The pods were harvested and percentages of crossed pods were calculated. In the next season, the F1 plants were used as pollen parents for the first backcross to each recurrent parent. Pods of BC1F1 generation from all crosses were harvested and grown in the next season. These plants were then used to make second backcrosses. The BC2F1s were grown and selfed thrice to produce BC2F4 population after three seasons (Fig. 1). Both amphidiploids were evaluated for component traits of rust and late leaf spot (LLS) resistances

using a detached leaf technique [18]. On the 40th DAS, tetrafoliate ALK targets leaves were excised from the pulvinous regions and arranged in plastic trays containing autoclaved sand in a randomized block design with two replications. In order to compare the disease severities, a susceptible check (variety “TMV 2”) was used for both the diseases. P. arachidis urediniospores and C. personatum conidia were Autophagy inhibitor chemical structure initially produced on susceptible cultivar TMV 2 and harvested with a cyclone spore collector. The concentrations of the spore suspensions were set to 20,000 spores mL− 1 using a hemocytometer by adding sterile distilled water containing a few drops of Tween-80 (polyoxyethylene sorbitan mono-oleate) in order to promote adhesion. Spore suspensions of both the pathogens were sprayed on to the leaves by using an atomizer, and the trays were kept in a growth chamber at 23–25 °C

immediately after the inoculation to ensure leaf moisture during the night. Two weeks after inoculation, leaves were inspected for symptoms and time to sporulation. Damage due to rust and LLS was determined after 30 days based on these parameters. Cultivar TMV 2 was used as the susceptible check and cultivar GPBD 4 was used as resistant check in all disease screening experiments. Plants of BC2F2 generation generated from each of the nine crosses were screened for disease Sclareol resistance during the rainy season

of 2011 following the protocol of Subrahmanyam et al. [19]. Seeds were treated with seed protectant and sown in the field with 45 cm and 10 cm inter- and intra-row spacing, respectively. The parental genotypes were sown once as controls and TMV 2 (susceptible variety for both diseases) was planted at every 10th row as well as a border around the field to maintain an effective inoculum load. Uniform inoculation across the field was performed in the evening of 45th DAS. Disease scoring for LLS and rust occurred on the 80th and 90th DAS using a 0–9 scale of disease severity (%) on the leaves for lesions and defoliation in the case of LLS, and on pustules and necrosis in the case of rust. Scores were as follows: (i) 1.0, no disease; (ii) 2.0, 1%–5% severity, lesions/pustules on lower leaves; (iii) 3.0, 6%–10% severity, lesions/pustules mostly on lower leaves and very few on middle leaves along with defoliation or necrosis of lower leaves; (iv) 4.