Genetic materials

Interspecific hybrids were made between the two diploid species A. ipaënsis K30076 (Ipa; female) and A. correntina 9530 (Cor; male) at North Carolina State University. Multiple plants of each diploid species were used for crossing. Cuttings from the sterile interspecific hybrids were established and shipped to the University of Georgia Tifton Campus for colchicine treatment. Sterility is expected in interspecific hybrids, and the hybrid nature of putative F₁s was confirmed by the presence of greater than 80% aborted pollen grains using the Alexander blue staining method (Alexander 1969).

For chromosome doubling, 300 cuttings of the diploid hybrid (∼20 cm long) were immersed in 0.2% colchicine solution for 12–14 hours at room temperature. The treated cuttings were rinsed under continuously running tap water for 1 hour to remove excess colchicine. The cuttings were shortened by 2 cm from the end of the existing cut. The 18 cm long piece was further cut in half yielding two approximately 9 cm long pieces. Fresh cut ends were dipped in Clonex rooting gel (Growth Technology, Western Way, TA, UK) before being planted in Jiffy peat pots (5.7 cm² sq. × 5.7 cm H) filled with Promix BX growth medium (Premier Tech Horticulture, Quakertown, PA, USA). The cuttings were kept in seed trays enclosed with a clear cover to reach 100% humidity. The colchicine-treated cuttings were grown under 16/8 hours light/dark condition at room temperature for 3–4 weeks to allow root formation before transplanting to a plastic tub (87 × 55 × 20 cm; LxWxH) filled with Promix growth medium in the greenhouse. Cuttings were periodically checked for the formation of pegs and marked with flags. Pods were harvested 40–50 days after peg identification.

S₀ plants of IpaCor^4x were used as male parents to cross with two advanced peanut breeding lines 13-2113 [(Tifguard × Florida-07) × C725-19-25] and 13-1014 [(Tifguard × Florida-07) × Georgia-06G]. Florida-07 (Gorbet and Tillman 2009), Tifguard (Holbrook et al. 2008a), and Georgia-06G (Branch 2007) are elite peanut cultivars adapted to US southeastern peanut growing regions. C725-19-25 is a high-yielding breeding line with resistance to tomato spotted wilt virus (Holbrook et al. 2008b). Two F₂ populations from F₁ hybrids (13-1014 × IpaCor^4x_S_0.2)_F_1.4 and (13-1014 × IpaCor^4x_S_0.5)_F_1.4 were grown in the greenhouse for tissue collection and DNA extraction. Each F₂ population consisted of 456 individuals.