Diversity of Spanish landraces of Cucumis sativus and Cucurbita ssp. 1

Diversity of Spanish landraces of Cucumis sativus and Cucurbita ssp. 1 C. Esteras, M.J. Diez *, B. Picó, A. Sifres, J.V. Valcarcel, and F. Nuez Institute for the Conservation and Breeding of the Agrodiversity, Universidad Politécnica de Valencia, 8E Building, CP 46022, Valencia, Spain * Corresponding author e-mail: mdiezni@btc.upv.es Keywords: Core Collection, Cucurbitaceae, morphological traits, seed analysis, Simple Sequence Repeats Abstract The Genebank of the COMAV at the UPV holds an important collection of cucurbits. It contains more than 1000 accessions belonging to the Cucurbita genus and about 180 accessions of Cucumis sativus. In this work a previously established core collection of 52 C. pepo landraces has been characterized with SSRs markers. Also the geographical variation and the utility of seed traits for classifying C. moschata accessions have been tested using a collection of 250 landraces. The variability of a collection of 63 accessions of C. sativus has been inspected using qualitative and quantitative traits. The SSR results support the idea that only a part of the great variability present in the area of origin of the species arrived into Europe. In fact some Spanish types share alleles with Central-American accessions, but others represent a more genetically distant group. The analysis allowed the selection of the accessions that better represent the extant Spanish variability in C. pepo. The Spanish accessions of C. moschata were highly variable, with the highest variability found in the Canary Islands and in the Mediterranean region within the Iberian Peninsula. Up to 9 morphotypes were identified, with variability for fruit size, rind and flesh color. A lack of correlation between fruit and seed traits was found in this species, in contrast to that found in C. pepo. Therefore, seed analysis is not useful for the management of germplasm collections in this species. The study of the variability conducted in C. sativus highlighted the variability existing in characteristics such as fruit predominant shape at stem and blossom end, fruit shape, brightness, stripes color and intensity of skin texture. The most uniform accessions were those from Castilla La Mancha and the most variable in shape and other characteristics of the fruits the ones collected in Valencia and Murcia. All this information is being used to establish core collections of Spanish landraces of these species. INTRODUCTION Squashes (Cucurbita spp.) and cucumbers (Cucumis sativus L.) are members of the economically important Cucurbitaceae family (Jeffrey 1980; Kirkbride 1993). Cucurbita genus is of American origin. Spanish landraces represent the great diversification of types that have arisen in Europe after this species arrived from America. Initial sites of domestication for cucumber were probably in Southern Asia. 1 Cucurbitaceae 2008, Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24 th, 2008 67

The Cucurbita collection at the Genebank of the Institute for the Conservation and Breeding of the Agrodiversity (COMAV), at the Polytechnic University of Valencia (UPV) is one of the most important collections in Europe. Two of the best represented species are C. pepo and C. moschata, with about 700 accessions. These are mostly landraces still cultivated under traditional cropping systems for selfconsumption or sale in local markets. The study of this collection is essential to understand the variability and potential of Spanish Cucurbita germplasm. In a previous study we analyzed in detail the variability within the C. pepo collection. In order to develop a core collection of Cucurbita Spanish landraces, accessions were grouped on the basis of seed characterization, as seed traits were highly correlated with fruit traits in this species (Paris and Nerson 2003; Picó et al. 2007). Seed length was positively correlated with fruit weight and fruit width. Also seed shape was negatively correlated with fruit shape. These relationships, which are very interesting for the management of large germplasm collections, have not been reported for the other species of Cucurbita. The geographical variation of the C. pepo landraces was also studied using morphological traits and different situations were found in Spanish areas with different agro-climatic conditions and traditional cultural practices. The seed analysis and the geographical study of the variation allowed us to select a sub-set of accessions (about 20 % of the original collection) that better represent the variation of the global collection. Also, previous studies show that a great diversity in fruit size, shape and color appears in the Spanish landraces of C. moschata (Ferriol et al. 2004). This diversity is comparable and even greater for some traits, to that found among landraces from other areas considered secondary centers of diversity for this species. However, these previous studies compare the variability of a sub-set of Spanish landraces with C. moschata landraces from different origins, but do not study in detail the variation within Spanish landraces. Landraces of cucumbers are cultivated in Spain in small farms for selfconsumption. The Spanish type of cucumber is short and small in size with spines and rough skin. The genebank of the COMAV holds a collection of 180 accessions of cucumber, mainly from the South Eastern of Spain. In spite of the greater uniformity of cucumbers compared with the great diversity in Cucurbita genus, there exist also some variability in Spanish landraces of this crop. No previous studies have been conducted about the variability of the landraces stored in the COMAV s Genebank. In the current paper we characterize the core collection of C. pepo, previously established, using molecular markers and initiate the construction of a core collection of C. moschata Spanish landraces by performing a seed analysis and studying the geographical variation in this species. The morphological variation of a group of accessions of cucumber is also studied in order to initiate the construction of a core collection on this crop. MATERIALS AND METHODS C. pepo In this study we used the accessions selected in a previous assay (Picó et al. 2007) that better represent the genetic diversity of the Spanish collection of C. pepo landraces. In Spain, there is no traditional consumption of the cultivar-groups of the subspecies ovifera, whose fruits are very popular in the USA. Thus, most of the 68

cultivated Spanish landraces belong to the ssp. pepo. We analyzed 52 accessions (approximately 20 % of the original collection) belonging to the different morphotypes [15 Pumpkins (P), 4 Bubango types (B) (Pumpkins typical from the Canary Islands), 5 Cylindrical shape (CS) (large pumpkins used for cattle consumption), 15 Vegetable Marrow (VM), 5 Cocozelle (C), 2 Zucchini (Z), 3 ornamentals (OR) and 3 Unclassified] coming from different Spanish regions: Mediterranean region, Valencia (V), Cataluña (C) and Murcia (MU); North of Spain, Asturias (AS) and Santander (S); Central Spain, Castilla y León (CL), Castilla-la Mancha (CM) and Aragón (A); South of Spain, Extremadura (E) and Andalucía (AN); and the Canary Islands (CA). Four additional accessions were included as controls of the extant variability of the area of origin of the species from Mexico (M), from Honduras (H), from Guatemala (G), and from Costa Rica (CR). Three plants of each accession were sampled for DNA isolation and analyzed with ten SSRs loci, previously selected for being polymorphic within C. pepo. These loci were mostly genomic SSR and EST-SSR detected in the Cucumis genus and transferred to Cucurbita. SSR analysis was performed as previously described in Picó et al. (2008). Results were analyzed using a Principal Coordinates Analysis (PCoA) (NTSYS 2.02). C. moschata The COMAV maintains a germplasm collection with about 250 C. moschata landraces, primarily from the Canary Islands and the Iberian Peninsula. Five seeds per accessions were characterized for seed length (SL, mm), seed width (SW, mm) and seed thickness (ST, mm). The ratio SL/SW was also calculated. Some of the accessions had been also characterized for fruit traits in different previous years. This characterization allowed us to classify the different landraces in horticultural types (Wessel-Beaver 2000; Ferriol et al. 2004). Seed traits were subjected to ANOVA to search for differences among seed traits between the different horticultural groups (Statgraphic Plus 5.1). C. sativus Sixty three accessions have been included in this study, most of them coming from the main cucumber producer regions of Spain: Andalucia (17 accessions), Castilla La Mancha (19), Murcia (7) and Valencia (15). A few accessions from Aragón, Asturias, The Canary Islands and Cataluña were also studied. Eighteen qualitative characteristics (reproductive system, plant growth habit, leaf size of blade, leaf intensity of green color, fruit predominant shape at stem end, fruit predominant shape at blossom end, fruit shape, spine color, predominating skin color at market stage, predominating skin color at physiological ripeness, brightness, speckled, stripped, stripes color, fruit surface, intensity fruit surface, hardness and uniformity) and five quantitative (fruit weight, fruit length, fruit width, number of flowers of the first ten nodes on the main stem, and number of flowers on the first ten nodes on the first secondary stem) were recorded for each accessions. A Principal Components Analysis (PCA) and Principal Coordinates Analysis (PCoA) were conducted using the quantitative and qualitative data respectively (NTSYS 2.02). 69

RESULTS AND DISCUSSION C. pepo All SSR loci detected variability within the Spanish collection of landraces. The Mexican accession M-8009 was the only that present unique alleles (two alleles that were not present in any of the other accessions). This result is coherent with the idea that only a part of the great variability present in the area of origin of the species arrived into Europe. Additionally, the Mexican and the other Central-American landraces presented alleles that only appear with a low frequency in the Spanish landraces. This indicates that part of the variability of the origin area still remains in some of the selected accessions. The first and second coordinates of the PCoA explained 14.7 % and 11.5 % of the total variation, respectively. The first coordinate defined three different groups (Fig. 1). The two more distant groups included accessions from Mexico and Guatemala along with 3 Spanish accessions (two pumpkins and one vegetable marrow all with primitive traits, hard rind and indeterminate growth habit) and on the opposite part of the axis most of the Spanish accessions. Despite the fact that all morphotypes are represented in this large group of Spanish landraces, is interesting to note that this group includes most of the vegetable marrow accessions (mostly from Southern Spain and the Canary Islands) and all the Zucchini accessions, which is consistent with the more recent origin of the Zucchini Group and with the fact that this group is the most dissimilar to the gourds of its subspecies (Paris et al. 2003). In the middle appear the other Central-American accessions (from Costa Rica and Honduras) with some accessions from Spain from different origins. This group also includes accessions of all morphotypes, except from Zucchini, but the best represented were the pumpkin types, bubangos and large pumpkins with cylindrical fruits. Also most of the unclassified types were in this group. The vegetable marrow accessions included in this group were those from the Mediterranean area. Some accessions showed a high level of intra-accession variability and displayed plants in different groups (2 bubangos, 1 pumpkin, 2 cylindrical shape, 2 vegetable marrow and one ornamental type). The pumpkin group was the more variable (including the bubangos and the cylindrical shape), as expected for this morphotype that is the more primitive among the subsp pepo morphotypes (Ferriol and Picó 2008; Paris 2008). Also there existed variability in the vegetable marrow and the cocozelle group, with cultivars mostly dispersed in two groups. Previous studies with molecular markers find similar results and do not clarify the relationships between these three morphotypes (Paris et al. 2003). This grouping confirms the idea that some Spanish types share alleles with Central-American accessions, but other represent a more genetically distant group. This analysis will allow to eliminate some accessions and to select those that better represent the extant Spanish variability in C. pepo for the core collection. C. moschata Results indicate that Spanish accessions of C. moschata are highly variable in fruit traits. As it has been reported previously (Andres 2004; Ferriol et al. 2004), the classical commercial groupings do not encompass all the fruit types that we can find in landraces. In fact the majority of the Spanish accessions could not be classified into the three types proposed by Castetter (1925). Most of them display particular 70

characteristics as they have been selected to meet regional consumer preferences as it occurs in other regions. The Spanish landraces varied in fruit size and shape, rind and flesh color, and presence and intensity of ribs and warts (Fig. 2). Accessions could be classified in 9 types (flattened, round, heart-shaped, oblong, cylindrical, pear-shaped, butternut, dumbbell, and crookneck) (Ferriol et al. 2004). The groups flattened and dumbbell were the more frequently found, followed by the butternut, round and crookneck. The group flattened was that more variable, with only some cultivars similar to the commercial Cheese pumpkin. Similarly, only some of the bell-shaped types fitted to the Butternut commercial type. The pear-shaped, cylindrical and oblong were less frequent. The heart-shaped type was only represented in the Canary Islands. This differential situation of the accessions from the Canary islands was studied previously with a sub-set of accessions (Ferriol et al. 2007) and the high variability and the distance of Canarian genotypes from genotypes of the Iberian Peninsula was confirmed by using molecular markers and was explained due to an earlier adaptation of C. moschata to the tropical climate of the islands, together with a differential germplasm introduction from America. Regarding the accessions of the Iberian Peninsula, most come from the Mediterranean area (Cataluña, Islas Baleares, Valencia and Murcia) and from Southern Spain (Andalucia and Extremadura). Differently from C. pepo there is no tradition of use of this crop in northern and central Spain, probably due to the poor adaptation of this vegetable to the cold climate of these areas. Diversity of types was higher in the Mediterranean area. Figure 1. Results of the PCoA with Spanish C. pepo accessions analyzed using SSRs. The grouping according to the first and the second coordinate is shown. Pictures of the main fruit types in each group have been included. Accessions from Central America [Mexico (M), Costa Rica (CR), Honduras (H) and Guatemala (G) are underlined. 71

The seed analysis (Tab. 1) revealed a different behavior in this species in comparison with C. pepo, species for which clear relationships between seed and fruit traits has been previously reported (Paris and Nerson 2003; Picó et al. 2007). This lack of correlation between fruit and seed traits could be due to the fact that groups in C. moschata are no so well defined as in C. pepo, and accessions segregating for fruits traits and with fruits displaying a continuous variation intermediate between morphotypes are frequently found. Only some aspects deserve to be noted. The weight of the flattened and globular fruits (round, heart-shaped and oblong morphotypes) is significantly higher than that of the elongated fruits (dumbbell, pearshaped, butternut and crookneck). This higher fruit weight is associated to a trend to have thicker seeds. A similar trend is observed for seed width and seed length, being always the dumbbell fruits those with smaller seeds and the heart-shaped those with larger seeds. However, differences are not clear in most cases. Therefore, seed analysis is not so useful in C. moschata as in C. pepo for the management of germplasm collections. Figure 2. Fruits diversity in Spanish landraces of C. moschata. The different morphotypes (flattened, round, heart-shaped, oblong, cylindrical, pear-shaped, butternut, dumbbell, and crookneck) are shown. Table 1. Seed characteristics (in mm) of the Spanish landraces belonging to different horticultural types of C. moschata Morphotypes Flattened Round Heart-shaped Oblong Cylindrical Butternut Dumbbell Pear-shaped Crookneck Seed thickness 2.8 bc 2.8 c 3.7 d 2.8 c 2.6 abc 2.7 abc 2.5 ab 2.4 a 2.7 abc Seed Width 0.96 b 0.95b 1.05 c 0.93 b 0.92 b 0.96 b 0.79 a 0.83 a 0.93 b Seed Length 1.67 bc 1.65bc 1.91 d 1.67 bc 1.59 bc 1.69 c 1.38 a 1.55 b 1.63 bc SL/SW 1.82 bc 1.79 b 1.83 bc 1.69 a 1.68 a 1.76 ab 1.78 ab 1.9 c 1.77 ab Mean is indicated for each trait. Numbers in the same column followed by the same letter are not significantly different according to the Duncan test for means comparison 72

C. sativus All the accessions tested belonged to the Spanish type. All plants were monoecious, with indeterminate growth type, medium life size, green fruits at market maturity and rough texture skin. The most variable characteristics were those relative to the fruit: fruit predominant shape at stem and blossom ends, fruit shape, brightness, stripes color and intensity of skin texture (Fig. 3). With few exceptions male flowers appeared consistently in the first ten nodes of the main stem. Only in one accession (MUC50) female flowers appeared in 9 of the first ten nodes of the main stem. In the first secondary stem some accessions developed female flowers, but only one or two on the first ten nodes. Again in the accession MUC50, 9 nodes developed female flowers. This characteristic can be of interest, as earliness in female flowering is a desirable characteristic in cucumber. The number of female flowers per node varied from one to two or more, being more frequent only one. Figure 3. Fruits diversity in Spanish landraces of C. sativus for fruit shape and size, skin texture, stripes and spines.from left to right and top to bottom (VC94, VC3, ANC9, ANC168, MUC51, MUC52, CMC21, CMC38). 73

The first, second and third components of the PCA explained the 40.9 %, 27.2 % and 19.7 % of the total variation, respectively. This analysis conducted with the quantitative traits grouped the accessions on the right size of the graphic (Fig. 4), indicating a small variability for these characteristics in the accessions tested. Accessions with larger fruits were separated from the rest. However, the accessions from Castilla La Mancha concentrated more than the others in the graphic and were consistently smaller and shorter, with a mean value of 14.04 mm in fruit length and 292 g in fruit weight in comparison with 17.74 mm and 351 g mean values, respectively, for the rest of accessions. Figure 4. Results of the PCA with Spanish C. sativus accessions analyzed using quantitative fruit traits. The grouping according to the first, second and third component is shown (Red colour for accessions from Andalucia, blue from Valencia, green from Castilla La Mancha, violet from Murcia and black from other origins). The first and the second coordinates of the PCoA explained 20.3 % and 11.9 % of the observed variation. In spite of the low variability found for quantitative traits, the group of accessions tested were highly variable for other qualitative characteristics as shows the dispersion of the accessions along the first and second coordinate axes (Fig. 5). Again, the accessions from Castilla La Mancha grouped on the right of the graphic, showing a greater uniformity with smaller, round shape at blossom and stem end of the fruit, and smooth skin (Fig. 4, CMC21, CMC38). Contrarily, the fruits from Murcia and Valencia were generally larger, irregular in shape and more variable for the rest of characteristics. 74

ACKNOWLEDGEMENTS The authors would like to thank to E. Martínez, R. Arrufat and M. Lairón for their technical assistance. This paper has been partially funded by the INIA projects RF2004-00003-00-00 and RF2004-00006-C10-01. 0.50 CMC38 CMC2 CMC4 0.29 VC27 MUC48 MUC52 MUC55 CC9 VC85 AC12 ANC26 VC129 VC94 CMC33 VC190 VC189 ANC84 CMC24 VC91 CMC37 Dim-2 0.07 ANC17 ANC67 VC65 VC1 CMC16 CMC34 ANC171 CMC12 ANC159 ANC98 ANC97 MUC54 VC144 VC107 VC3 VC59 MUC53 AC15 CMC21 ASC1 VC49 ANC169-0.15 ANC165 ANC21 CAC1 ANC167 MUC49 CMC19 ANC95 ANC99 ANC83 CMC47 ANC166 ANC170-0.36 VC106 ANC168-0.47-0.25-0.03 0.19 0.41 Dim-1 Figure 5. Results of the PCoA with Spanish C. sativus accessions analyzed using qualitative fruit traits. The grouping according to the first and second coordinates is shown (Red colour for accessions from Andalucia, blue from Valencia, green from Castilla La Mancha, violet from Murcia and black from other origins). Literature cited Andres TC (2004) Diversity in tropical pumpkin (Cucurbita moschata): cultivar, origin and history. In Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding, (Lebeda A, Paris HS, eds), Palacky University, Olomouc (CZ) pp 113-118 Castetter E (1925) Horticultural groups of cucurbits. Proc Amer Soc Hort Sci 22: 338-340 Ferriol M, Picó B, Fernández de Córdova P, Nuez F (2004) Molecular diversity of a germplasm collection of squash (Cucurbita moschata) determined by SRAP and AFLP markers. Crop Sci 44: 653-664 75

Ferriol M, Picó B, Nuez F (2007) Genetic diversity of Cucurbita spp. in the Canary islands: a bridge between America and Europe. In Plant genetic resources of geographical and other islands. Conservation, evaluation and use for plant breeding. Cap 1. The importance of islands for the conservation of plant genetic resources. pp 25-32 Italy (ISBN 88-901771-3-6) Ferriol M, Picó B (2008) Pumpkin and Winter Squash. In Handbook of Plant Breeding.Volume 1. Vegetables I. (Prohens J, Nuez F, eds) Springer, Heidelberg (DE) pp 317-349 Jeffrey C (1980) A review of the Cucurbitaceae. Bot J Linnean Soc 81: 233-247 Kirkbride JH Jr (1993) Biosystematic Monograph of the Genus Cucumis (Cucurbitaceae). Parkway Publishers, Boone (NC, USA) Paris HS, Nerson H (2003) Seed dimensions in the subspecies and cultivar-groups of Cucurbita pepo. Genet Res Crop Evol 50: 615-625 Paris HS, Yonash N, Portnoy V, Mozes-Daube N, Tzuri G, Katzir N (2003) Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using AFLP, ISSR, and SSR markers. Theor Appl Genet 106: 971-978 Paris HS (2008) Summer Squash. Handbook of Plant Breeding.Volume 1. Vegetables I. (Prohens J, Nuez F, eds) Springer, Heidelberg (DE) pp 351-379 Picó B, Díez MJ, Nuez F (2007) A core collection of Cucurbita Spanish landraces. In 18th EUCARPIA Genetic Resources Section Meeting Session 1, (Hauptvogel P, Benediková D, Hauptvogel R, eds). Slovak Republic (ISBN 978-80-88872-63-4) p.78 Picó B, Sifres A, Esteras C, Nuez F (2007) Cucumis SSRs Markers Applied to the Study of the Genetic Diversity in the Cucurbita genus. Cucurbits Genetics Coop Rep 30 (in press) Wessel-Beaver L (2000) Evidence for the center of diversity of Cucurbita moschata in Colombia. Cucurbit Genet Coop Rep 23: 54-55 76