Brabbs et al. Supplementary Methods
Identification of M1 SNPs
The 142S and M1 DNA samples were sequenced initially using the Illumina
HiSeq 2000 platform at the University of Exeter (U.K). The M1 and 142S DNA
samples were run in separate columns on an Illumina flowcell and produced a
total of 129,533,529 reads for M1 and 66,098,454 reads for 142S. To identify
and map the mutation sites in M1 compared to 142S, SHORE analysis
pipeline was used (Ossowski et al., 2008, Schneeberger et al., 2009).
Following the pipeline, a SHORE-specific predefined directory structure was
created to store raw data and alignment results. Initially the C24 reference
genome, obtained from the 1001 genomes website
(www.1001genomes.com), was translated into the SHORE reference format
(Schneeberger et al., 2011). The sequence data was quality checked and
processed into the SHORE flat file format. For both 142S and M1, the reads
from the first and second sequencing runs were mapped to the C24 reference
genome separately before being merged. Consensus analysis was then
performed between 142S and M1 to identify SNPs. SHORE identifies all
SNPs between the C24 reference, 142S and M1, and the SNPs were filtered
to select for homozygous SNPs and ones that are present in M1 but not in
142S. An in-house script was used to compare the SNPs and identified 69
putative homozygous SNPs on the left arm of chromosome 1, which is the
region indicated from the rough mapping studies. The location of each SNP in
the genome was determined and exonic SNPs that gave rise to amino acid
changes were identified (Table S1).
Flow Cytometry Protoplast Counting and Sorting
Protoplasts suspended in protoplast resuspension buffer were sorted into
GFP positive and negative populations using a MoFlo Astrios cell sorter
(Beckman Coulter – Fort Collins, Denver, USA). As the protoplasts were
estimated 20-30 µm, a 200 µm MoFlo nozzle tip was selected for the sort to
prevent shear force. A sheath pressure of 6.0 psi and drop drive frequency of
6670 Hz (6670 independent drops per second) was used. A sample of the
resuspension buffer was run to ensure the mannitol content did affect the
stability of the last attached drop. The fluorescence signal from the 488 nm
laser and signal in to the 664/22 filtered photo multiplier tube detector (PMT)
was used as the trigger signal and threshold set using the above buffer.
Protoplasts were positively identified from sample debris using native
fluorescence emitted by the chloroplasts when excited by the 488nm laser
and detected in the 710/45 PMT. Intact protoplasts were typically 5 times
brighter than individual, free-floating chloroplasts resulting in 2 obvious
fluorescent peaks. (Figure 1 below). GFP signals were detected by 488 nm
laser excitation and emission collected via a 526/52 filtered PMT (Figure 2
below). Boolean logical gating strategies were used to select intact
protoplasts and the GFP positive or negative populations, identified using
bivarent plots were sorted into independent tubes containing an excess of
resuspension buffer.
Figure 1 – Identification of intact protoplasts in line morc6-7 by flow cytometry
using native fluorescence of chloroplasts, due to the presence of chlorophyll,
excited by the 488nm laser and detected in the 710/45 PMT. The region R2
denotes the peak of higher fluorescence emitted from intact protoplasts.
Figure 2. Fluorescence signals from intact protoplast of line morc6-7, as
selected using R1 in Figure 1, by flow cytometry. Fluorescence is excited
using the 488nm laser and detected in the 526/52 filtered PMT channel. R3
represents GFP negative cells and the higher fluorescence peak denoted R4
represents GFP positive cells.
Table 1: Primers used in this study.
Name
Sequence
Primers used to generate probes
GFP 1
CAAGGAGATATAACAATGAAG
GFP 8
CATGACGAACTCTAAGAGCTA
Actin F
CATGGTTGGGATGAACCAGAAGGA
Actin R
GTCTCTTACAATTTCCCGCTCTGC
soloLTR F
AATGCATTACAAAAACCTTCTGA
soloLTR R
GGATTCACGATTAGAGAACGTAGA
35S F
GTCTCAGAAGACCAAAGGGCTA
35S R
GGTCTTGCGAAGGATAGTGG
5s F
GGATGCGATCATACCAG
5s R
GAGGGATGCAMCACSAG
AtMuF
GTGGATATACCAAAAACACAA
AtMuR
CTTAGCCTTCTTTTCAATCTCA
MEA-ISR-F
AAACCTTTCGTAAGCTACAGCCACTTTGTT
MEA-ISR-R
TCGGATTGGTTCTTCCTACCTCTTTACCTT
Primers used for rtPCR and q-rtPCR
rtMOR6 F
TCTCATGCATCCGCAGTACATC
rtMOR6 R
GCTGCACTTGCAACAGAATCAG
q-rtMORC6 F
ACAAGAAGCTCCGAGCAAAA
q-rtMORC6 R
CTGTGCTCGTCTTTCACCAA
rtActin F
TGAGAGATTCAGATGCCCAGAA
rtActin R
TGGATTCCAGCAGCTTCCAT
rt-soloLTR F
ATCAATTATTATGTCATGTTAAAACCGATTG
rt-soloLTR R
TGTTTCGAGTTTTATTCTCTCTAGTCTTCATT
AtMu F
GTGGATATACCAAAAACACAA
AtMu R
CTTAGCCTTCTTTTCAATCTC
Primers used for bisulfite sequencing
BS14
CACACTTRTCTACTCCAAAAATATC
BS22
GAGGATAATGATAGGAGAAGTG