Horizontal gel electrophoresis system

The Eppendorf ThermoMixer (RTM F1.5, 220–240
V/50–60 Hz) was purchased from Eppendorf. The horizontal gel
electrophoresis system was purchased from Bio-Rad. Gel images were
obtained with a GelDoc Go system, Bio-Rad. PCR was performed using
a Proflex 3 × 32-well PCR thermal cycler system (Thermo Fisher
Scientific). qPCR was performed using a QuantStudio 7 qPCR instrument
(Applied Biosystems). HPLC was performed using an Infinite 1260 HPLC
with C 18 column, Agilent.

Total 45 SSR markers, reported earlier (22–25 (link), 39 (link)) in aroid species were used for the initial screening, 34 shown amplification and except marker Xuqtem-110, remaining 33 were found polymorphic (Table 2). Moreover, markers Ce1B-12, Taro-5, Taro-7, Taro-9, Taro-10, Taro-15, Taro-16, Taro-17, and Taro-18 failed to amplify. Marker Taro-2 and Taro-6 shown amplification in few genotypes only. The PCR analysis was carried out in 20 μL volume containing 40 ng template DNA, 0.5 U TaqDNA polymerase, 0.2 mM each dNTP, 0.2 μM forward and reverse primer each in (1×) reaction buffer that contained 10 mMTris–HCl (pH 8.3), 50 mMKCl, and 2.5 mM MgCl₂ (Thermo Scientific, Bangalore, India). Amplification conditions (Applied Biosystems Veriti™, Singapore) were initial denaturation at 94°C for 5 min and 35 cycles at 94°C for 60 s and then 50 – 66°C for 60 s, and extension at 72°C for 2 min, followed by 10 min at 72°C and indefinite soak at 4°C. Amplified products were resolved on 3.5% SFR agarose gel containing ethidium bromide (10 mg/mL) at a constant voltage of 80 V for 3 h using a horizontal gel electrophoresis system (Biorad, Singapore). The gel was run in 1 × TBE buffer. A 50 bp DNA ladder (MBI Fermentas) was run alongside the amplified products to determine their approximate band size. Similarly, the amplified products were visualized under UV by image analyses.