A total of 219 accessions consist of eight Vigna species (including 149 accessions of seven wild species) were provided from the genebank in NIAS (National Institute of Agrobiological Sciences) in Tsukuba, Japan. See details in Table 1 and Electronic Supplementary Materials (ESM) Table 1.
Salt tolerance screening in soil culture
Primary screening for salt tolerance was conducted at NIAS in Tsukuba, Japan (36°03′N, 140°10′E). The 219 accessions were grown in plastic pots (7 cm in diameter × 9 cm in height) under natural light condition in a greenhouse from June in 2009. The soil used in the pot was KUREHA-Engei-Baido (KUREHA Inc, Japan). Twenty-one days after sowing, an adequate amount of salt solutions was applied in the pool and then the pots were immersed for 2 weeks. NaCl concentration was set to 0, 50, 100, 150 or 200 mM. Two plants with two replications were tested in each treatment.
Tolerance was evaluated at 1 and 2 weeks after treatment started. Each plant was visually scored by percent of wilted leaves as follows; 1 = No leaves wilted, 2 = 1–25 % of leaves wilted, 3 = 26–50 % of leaves wilted, 4 = 51–75 % of leaves wilted, 5 = 76–99 % of leaves wilted and 6 = all leaves wilted (ESM Fig. 1).
Salt tolerance screening in hydroponic culture
A total of 30 accessions from eight species (see Figs. 1, 2) was selected based on the result of primary screening (soil culture) and was sown in plastic pots with KUREHA-Engei-Baido supported and kept in green house for 21 days. Each accession was transplanted to a hydroponic culture in green house from August 2010. One culture pool (62.5 cm × 100 cm × 17 cm height) contained 64 plants (8 × 8 plants). The culture pool contained diluted nutrient solution of a 1:1 ratio of Otsuka house No. 1 (1.5 g/L): Otsuka house No. 2 (1 g/L) (Otsuka Chemical Co., Osaka, Japan: N, P, K, Ca and Mg = 18.6, 5.1, 8.6, 8.2 and 3.0 mEq/L, respectively). The nutrient stock solution was diluted to reach EC at 100 mS/m with water. Ten days after transplanting, salt stress was initiated. NaCl concentration in the hydroponic culture was 50 mM for the first week, 100 mM for the second week, and then 200 mM from the third week to the eighth week. Wilt scores were evaluated every week after the initiation of salt stress.
Measuring Na+ accumulation in plants
Four days after the NaCl concentration reached 200 mM, leaves, stems and roots were separately harvested from the three plants of V. angularis (JP233136, ‘Kyoto Dainagon’), V. nakashimae (JP107879, ‘Ukushima’) and V. riukiuensis (JP235833, ‘Tojinbaka’). The harvested samples were dried at 62 ºC for 24 h, ground into a powder and were reduced to ash for sodium extraction with 100 μL of 1 N HNO3. The cation concentrations in the leaves, stems and roots were determined using an ion chromatograph with a conductivity detector (Shimazu, Japan). Oxalic acid (3.3 mM) was used as the mobile phase. The mobile phase was degassed by degasser (DGU-12A) and pumped with liquid chromatograph pump (LC-9A) at speed 1 μL/min. This mobile phase was flown to the auto injector (SIL-6B) and mixed with 10 µL of ample solutions to be homogenized, which was controlled by the system controller (SCL-6B). The Na+ concentration was detected through the analytical column (IC-C3) in the column oven (CTO-10A) at 40 ºC. The result was printed by a chromatopac (C-R 6A). The standard solution of the Na+ concentration (for 100 % was equated with 2 ppm) was measured for wring a standard calibration to calculate the correct ion concentrations of the sample solution.
Evaluation of photosynthetic activity and stomatal conductance
Seeds of V. angularis (JP233136, ‘Kyoto Dainagon’), V. nakashimae (JP107879, ‘Ukushima’) and V. riukiuensis (JP235833, ‘Tojinbaka’) were germinated on Seramis (Effem GmbH, Verden, Germany) clay and hydroponically grown in a greenhouse for 4 weeks before measuring photosynthetic rate and stomatal conductance. To minimize the osmotic shock, 50 mM NaCl was added to the nutrient solution 3 days before the NaCl concentration was raised to 100 mM. The salt treated plants were grown for 7 days in 100 mM NaCl conditions. The control plants were grown in salt-free nutrient solution during the experiment. The photosynthetic rate and stomatal conductance of the topmost fully expanded leaves were measured using a portable photosynthesis system (LI-6400, LI-COR Inc., Lincoln, NE, USA). The measurements were obtained from 10:00 am–14:00 pm at 1 day before salt treatment, 3 days after 50 mM NaCl treatment, 3 and 7 days after 100 mM NaCl treatment. The measurement was done for 10 replications in each of salt treated and control plant under 1200 μmol photon m−2 s−1 photosynthetic photon flux density, at 31.9 ± 3.2 °C leaf temperature, 38.8 ± 4.7 % relative humidity and 399.9 ± 0.2 ppm CO2 concentration. The measurements were performed after approximately 3 min of light exposure when the CO2 gas exchange rate reached a steady state.
Growth test in a salt-damaged field
We borrowed a salt-damaged field and a de-salted field from a farmer in Soma-city, Fukushima prefecture, Japan, where tsunami covered his field by the East Japan Earthquake on March 11, 2011. We sowed seeds of V. nakashimae (JP107879, ‘Ukushima’) and V. riukiuensis (JP235833, ‘Tojinbaka’) and soybean (Glycine max) (JP67666, cv. ‘Tachinagaha’) on June 11th, 2013, and harvested the whole plant shoot above ground on October 31th, 2013. We cultivated 32 plants for each strain/cultivar in a salt damaged and a de-salted field, and measured dry weight of each plant to evaluate plant growth.