DNA extraction
Total DNA was extracted from individual leafhoppers using a
non-destructive method to preserve the specimen exoskeletons as vouchers
and for subsequent morphological study. For each specimen, the abdomen
was dissected, transferred to a 1.5 ml tube containing 400 µl 1X TES pH
7.8 buffer (20 mM Tris, 10 mM EDTA, 0.5% SDS) and 4 µl Proteinase K (20
mg/µl) and incubated at 56°C overnight. The abdomen was then removed and
preserved in ethanol for morphological study. The buffer solution was
then blended for 10 minutes using a mixer (MixMate) and the solution was
transferred to a new 1.5 ml tube with 400 µl of chloroform, mixed and
centrifuged 10 min at 4°C at 11,000 rpm. The supernatant was transferred
to a new tube and the chloroform wash was repeated. DNA was then
transferred to a new tube and 400 µl of ice-cold isopropanol was added
followed by mixing and centrifuging for 15 min at 4°C at 12000 rpm.
Supernatant was discarded and the DNA pellet was washed twice using 500
µl of ice-cold 96% ethanol. The DNA pellet was then dried for 20 min
and re-suspended in 50 µl of TE buffer (pH 7.8). To each leafhopper
sample a molecular code was assigned: e.g., LH078 stands for LeafHopper
followed by an ordinal number indicating the collection event.