Identification of novel genes involved in phosphate accumulation in Lotus japonicus through Genome Wide Association mapping of root system architecture and anion content

<p>130 Lotus japonicus accessions were used. The names and accession numbers are<br> listed in S6 Table. Seeds were scarified with sandpaper and then sterilized 14 minutes in 0.05%<br> sodium hypochlorite. Subsequently, seeds were rinsed and washed 5 times in sterile distilled<br> water. For the germination, seeds were positioned in imbibed filter paper, in sterile Petri dishes,<br> and wrapped in aluminium foil. After 3 days at 21&deg;C, young seedling were transferred to square<br> plates (12 x 12 cm) containing growth medium. Both media used in this<br> study were based on Long-Ashton solution (with two levels of phosphate concentration -20 or<br> 750 &mu;M, LP or HP, respectively) with 0.8% MES buffer (Duchefa Biochemie,<br> Haarlem, The Netherlands), 0.8% agarose (to minimize phosphate contamination), and adjusted<br> to pH 5.7 with 1M KOH. After adding the medium, plates were dried, closed, overnight in a<br> sterile laminar flow hood. Two accessions, with four replicates per each accession, were placed<br> on each plate. Each plate was replicated, with mirrored position of each accession to minimize<br> any positional growth effects. Plates were placed vertically, and plants grown under long-day<br> conditions (21&deg;C, 16 h light/8 h dark cycle) with white light bulbs emitting 50 &mu;mol/m 2 /s and<br> roots were exposed to light. Every day at the same time, the racks were transported to the image<br> acquisition room where images of each plate were acquired with eight Epson V600 CCD flatbed<br> color image scanners (Seiko Epson) and then immediately returned to the growth chamber.</p>