Development of Advanced Interspecific-bridge Lines among Cucurbita pepo, C. maxima, and C. moschata

Interspecific hybridization among the three most economically important cultivated species of Cucurbita spp., Cucurbita pepo, C. moschata, and C. maxima can be made but not readily. By means of various pollination measures, different mating systems, and varying selection methods, nine advanced interspecific-bridge lines were developed, in which the crossing barrier among the species and the male sterility of the F1 and subsequent generations were overcome over a 12-year period from 1999 through 2011. Despite the considerable influence of parental cultigens and environmental factors on the incompatibility of interspecific crosses, the plant and population compatibility significantly increased when a backcross with a recurrent parent in the same species or a multiple-way cross with a parent in the different species was made. As the generations advanced, the percentage of fertile seeds (PFS) significantly increased in all the siband self-families. The four advanced interspecific-bridge lines out of nine not only have gained the normal crossability of interspecific hybridization, but also could eliminate the sexual obstacles of the subsequent generations. The results demonstrate that a twoor three-species bridge line with crossing compatibility can be created by twoor threespecies recombination and continuous selection. More importantly, the breakthrough of the advanced interspecific-bridge lines could provide a powerful platform for breeders to transfer favorable traits freely among the species and create more valuable and unique types or varieties through a conventional breeding process. Cucurbita pepo, C. moschata, and C. maxima are the most economically important three (out of five) cultivated species within the Cucurbita genus that include squashes, pumpkins, and gourds, which represent several species in the same crop (Blanca et al., 2011; Robinson, 1995). These species are remarkably diverse in morphology, disease resistance, and environmental adaptability (Loy, 2004; Saade and Hernandez, 1994; Whitaker and Bemis, 1964). For a long time, breeders have attempted to use variability in the genus for crop improvement through interspecific breeding yet overcoming crossing barriers, the male sterility, and incompatibility of the interspecific F1 and early succeeding generations of distant crosses has been a major challenge for Cucurbit breeders (Chekalina, 1974; Hiroshi, 1963; Rhodes, 1959; Shifriss, 1987; Wall, 1961). Based on the species crossability, Whitaker and Davis (1962) concluded that C. moschata occupies a central position among the annual species and can be crossed with difficulty with C. maxima, C. pepo, and C. mixta. Fertile seeds from a series of interspecific crosses were successfully obtained in the past few decades (Baggett, 1979; Castetter, 1930; Erwin and Haber, 1929; Kanda, 1984; Shifriss, 1987; Wall, 1961). While making the crosses, fruit set is generally quite low for many crosses and the occasional fruit produced may have few seed or none (Baggett, 1979; Cheng et al., 2002; Robinson, 1999). To obtain fruits and fertile seeds from the F1 plants of interspecific crosses, additional techniques like repeated pollination, bud pollination, mixed pollen pollination, embryo culture and /or amphidiploidy, and the adjustment of florescence and environmental conditions are frequently used (Bemis, 1973; Cheng et al., 2002; Hiroshi, 1963; Shifriss, 1987). To overcome species barriers, a wild species (for example, C. argyrosperma) with a wide cross compatibility have been used as a genetic bridge to transfer genes between other less-compatible cultivated species (McCandless, 1998; Wessel-Beaver et al., 2004) or used to create genetic bridge lines by crossing with an interspecific F1 (Chetelat and DeVerna, 1991; Finkers et al., 2007). A sterile F1 from two distant species can be retrieved by embryo and ovule culture and directly used as a bridge line for gene transfer (Pico et al., 2000; Poysa, 1990; Wang et al., 2002) or subsequently chromosome doubled to produce a fertile amphidiploid. This amphidiploid or the derivatives therefrom offer a possible genetic bridge between the incompatible species (Chen et al., 2011; Parisi et al., 2001; Staub, 2002). However, although a wild species, interspecific F1, amphidiploidy, or induced polyploidy as a genetic bridge plays an important role in overcoming species barriers and the male sterility of interspecific F1 for gene transfer, none of these genetic bridges can solve the male sterile, incompatible, and infertile problems in the later generations (Stebbins, 1956; Wang et al., 2002). Moreover, during the transfer of important characteristics with the bridges, unfavorably species-specific traits are frequently carried along to subsequent populations from initially interspecific hybridization (Whitaker and Robinson, 1986). Nevertheless, the disadvantages may be removed by intervarietal hybridization and selection (Munoz et al., 2004; Singh et al., 2009; Stebbins, 1956). The objective of this study is to develop interspecific inbred lines with normal compatibility by varietal recombination among the three species and successive selection through different mating and selection methods. Meanwhile, some important traits such as plant habits, fruit types, multiple disease resistance, and heat and cold tolerance are integrated into the lines for the purpose of developing new Cucurbit types or varieties. To realize the objective, the removal of the male sterility and sexual incompatibility of interspecific F1 and subsequent generations was determined as a main task in this study. Materials and Methods The breeding materials used in this study included S179 (C. pepo, spp. pepo), 3112 PMR (C. pepo, spp. pepo), H7B (C. pepo, spp. pepo), Sugar Loaf (C. pepo, spp. ovifera), Neck Pumpkin (C. moschata), Argonaut (C. moschata), Buttercup (C. maxima), and Rouge Vif D’Etamps (C. maxima). Among the first four, S179 is a long, straight Lebanese marrow line that was used as an interspecific donor of the neck straightness to correct the curviness of the long butternut cultivars; 3112PMR represents a short marrow zucchini line with zucchini yellow mosaic virus, watermelon mosaic virus, and PMR. H7B is a PMR pumpkin, which also tolerates Charcoal Rot (Macrophamina phaseolina), Pythium Root Rot (Pythium aphyanidermatum), and fusarium wilt (Fusanrium solani f. sp. cucurbitae). Both 3112PMR and H7B were designed to provide multiple disease-resistant background for the interspecific crosses; and Sugar Loaf is a winter squash that has a very fine flesh texture. The next two winter squash cultigens, Neck Pumpkin and Argonaut, have very strong vines, a large root system, and high heat and humidity tolerance, which were integrated into the interspecific-bridge lines for rootstocks. These two very long butternut varieties have a polymorphic curved neck problem, which was expected to be solved by the allelic introgression with an interspecific recombination. Within the last two maxima Received for publication 10 Jan. 2012. Accepted for publication 28 Feb. 2012. Plant Breeder. Research Assistant. President of Hollar Seeds. To whom reprint requests should be addressed; e-mail [email protected]. 452 HORTSCIENCE VOL. 47(4) APRIL 2012 Fig. 1. Scheme of the development of interspecific-bridge lines. S179, 3112PMR, H7B, and SgLf (Sugar Loaf) are C. pepo squashes; Arg (Argonaut) and NK (Neck Pumpkin) are C. moschata winter squashes; Bcup (Buttercup) and RVif (Vif D’Etamps) are C. maxima pumpkins. ‘‘BC/w’’ and ‘‘X/w’’ mean ‘‘backcrossed with’’ and ‘‘crossed with,’’ respectively. ‘‘Self’’ and ‘‘sib’’ represent ‘‘self-selection’’ and ‘‘sib-mating selection.’’ , , , and are newly developed inbred lines with stable crossing compatibility. ‘‘h’’ means continuous selection. HORTSCIENCE VOL. 47(4) APRIL 2012 453 pumpkins, Buttercup is a green fruit variety and Rouge Vif D’Etamps is a red fruit type that is not present in C. pepo and C. moschata. These two pumpkins also have a strong root system and full vine. Both of them have a flat, round fruit with a rich, sweet, and flavorful orange flesh that is fine-textured and dense in consistency. For the convenience of formalizing breeding pedigree, Sugar Loaf, Neck Pumpkin, Argonaut, Buttercup, and Rouge Vif D’Etamps are henceforth symbolized as ‘‘SgLf,’’ ‘‘NK,’’ ‘‘Arg,’’ ‘‘Bcup,’’ and ‘‘RVif’’ for all pedigrees. The study was conducted with conventional breeding measures over a 12-year period from 1999 through 2011 (Fig. 1) at the research station of Hollar Seeds Company in Rocky Ford, CO. Multiple-parent populations and the advanced families or lines derived from them were largely created by interspecific crossing, backcrossing, sib-mating, and selfing for better fertility, self-compatibility, and a broader genetic background during the long-continued breeding process. To overcome crossing barriers including the time mismatch between pollen germination and pistillate receptivity, and individual differences in sexual incompatibility, repeated pollination and mixed-pollen pollination were adopted for the first two-way crosses and subsequent threeand four-way base populations. The pollination was repeatedly made by hand with bulked pollen from the plants of a male parent at 0700 HR and 0900 HR on the day of anthesis in Hollar’s greenhouse in the fall and the spring seasons of 1999–2003. To surmount the male sterility and incompatibility of the interspecific F1 and its later generations, backcross and sib pollinations were used in the early generations, and pedigree and sib selections were alternatively implemented as the fertility and compatibility became higher in later generations. Four two-way crosses that were made first consisted of S179 · NK, SgLf · Arg, Arg · Bcup, and S179 · Bcup. After the seed of the four two-way crosses were obtained, six three-way and three four-way crosses were created as multiple-parent populations for the program to start the cycles of selection, which included: [(S179 · NK) · Arg], [(SgLf · Arg) · NK], [(S179 · NK) · Bcup], [(SgLf · Arg) · Bcup], [(Arg · Bcup) · RVif], [(S179 · Bcup) · Rvif], {H7B · [(S179 · NK) · Arg]}, {H7B · [(S179 ·NK) · Bcup]}, and {3112PMR · [(Arg · Bcup) · RVif]} (Table 1), where [.. ..] and {.. ..} represent the threeand four-parent base populations, respectively. Half-sib selection of male sterile plants and pedigree selection of fertile plants were conducted within the multiple-parent populations planted in the greenhouse during the spring or fall seasons of 2001 to 2003, and the same selection methods plus full-sib selection with open pollination were applied in an isolated field during the summers of the years and thereafter. The main systematic characteristics of the three species (Baggett, 1987) were taken as selection markers while selecting interspecific-derived plants or families. In accordance with the markers, plants with recombined traits were selected and carried forward during the breeding procedure. Figure 2 shows the intermediate plant and fruit characters of [(S179 · NK) · Bcup]Sib12, one of those interspecific plants derived from the second individual in the first sib generation of the three-parent base population [(S179 · NK) · Bcup]. This plant was selected based on the intermediate vine of S179 · NK, intermediate fruit of S179 · Bcup, and the color and peduncle of S179 in the summer of 2001. No attempt was made to determine the genetic mechanism of genes or traits involved, and no disease-screening was conducted to avoid losing limited interspecific seeds in the study. However, the fertility, cross-, and self-compatibility of advanced families or lines were considered the main acquired characteristics for selection with the integration of other important traits as Table 1. Intermating effect among three cultivated species of Cucurbita on the seed setting of interspecific