The cucumber (
Cucumber (
Spanish cucumber accessions are mainly held at the ‘Banco de Hortícolas de Zaragoza’, BGHZ (Vegetables Genebank, Zaragoza, Spain) and at the Genebank of the ‘Instituto de Conservación y Mejora de la Agrodiversidad Valenciana’, COMAV (Institute for Conservation and Improvement of Agrodiversity, Valencia, Spain). All these accessions are landraces, collected from local farms. A morphological characterization of the collection maintained at the COMAV has been recently reported, including 195 Spanish accessions held in this genebank (
Initial molecular diversity studies in cucumber were carried out using isozyme loci, and included a high number of accessions from the NPGS (
Given the availability of high-density molecular markers, the key point in diversity studies is the sampling of the genetic variability. In this sense,
Efficiency of the management of genebanks can be increased with rationalization of their collections (
In this study, the molecular characterization of the cucumber collection held at the COMAV has been carried out. The final objective is to obtain information to rationalize the collection. The 23 highly polymorphic SSRs described by
Young, healthy, and fully-expanded leaves from the 627 plants representing 131 Spanish accessions as well as leaves belonging to three foreign accessions, were analyzed (Table S1 [suppl.]). Four or five plants per accession were used. This number was considered enough as populations in cucumber (and other cucurbits) are generally small, not only at farms but also in wild populations, favoring endogamy. The Spanish accessions included in the study were all landraces, collected from local farms. Thus, these accessions derive from a low number of plants, since local farmers usually grow small populations for self-consumption.
One hundred and twenty-seven Spanish accessions come from the COMAV and the other four accessions were provided by the BGHZ. Some of the accessions conserved at the COMAV genebank were collected by the holder institution and some others were collected by the BGHZ and maintained as duplicates in the COMAV. All these landraces are routinely reproduced using 10 plants per accession. Considering the origin of the Spanish accessions, 31 accessions came from Andalusia, 26 from Valencian Community, 21 from Castilla-La Mancha, 16 from Aragon, nine from Castilla-Leon, seven from Extremadura, six from Murcia, four from Canary Islands and three from the Basque Country, two accessions from Cantabria, Catalonia and Navarra, and one accession from Galicia and Asturias. Besides, three accessions provided by the Center for Genetic Resources (CGN, The Netherlands) and by the Chinese Academy of Agricultural Science (CAAS, China) were used as outgroups in the analysis. The phenotypic characterization of the complete collection of cucumber hold at the genebank of the COMAV has been recently conducted (
DNA was extracted using the protocol proposed by
Polymorphic SSR markers were used to analyze the genetic diversity of 131 Spanish cultivars and the three foreign accessions considered as outgroups. The number of alleles (Na), the number of genotypes (Ge) and the effective number of alleles (Ne) was determined for each SSR locus in the Spanish collection using the PowerMaker software (
Pearson’s correlation coefficients were computed between the number of alleles per locus found by
A total of 627 plants belonging to 131 accessions were analyzed using 23 SSRs. Two of them, SSR02895 and SSR07543, yielded amplification in less than 50% of the plants. SSR20852 and SSR23370 were monomorphic in all samples analyzed, while SSR16068 was monomorphic except for two plants of outgroup R41, homozygotes for the alternative allele. These five markers were not included in subsequent analysis.
The remaining 18 polymorphic SSR markers generated a total of 58 alleles in the Spanish collection (
Polymorphism levels and mutation rate were correlated with the number of repeat units, with different results depending on the species. A positive correlation was found in grapevine (
Differences were also found depending on the study when comparing the relative frequencies of the alleles. In our study, among the 58 alleles found in the Spanish collection, 34 (58%) were ‘common’ alleles, as their frequency was higher than 5% (
Accession-specific alleles were found for SSR19998, SSR22653 and SSR31399. SSR19998 allele 339 bp was present in two of the plants of accession Sty17, in homozygosis in one of them and in heterozygote state for the other one. Allele 401 bp for SSR22653 was specific for three plants of accession Lti113, in homozygosis in one of them and combined with allele 411 bp in the other two. SSR31399 allele 202 bp was the only ‘very rare’ allele of the collection. As previously stated, it was only present in heterozygote state in one plant, which belonged to accession Sty157. Moreover, some alleles appeared either exclusively or almost exclusively in some cucumber types. SSR16695 allele 172 bp and SSR10018 allele 137 bp were only present in accessions belonging to the short type. SSR10018 allele 147 bp, SSR19998 allele 344 bp and SSR22653 allele 409 bp were mainly present in short type accessions. On the other hand, some of the ‘rare’ alleles and the ‘very rare’ alleles were in many cases specific of concrete geographical origins. As an example, allele 143 bp of SSR16056 was only identified in accessions from Andalusia or SSR14861 allele 382 bp was exclusive of accessions from Andalusia or Castilla-La Mancha. All these results must be considered when using this information to rationalize management of the collection.
Most of the possible genotypes for each of the SSRs were identified in the Spanish collection, concretely, 105 out of the 133 genotypes (
The values of H
0 ranged from 0.00 (SSR05723) to 0.22 (SSR16056), with a mean of 0.07. All individuals analyzed in the present study presented the SSR05723 in homozygosis, corresponding to the lack of heterozygosity observed for this marker. The highest value corresponded to SSR16056, the SSR with the highest number of alleles identified in our collection, two of them ‘common’ (with similar frequency), other two ‘less common’ and only the other two with frequencies lower than 1%. Results for this SSR were similar for the three groups that include more than one accession,
The values of H
e ranged from 0.04 (SSR19998) to 0.58 (SSR16056), with a mean of 0.261. These values were lower than those obtained by
Taking into account the classification performed by
The AMOVA of the distance matrix for all the analyzed plants permitted a partitioning of the overall variation into two levels. The proportion of variation attributable to within-accessions differences was high, 45%, whereas 55% occurred among accessions. The intra and inter-accession variability’s distribution has implications for the management of conserved accessions when regeneration has to be done in the genebank. When similar intra and inter-accessions variability is found, the number of plants to be regenerated must be high, in order not to lose the variability among plants of the same accession and to avoid the influence of genetic drift and selection, resulting in changes in genetic composition. The effective population size and the methods of pollination and harvesting seeds have to be also taken into account. However, when variability is higher among accessions, the effort must be devoted to increase the number of regenerated accessions.
The mean similarity coefficient of the 131 cucumber accessions was 0.14, ranging for 0.01 (between Ltp8 and Ltp48) to 0.44 (between Shs101 and Fty183). Diversity in Spanish cucumber is lower than that found by
None of the accessions showed the same genotype for all the analyzed plants. Sty122 accession showed the highest number of plants sharing the same genotype, four plants. The other plant showed three different alleles. Shs101 and Sty116 accessions had three plants with the same genotypes, and the other plants showed different genotype inter and intra-accessions. Lti47, Lhs121 and Stp144 accessions had two groups of two plants with the same genotype differing among genotypes in two or three SSRs, depending on the accessions. A total of 12 accessions (Ltp8, Stp89, Lti131, Ltp147, Sty165, Sty180, Sel79, Sty146, Stp56, Sty94, Stp59 and Stp62) showed two plants with the same genotype. The rest of the Spanish accessions showed plants with a unique genotype within accession.
Discrimination between closely related individuals has been reported using SSRs, even when few loci were employed (
The clustering pattern of cucumber genotypes based on SSR markers was not in consonance with the groupings based on morphological traits or geographical area either including (
Dendrogram for the specific main groups (‘Short’, ‘Long’ and ‘French’) were performed, and a lack of agreement among subgroups were also found. Similar results were found when the geographical origin was taken into account (data not shown). Although environmental conditions and market orientation in a specific geographical area may select a specific morphological appearance, which can explain the high similarity among accessions from the same area, several accessions collected in different geographical areas could have at least some common genetic bases as a consequence of gene flow.
Given the low molecular diversity among the Spanish cucumber accessions found in the present study, a combined strategy was followed to perform the rationalization of the collection, including phenotypic traits, origin and molecular data. According to the groups established using morphological traits (
According to the results obtained, the use of the stratified method, first constructing groups based on phenotypic traits, maximizing the origin and uniqueness of the accessions, and checking then the genetic distances among the groups and subgroups established, seem to be useful for rationalizing the collections of this crop. The rationalization of the collection done will be useful for optimizing the management of the cucumber collection conserved at the COMAV genebank.
The authors would like to thank Patricia Muñoz, Ángel Rodríguez and Sebastian Zahn for their technical assistance.