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