Molecular Identification of Pollen
Pollen DNA was extracted using a modified glass fibre protocol (Ivanova
et al. 2008). The remaining 200 μL of ethanol suspended pollen samples
were dried under a sterile hood and resuspended in 300 μL of insect
lysis buffer. Samples were transferred into 96 well plates with
microbeads (MP Biomed, lysis matrix E, OH, USA). Samples were randomly
assigned a location in the plate matrices. In order to detect
contamination, 116 negative controls were added into the matrices,
randomly assigned with at least one negative control per column in the
96 well plate matrix. Pollen grains were pulverized by shaking samples
at 28 Hz for two minutes. Samples were incubated at 56°C for 2 hours,
followed by 1 hour at 65°C. Following incubation, 6M GuSCN buffer was
added to lysate in a (2:1 to lysate, 400 μL to 200 μL), mixed briefly by
vortexing, centrifuged at 1000 g for 20 seconds. The lysate was
transferred to a glass fibre filter plate (PALL Corp) and centrifuged at
5000 g for 5 mins, followed by the addition of 300 μL of binding mix and
centrifuged at 5000 g for 2 mins. The glass fibre plate was then washed
twice with 600 μL of wash buffer and spun down at 5000 g for 5 mins. The
plate was spun for an additional 5 mins at 5000 g and incubated at 56°C
for 30 mins to dry the plate. DNA was eluted into a PCR plate with 25 μL
of elution buffer and incubated at 56°C for 1 minute and then
centrifuged at 5000 g for 5 mins.
To assess plant diversity, we amplified a 184 bp fragment of rbcL(large subunit of RuBisCo) using rbcL1 and rbcLB (Palmieri et al., 2009)
(Table 1). A total of 284 samples w selected for sequencing. The Qiagen
multiplex plus master mix (QIAGEN, Hilden, Germany) was used for PCR.
Amplification was performed under the following thermal conditions: 5
mins at 95°C; 35 cycles of 30 s at 95°C, 30 s at 50°C, and 1 min at
72°C; 5 min at 72°C; then held at 4°C. The 25 μL PCR reaction mix
included 12.5 μL of Master Mix, 1.25 μL of each 10X PCR forward and
reverse rbcL primer and 10 μl of DNA template (Palmieri et al.
2009, Little 2014). PCR amplicons were visualized on a 1.0% agarose gel
using GelRed® Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA).
Samples were indexed with a secondary PCR using fusion primers and run
under the same thermal conditions (Elbrecht and Steinke 2018). Fusion
primers were used to attach unique molecular identifiers (UMIs) along
with TruSeq sequencing adaptors for Illumina MiSeq sequencing
(Supplemental Table S2). The PCR mix included 12.5 μL of Master Mix, 9
μL of molecular grade water, 1.25 μL of each 10X PCR forward and reverse
primer with custom tags (Elbrecht and Steinke 2018) and 1 μL of DNA
template. The samples were cleaned and normalized using the SequalPrep™
Normalization Plate Kit (Invitrogen, Thermo Fisher Scientific Inc., MA,
USA) following manufacturer’s instructions. Libraries were pooled and
subsequently went through clean up using the SPRIselect Kit (Beckman
Coulter) and the Left Side Size Selection procedure with a
sample-to-volume ratio of 0.75. Final quantification was done using a
Qubit Fluorometer with the Qubit dsDNA HS Assay Kit according to
manufacturer’s instructions. Sequencing was done using an Illumina MiSeq
with the 600 cycle Reagent Kit v3 (2 × 300) at the Advanced Analysis
Centre at the University of Guelph.