Electronic Supplementary Material (ESM) S1: Material and methods
Eight Eurema mandarina females were captured on Tanegashima Island, Japan,
from populations previously reported to be infected with both Wolbachia strains wCI
and wFem [1]. These females were kept in isolation for egg-laying. Five females
showed a female-only offspring ratio, whereas three had unbiased offspring ratios
(ESM S1 Table 1). For the qPCR experiment we used 11 daughters of all-female
broods; for unbiased broods we used eight males and eight females. The unbiased
broods were either infected with wCI (a total of five females and five males from
mothers 6-8) or cured from infection after tetracycline treatment (three females and
three males from mother 6).
ESM 1 Table 1. Eurema mandarina from Tanegashima and their offspring included in
this study
Mother Daughters
Daughters
Sons
#
n
used in qPCR
n
1
8
3
-
Sons
used in qPCR
-
2
8
3
-
-
3
2
2
-
-
4
3
3
-
-
5
6
0
-
-
6
7
5
6
5
7
1
1
1
1
8
3
2
4
2
As control we included three female and three male E. mandarina from Hachijō-jima
Island, Japan, from a population previously found to be infected only by wCI. We also
included five females and five males of each wCI infected E. hecabe and uninfected
E. smilax from various locations in Australia (ESM S2).
DNA isolation
Genomic DNA was extracted from single legs (the standard tissue used for
Wolbachia detection in E. hecabe [1]) using the GenElute Mammalian Genomic
Miniprep kit (Sigma) according to the manufacturer’s protocol. DNA was eluted in 100
µL elution solution and then stored at 4 °C.
Wolbachia screening PCR
PCR-based screening of Eurema individuals was performed using strain specific wsp
primers for wCI and wFem [1]. Cycling conditions were 3 min at 94 °C, then 35 cycles
of 94 °C for 30 s, 60 °C for 45 s, 72 °C for 60 s, and a final step at 72 °C for 10 min,
and the master mix was as listed in ESM 1 Table 2.
PCR of the Tpi gene
The Tpi gene is located on the Z chromosome of lepidopteran species. We amplified
a highly variable intron using primers that were positioned in the conserved exon
region of Tpi and previously used in [2]. The PCR cycling conditions were 3 min at 94
°C, then 40 cycles of 94 °C for 30 s, 50 °C for 45 s, 72 °C for 1 min 20 s, and a final
step at 72 °C for 10 min. PCR products were cleaned for direct sequencing by
treatment with a combination of 0.5 U Exonuclease I (New England Biolabs, Ipswich,
MA) and 0.25 U Shrimp Alkaline Phosphatase (Promega), with incubation at 37°C for
30min, then 95°C for 5min, prior to capillary sequencing by Macrogen (Seoul, Korea).
ESM 1 Table 2. PCR conditions for Wolbachia detection and the Tpi gene
wsp (wCI, wFem)
Tpi
10 µL
20 µL
1x
1x
Primer 1
0.4 µM
0.8 µM
Primer 2
0.4 µM
0.8 µM
0.5 U
1U
1 µL
1 µL
PCR protocol
Total volume
5x MyTaq Red Reaction
Buffer
MyTaq™ Red DNA
Polymerase (Bioline)
DNA Template
Sequence data analysis
In total we direct sequenced Tpi from 57 specimens of 8 mothers and their offspring
(11 males and 38 females, see ESM S3). No indels or SNPs were observed in
sequence chromatograms of females; some males where heterozygous due to
detected double peaks and shifts of sequence reads. For this reason we sequenced
males form both sides and assigned the ambiguous positions using the IUPAC code.
We used PHASE [3] algorithm as implement in DnaSP v5.10.01 [4] to infer putative
Tpi alleles (coded as Allele 1 and Allele 2 in ESM S3). The sequences were then
trimmed and edited in Geneious version 8.0.4 [5]. The 485 bp alignment consisted of
13 parsimony informative sites, representing 17 haplotypes. The overall haplotype
diversity was 0.914. We conducted a network analysis using the median-joining
method [6] implemented in PopART [7]. The network (ESM S4) shows the high
haplotype diversity. Of the 17 haplotypes only 5 haplotypes were shared between
families. There was also no clear divergence of alleles found in wCI and wFem coinfected individuals. We also confirmed the presence of the qPCR primer sequences
within individual sequences and thus excluded the presence of any pseudogenes in
our sequence alignment.
qPCR primer design
Tpi primers were designed in an exon region of previously published sequences of E.
mandarina (GenBank accession numbers: AB231163-AB231194) [8]. Primers
amplifying an exon region of the kettin gene were designed based on an RNAseq
sequence of E. mandarina (D Kageyama, personal communication) and sequenced
from two females each of E. mandarina, E. hecabe
and E. smilax (GenBank
accession numbers KM502545-KM502550). Primers for EF-1α were designed from
previously published E. mandarina sequences (GenBank accession numbers:
AB194749-AB194754). All primers are shown in ESM 1 Table 3 and were designed
using
the
software
PrimerQuest
(http://www.idtdna.com/SCITOOLS/Applicati
ons/PrimerQuest/).
ESM 1 Table 3. qPCR primers used for the amplification of Tpi, kettin and Ef-1α
Name
Primer sequence (5’  3’)
TPI-F
GGC CTC AAG GTC ATT GCC TGT
TPI-R
ACA CGA CCT CCT CGG TTT TAC C
Ket-F
TCA GTT AAG GCT ATT AAC GCT CTG
Ket-R
ATA CTA CCT TTT GCG GTT ACT GTC
EF-1F
AAA TCG GTG GTA TCG GTA CAG TGC
EF-1R
ACA ACA ATG GTA CCA GGC TTG AGG
Tm, primer melting temperature
Amplicon length (bp) Tm (℃)
60
73
60
60
73
60
60
73
60
qPCR conditions
qPCR reactions were performed on a Rotor-Gene Q PCR system (Qiagen). Using 20
µL reactions, each qPCR reaction contained 1x SensiFAST™ SYBR No-ROX
(Bioline), 0.8 µL of each primer and 1 µL of template DNA. Each DNA template was
diluted 1:10 prior to reaction set up. Cycling conditions for all qPCR reactions were 2
min at 95 °C, then 40 cycles of 5 s at 95 °C, 10 s at 60 °C and 10 s at 72 °C. A melt
curve for each primer pair was conducted to clarify that there was no primer dimer
formation. Every run was performed analysing each specimen in three technical
replicates with all three primer sets including a no-template control. One E.
mandarina male was selected as a run calibrator in all runs in order to compare runs.
Quantitative Analysis of Gene Dose Ratio
PCR amplification efficiencies were measured for each individual reaction using
LinRegPCR [9] and tested that they were approximately equal [9]. The relative copy
number of the Z-linked genes was obtained as gene dose ratio (GDR) using the 2ΔΔCt
method, ΔCt = Cttarget - Ctreference [10]. The target and the reference PCR
efficiencies where approximately equal with less than 10 % difference and the ratios
were calculated without efficiency correction [10].
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