolates to other DWV and VaDV-1 sequences on

6

Genbank was determined using the BLASTn algorithm and the range of similarities

against both DWV and VaDV-1 presented. Using MEGA-5 (32) we re-constructed

the phylogram for the Lp region (15) before adding in our Oahu sequence data and

new VaVD-1-DWV recombinants data (22, 33). Minimum Evolution criteria was

used and statistical strength is based on 1000 replicate bootstrap analysis.

7

Fig. S1 Viral load (∆CT) and prevalence of Kashmir bee (KBV), Acute bee paralysis (ABPV), Israeli acute paralysis (IAPV) and

Deformed wing viruses (DWV) among the 293 Hawaiian colonies across the four islands in 2009 where Varroa had been present for

different lengths of time. The pie diagrams illustrate that Varroa has only had a significant effect of the viral prevalence and load of

DWV, with red showing the clear positives (supported by two or more positive tests). In Varroa-free areas the viral load of DWV,

KBV or ABPV were not significantly different (One-way ANOVA: F = 1.373, df = 2, p = 0.3) and remained low, even in the presence

of Varroa, since the viral load of ABPV (ANOVA: F = 0.229, df = 4, p = 0.91) and KBV (F = 0.274, df = 4, p = 0.89) did not

significantly vary across the five honeybee populations that had been exposed to Varroa, for different lengths of time. Slow paralysis

virus (SPV) was not detected. KBV and SPV have retained their defined geographical distribution. That is SPV is rare, detected in

only three countries (36, 37), whilst KBV is highly prevalent (90%) in North America (6). The mean viral load of IAPV was

significantly higher than all of the other four viruses in Varroa-free areas (ANOVA: F = 11.030, df = 3, P<0.0001), although this

result was based on only three colonies.

8

Fig. S2

High Resolution Melt profiles for DWV variants from different colonies analysed from

the four Hawaiian Islands during 2009 and 2010 where peaks at different temperatures

correspond to unique putative genotypes (fig. S5). This indicates a lost of DWV diversity

in the Oahu population and parallel loss of DWV diversity on Big Island between 2009

and 2010 as Varroa became established, as demonstrated by the similarity of their HRM

profile clusters.

deg.

75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

1.2

1.0

0.8

0.6

0.4

0.2

0.0

deg.75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

deg.73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.20

0.15

0.10

0.05

0.00

deg.73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.25

0.20

0.15

0.10

0.05

0.00

deg.

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.25

0.20

0.15

0.10

0.05

0.00

B: Big Island 2009

C: Maui 2009

D: Kauai 2009

B: Big Island 2010

A: Oahu 2009

deg.75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

2.75

2.50

2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00

A: Oahu 2010

deg.75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

1.2

1.0

0.8

0.6

0.4

0.2

0.0

deg.75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

deg.73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.20

0.15

0.10

0.05

0.00

deg.73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.25

0.20

0.15

0.10

0.05

0.00

deg.

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

dF

/dT

0.25

0.20

0.15

0.10

0.05

0.00

B: Big Island 2009

C: Maui 2009

D: Kauai 2009

B: Big Island 2010

A: Oahu 2009

deg.75.5 76.0 76.5 77.0 77.5 78.0 78.5 79.0 79.5 80.0 80.5 81.0 81.5 82.0 82.5 83.0 83.5 84.0 84.5 85.0 85.5 86.0 86.5 87.0 87.5 88.0 88.5 89.0 89.5 90.0

dF

/dT

2.75

2.50

2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25

0.00

A: Oahu 2010

9

A

B

A

B

0.1

L=Big Island

M=UK, Big Island

N=UK, Big Island

D=UK

K=Europe*, Oahu, Big Island, Kauai

O=UK, Big Island

J=UK

F=UK

G=UK

H=UK

I=UK, USA

A=UK

Y=Maui

S=UK

V=UK

W=UK

U=UK

C=UK, Kakugo

P=UK, Uruguay, Kauai

E=UK

Q=UK, Kakugo, Spain

B=UK, Turkey, Big Island

X=UK

R=UK

T=UK

Z=Kauai

AB=UK, Kauai

AA=VaDV-1

52

51

Group A

Group B

0.1

L=Big Island

M=UK, Big Island

N=UK, Big Island

D=UK

K=Europe*, Oahu, Big Island, Kauai

O=UK, Big Island

J=UK

F=UK

G=UK

H=UK

I=UK, USA

A=UK

Y=Maui

S=UK

V=UK

W=UK

U=UK

C=UK, Kakugo

P=UK, Uruguay, Kauai

E=UK

Q=UK, Kakugo, Spain

B=UK, Turkey, Big Island

X=UK

R=UK

T=UK

Z=Kauai

AB=UK, Kauai

AA=VaDV-1

52

51

Group A

Group B

Fig. S3 DWV sequence alignment to the 100 bp region of the replicase polyprotein(RdRp) of Hawaiian sequences generated in this study and those available from Genbank. A total of 282 sequences were used. This region corresponds to the bases between 22 and 122 in fig. S5. Variants (A-AB) were designated as a variant if a nucleotide substitution was detected in >3 sequences, meaning that random substitutions or those occurring in <3 sequences were ignored. Numbers in brackets correspond to the number of different sequences comprising the variant sequence (i.e. 1 means all sequences within this variant group are identical etc). Dots correspond to identical nucleotides and letters to any base substitutions according to the degenerate nucleotide code. DWV group A are shaded blue, and DWV group B shaded pink. * corresponds to Italy, Denmark, Spain, France and UK. The resulting neighbour-joining tree is given below for these consensus sequences where genotype Z, AA and AB are the out-groups. Numbers at nodes correspond to the bootstrap values derived from 1000 replicates.

10

Fig. S4

Rarefaction curves based on putative DWV variants (OTUs) as detected by High

Resolution Melt analysis for colonies from the four Hawaiian islands in 2009 (solid lines)

and 2010 (dashed lines). The rarefaction curves did not reach saturation for the majority

which is typical of highly diverse systems. The Oahu 2009, 2010 and Big Island 2010

groups were the most curvilinear. V+ and V- indicated the presence or absence of Varroa

in that population.

Big Island V+ 2009

40

30

25

20

15

10

5

0

35

Cum

ula

tiv

e nu

mb

er o

f O

TU

sdet

ecte

d (

DW

V v

aria

nt

seq

uen

ces)

Number of analysed sequences

302010 60 80 90 9640 700 50

Big Island V- 2009

Maui V- 2009

Kauai V- 2009

Big Island V+ 2010

Oahu V+ 2009

Oahu V+ 2010

Big Island V+ 2009

40

30

25

20

15

10

5

0

35

Cum

ula

tiv

e nu

mb

er o

f O

TU

sdet

ecte

d (

DW

V v

aria

nt

seq

uen

ces)

Number of analysed sequences

302010 60 80 90 9640 700 50

Big Island V- 2009

Maui V- 2009

Kauai V- 2009

Big Island V+ 2010

Oahu V+ 2009

Oahu V+ 2010

11

A

B

A

B

BI-B BI-M

BI-O

BI-L

M-Y

K-P

K-Z K-ABK-AB

O-K

BI-NK-K

BI-K

BI-B BI-M

BI-O

BI-L

M-Y

K-P

K-Z K-ABK-AB

O-K

BI-NK-K

BI-K

Fig. S5 DWV sequence alignment of a 145 bp region of the replicase polyprotein (RdRp) of 136 clones from four

Hawaiian colonies collected in 2009 (upper Table), with the number of clones given in brackets. The lower graph

indicates the HRM curves for each distinct DWV clone showing that the method is capable of discriminating

between subtly different sequences. The clone HRM codes begin with the island of origin (Big Island = BI-; Kauai

= K-; Oahu = O-; Maui = M-) and end with designated sequence variant as described in the table S1 and fig. S3 (A

to AB). Blue curves belong to the ‘A’ group while the pink curves correspond to the ‘B’ group (fig. S3).

12

Fig. S6

Similarity of six Oahu variants compared to all known DWV and VaDV-1 variants at

four different sites on the DWV genome. Fragment sizes of the sequenced sections are

given and the proposed recombination points (22) are indicated by the dotted lines.

5’IRES

Lp VP3 VP1 VP2 helicase 3C-pro RdRpVP

4

VP

g

AAAAAA3’

Recombination points

DWV 95-97% 99% 87-97% 99-100%

693bp 354bp 408bp 450bp

VaDV-1 85-86% 85% 0% 82-83%

5’IRES

Lp VP3 VP1 VP2 helicase 3C-pro RdRpVP

4

VP

g

AAAAAA3’

Recombination points

DWV 95-97% 99% 87-97% 99-100%

693bp 354bp 408bp 450bp

VaDV-1 85-86% 85% 0% 82-83%

13

Fig. S7

Phylogram of the Lp (693 bp) region of DWV and VaDV-1 genomes adapted from (15)

and updated with variants from the UK (22) & Israel (33) (VaDV-1) and Hawaii. The six

Hawaiian Oahu variants all clustered with other DWV variants indicating that they are

not DWV/VaVD-1 hybrids, since the RdRp region (fig. S3) also clustered with other

DWV variants. The phylogram was constructed by MEGA-5 (32) using Minimum

Evolution criteria. The nodes are shown as the percentage of correct partitions in a 1000

replicate bootstrap analysis; branches with less than 40% bootstrap support were

collapsed.

UK (1)

98

96

95

95

85

40

85

53

52

44

41

81

47

46

82

Sweden (40)

Sweden(22)

Ireland (2)

USA (3)Hawaii (6)France (2)Germany (1)Greece (3)Ireland (2)Italy (3)Netherlands (2)Spain (3)Switzerland (3)Sweden (2)Yugoslavia (2)

UK (1)

Netherlands (1)Israel (1)

Israel (1)

1

VaDV-1

DWV

Japan (3)

Kakugo virus

China (2)

China (9)

China (3)

China (2)

100

100

100

100

UK (1)

98

96

95

95

85

40

85

53

52

44

41

81

47

46

82

Sweden (40)

Sweden(22)

Ireland (2)

USA (3)Hawaii (6)France (2)Germany (1)Greece (3)Ireland (2)Italy (3)Netherlands (2)Spain (3)Switzerland (3)Sweden (2)Yugoslavia (2)

UK (1)

Netherlands (1)Israel (1)

Israel (1)

1

VaDV-1

DWV

Japan (3)

Kakugo virus

China (2)

China (9)

China (3)

China (2)

100

100

100

100

14

Table S1.

Primers used in this study. The 5′-terminal reporter dye for each TaqMan® probe was 6-

carboxyfluorescin (FAM) and the 3′ quencher was tetra-methylcarboxyrhodamine

(TAMRA) or Minor groove binding (MGB) as indicated.

* This study

Target Primer name Sequence (5'-3')

SYBR DWV (7) DWVQ_F1 TAGTGCTGGTTTTCCTTTGTC

DWVQ_R1 CTGTGTCGTTGATAATTGAATCTC

Actin (7) Actin_F1 CCTGCAATCGCAGATAGAATGC Actin_R1 AAGAATTGACCCACCAATCCATAC

ABPV (16) A AATGGGCCTATGGACTTTTCTA B AAATCTCCTGCAATAACCTTGG

TaqMan® DWV (34) DWV CCT GGACAAGGTCTCGGTAGA A

DWV 9711R ATTCAGGACCCCACCCAAAT

DWV 9627T CATGCTCGAGGATTGGGTCGTCGT

DWV (+VDV1)* DWV F2 GTAGAAGGAACGACGAATTGGAC DWV R2 GCTTGTTTAGCATTTTCTATGGTTGC DWV 9627T CATGCTCGAGGATTGGGTCGTCGT

KBV (34) KBV83F ACCAGGAAGTATTCCCATGGTAAG KBV161R TGGAGCTATGGTTCCGTTCAG KBV109T CCGCAGATAACTTAGGACATCAATCAC

IAPV (35) IAPVB4S0427_R130M RCR TCAGTCGTCTTCCAGGT IAPV B4S0427_L17M CGA ACT TGG TGA CTT GAR GG

IAPV Probe TTGCGGCAATCCAGC CGTGAAAC

ABPV (34) APV 95F TCC TATATCGACGACGAAAGACAA APV 159R GCG CTT TAA TTCCATCCAATTGA

APV 121T (MGB) TTTCCCCGGACTTGAC

ABPV * ABPV 5436F TAACCAATGAAGTRTCCATAGGAACTA ABPV 5481R TCTCCTGCRATAACCTTGGGT ABPV 5515T (MGB) TGTTTATTCCCAAGATTG

SPV major (36) SPV 8383F TGATTGGACTCGGCTTGCTA SPV 8456R CAAAATTTGCATAATCCCCAGTT SPV 8407T CCTGCATGAGGTGGGAGACAACATTG

SPV minor* SPV Minor F1 ATAGCGCTTTAGTTCAATTGCCAT SPV Minor R1 CTGGAATATGACCATCACGCAT

SPV Minor T1 ACCCCCATGGATTGAAAATACAGCC

EF1* EF 1 F CTGGTACCTCTCAGGCTGATTGT EF 1 R GCATGCTCACGAGTTTGTCCATTCT

EF1 TGCTTCGAACTCTCTCCAGTACCAGCAG

1

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