Phenotyping

Rust phenotyping:
The recombinant inbred lines (RILs) population and the parents were phenotyped for resistance to P. arachidis. Arachis hypogaea cv. Runner IAC 886 was included as susceptible control. The population was evaluated on F6 and F7 generations. Phenotyping was performed using the detached leaf technique (Moraes and Salgado 1982; Leal-Bertioli et al. 2009). Field assays would not be suitable because of the architecture of the wild-derived diploid plants. Rust spores were collected from infested peanut plants in Pindorama, São Paulo State, Brazil (coordinates 21.1858° S, 48.9072° W). Two bioassays were done, one in 2012 and the other in 2013. In the bioassay of 2012, leaves were inoculated with ca. 4 × 105 urediniospores/mL in 0.05% Tween 20 fungal spores and maintained at 26–28° and photoperiod of 10-hr light and 14-hr dark. In the bioassay of 2013, ca. 2 × 105 urediniospores/mL were used. Four replicates of each individual were analyzed 25 d after inoculation. Susceptibility was measured using the following parameters: total number of lesions/leaf area (cm) (TL/LA), number of sporulated lesions/leaf area (cm) (SL/LA), Incubation period (time for appearance of first lesion in number of days after inoculation) (IncPer), and susceptibility index (SI). SI was calculated with the scale of Savary et al. (1989), with the following modifications: index was the number of lesions times a number that reflected lesion size/reaction. I = ∫(s * n)/LA, where s = lesion size (1 = necrotic aborted lesion, 2−6 = ruptured, sporulating pustules, varying between 0.5 and 3 mm in diameter), n = number of lesions of a particular size, LA = leaf area (mm2). Sporulation was evaluated with the aid of a stereoscope microscope. LA was calculated with the software Quant (Vale et al. 2001). In genotypes that did not present symptoms and therefore did not have incubation period, for QTL analyses, this trait was artificially tabulated as 200.

Other agronomic/domestication traits:
Plants were grown in long trays (1 m × 30 cm × 30 cm), with enough space for lateral branch trailing and seed set. Branches were regularly trailed back to the pots to ensure that pegs would penetrate the soil. At between 40 and 60 d after planting, height of main stem of up to 10 plants of each RIL was measured (main stem height; MSH). At harvest (approximately 120 d after planting), peg length (PL) and pod constriction (PC) was measured with six replications. Harvested seeds were counted (seed number), dried at 20° at 15% relative humidity (RH) for 15 d, and 10 seeds, randomly selected, were weighted (10-SW). Evaluations were performed in three years, 2011, 2012 and 2013, except for PL and PC, which were evaluated only in 2012.