Enduro gds touch imaging system

Around 200 mg of plant tissues from each banana samples were homogenized in liquid nitrogen in double sterilized mortar and pestle. Total RNA from youngest leaf tissues of three biological replicates of wild M. balbisiana and M. acuminata ‘Lakatan’ (BBTV-inoculated and mock-inoculated) were extracted using the modified RNeasy Plant Mini Kit Protocol (QIAGEN, GmbH, Hilden, Germany).
RNA concentration and quality were determined by gel electrophoresis in 1% UltraPure™ agarose (Invitrogen Corp., Carlsbad, California, USA) in 1X TBE running buffer at 100 V for 30 min. The gel was stained with 0.1 µL Gel Red (Biotium, CA, USA) visualized under UV illumination at 300 nm using the Enduro GDS Touch Imaging System (Labnet International, Inc, Edison, New Jersey, USA). RNA was quantified using Qubit™ RNA HS Assay Kit (Life Technologies, Thermo Fisher Scientific Inc.) on Qubit™ 3.0 Fluorometer (Life Technologies, Thermo Fisher Scientific Inc.) following the manufacturer’s instruction.

A total of eight individuals/palms of the coconut genotypes were collected (Table 1). Genomic DNA was extracted following the procedure adapted from Doyle and Doyle [3 ] with modifications. DNA quality and yield were determined by electrophoresis in 1% UltraPure™ agarose (Invitrogen Corp., Carlsbad, California, USA) in 1× Tris-borate EDTA (TBE) running buffer at 100 V for 40 min, 0.5 ug mL⁻¹ ethidium bromide staining, and UV illumination at 300 nm using the Enduro GDS Touch Imaging System (Labnet International, Inc, Edison, New Jersey, USA). DNA concentration was estimated by visual comparison of gel fragments with known concentrations of lambda (λ) DNA molecular weight standards (Sigma-Aldrich Inc., St. Louis, Missouri, USA).Table 1

Coconut genotypes used in the study for screening the SSR markers

Entry number	Coconut cultivars	Code	Palm number	Origin
1	Catigan Green Dwarf	CATD	1715	Davao City
2	West African Tall	WAT	0519	Ivory Coast
3	West African Tall	WAT	0610	Ivory Coast
4	West African Tall	WAT	0704	Ivory Coast
5	West African Tall	WAT	0720	Ivory Coast
6	Laguna Tall	LAGT	0107	Davao City
7	Laguna Tall	LAGT	0508	Davao City
8	SYNVAR (LAGT × WAT)	AN17	4017	Zamboanga City

PCR was carried out with 10 uL reaction volume (15 ng genomic DNA, 1× PCR buffer (10 mM Tris pH 9.1 at 20 °C, 50 mM KCl, 0.01% Triton™ X-100); Vivantis Technologies, Malaysia), 1.5 mM MgCl₂, 0.2 mM dNTPs (Promega Corporation, Madison, Wisconsin, USA), 0.2 μM forward and reverse primer (Integrated DNA Technologies Pte. Ltd., Singapore), and Taq DNA polymerase (Vivantis Technologies, Malaysia). The temperature profile used is as follows: initial denaturation at 95 °C for 3 min, 30 cycles of denaturation (95 °C, 30 s), annealing (45–60 °C depending on the primer pair, 45 s), extension (72 °C, 1 min), and final extension at 72 °C for 5 min. Amplifications were carried out in the Applied Biosystems Veriti™ 96-well Thermal Cycler (Thermo Fisher Scientific, Madison, Wisconsin, USA). PCR products were resolved with electrophoresis using 8% non-denaturing polyacrylamide gel in 1× Tris-borate EDTA buffer at 100 V for 60–75 min in the C.B.S. Scientific Triple Wide Mini-Vertical System™ (C.B.S. Scientific Company San Diego, California, USA) and visualized using 0.5 ug mL⁻¹ ethidium bromide staining and UV illumination using the Enduro GDS Touch Imaging System (Labnet International, Inc, Edison, New Jersey, USA). Gels were scored manually for the presence or absence of bands.