Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.
Elisabet Claveria, Jordi Garcia-Mas, and Ramon Dolcet-Sanjuan
Mónica Morales, Marisol Luís-Arteaga, José María Álvarez, Ramon Dolcet-Sanjuan, Amparo Monfort, Pere Arús, and Jordi Garcia-Mas
The recessive allele (nsv) of the NSV gene confers resistance to the Carmovirus melon necrotic spot virus (MNSV) in melon (Cucumis melo L.). Using an F2 population obtained from the cross between the resistant Korean accession PI 161375 and a susceptible line of `Piel de Sapo', we have mapped the NSV locus to linkage group 11 (G11) of the melon genome. Additional markers closely linked to NSV were developed by bulked segregant analysis (BSA) using a doubled haploid progeny population derived from the same cross. A detailed map of the NSV region was constructed containing 10 markers spanning a distance of 17.7 cM. The nearest flanking markers to NSV were two amplified fragment length polymorphisms (AFLPs) (CTA/ACG-115 and CTA/ACG-120) and one random amplified polymorphic DNA (RAPD) (OPD08-0.80) separated by 5.9 cM. Two more markers, ACC/ACC-110 and OPX15-1.06, cosegregated with NSV.