SSR markers for the genus Cucurbita

In a previous FWF project (No. P15773) we have constructed the first consensus map for Cucurbita pepo, using mainly RAPD and AFLP markers (Zraidi et al. 2006), and have produced the first 22 Cucurbita-specific SSR markers. Inspired by the results of this project and by the needs of our ongoing Cucurbita improvement program, the present proposal pursues three main goals: (1) About 200 polymorphic SSR markers for the genus Cucurbita will be isolated from partial genomic libraries, in approximately equal numbers from C. pepo and C. moschata. Successful completion of this objective will lead to the establishment of markers linked to agronomically important traits, especially to quantitative trait loci (QTL), for marker assisted selection (MAS). Further improvement of understanding the phylogenetic relationship within the genus may result. (2) Two genetic maps will be created using three C. moschata genotypes. After that, those markers, which could be mapped in C. moschata and show polymorphism between the parents of a C. pepo mapping population created in the previous project, will be mapped in this C. pepo population. Genetic maps are primarily used to discover the position of genes in relation to markers, especially of QTLs controlling important traits. Comparing the C. moschata and C. pepo map will reveal synteny between the genomes of the two species. SSR markers may disclose the assumed polyploid nature of the genus. (3) Polymorphism Information content (PIC) of the SSR markers will be estimated, using two sets of selected Cucurbita genotypes. Markers with high PIC values will primarily be valuable to assess germplasm for improved germplasm management, e.g. for a hybrid breeding program. Furthermore PIC values will allow the Cucurbita community to choose the appropriate markers for specific applications.

SSR or `simple sequence repeat` markers are short stretches of genomic DNA, composed of tandemly repeated short motives of one to six nucleotides bordered on both ends by unique sequences. They are large in number and occur in both repetitive and non-repetitive sequences in a fairly even distribution. The two primers, synthesized to the border sequences, allow their amplification, making them easy to locate in genome, acting as physical pin marks. Due to a relatively frequent error during replication (slippage), the number of motive repetition may change, creating alleles in such an SSR locus. These properties make an SSR locus an ideal, easy to handle, multipurpose marker with many applications, both in practical breeding, as well as scientific investigations, their only disadvantage being the high costs of finding them. Nevertheless, hundreds, in some species like maize several thousands, of SSR markers have been developed and used for relationship studies, for genotype identification, for constructing marker maps, for map based cloning of genes. Linking them to agronomic traits makes marker assisted selection possible. No SSR markers existed so far for the genus Cucurbita, a situation this project aimed to change. Using one genotype of C. pepo and one of C. moschata we isolated from genomic DNA some 560 functional SSR markers. With these markers we then produced marker maps for both species, which revealed the close genetic relationship of both. We made a relationship study of C. pepo, the most important cultivated species of the genus, using more than 100 accessions, including mostly cultivars, but also some wild and ornamental accessions. This study sheds light onto the origin, history, and worldwide distribution of the species. Due to the high transferability of SSR markers between Cucurbita species, we have done a second relationship study, including eight wild and cultivated species represented by a varying number of accessions. This study is still in the stage of statistical evaluation. Finally, we ventured into the field of genome analysis of Cucurbita based on sequencing genomic DNA of C. pepo. We received 2,350,074 sequenced fragments (reads) with an average fragment length of 330 bp, meaning in total 774,441,382 bp, covering ca. 1.5x the total pumpkin genome. These sequences will be analyzed with tools of bioinformatics, comparing the reads with sequence information of other plants already available in international databases.