Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/6/2004
Publication Date: 1/1/2005
Citation: Lewers, K.S., Bassil, N.V., Styan, S.M., Hokanson, S.C. 2005. Developing simple sequence repeat (ssr) markers from genbank sequences for strawberry, blackberry, and raspberry. Journal of American Society for Horticultural Science. 130(1):102-115.
Interpretive Summary: The genetic research tools available to a plant researcher vary significantly depending on the species studied. Some plant researchers have an enormous amount of information available from which they struggle to extract meaning and value. Many more struggle to study and manipulate valuable traits with no genetic tools or are just beginning to develop them. Our objectives were to develop genetic tools for strawberry, blackberry, and raspberry breeding and compare the results to determine which methods were most successful. In addition to the conventional technique, two Internet based data search methods were used to identify useful DNA sequences from an international database, and the resulting genetic tools were tested for efficacy with several strawberry, blackberry, and raspberry plants. The genetic tools developed from both Internet methods were as useful as those developed using the conventional technique. One of the Internet methods was significantly better than the other at searching the database. Genetic tools developed from the database's strawberry information were somewhat useful with blackberry and raspberry plants in addition to strawberry plants, and the reciprocal was also true. However, genetic tools developed from information from more distantly related plants were not useful with strawberry, blackberry, or raspberry. This information will be useful to researchers developing genetic tools for minor crops and should substantiate the need for obtaining information from those crops rather than trying to depend on model plants for which large amounts of information have been obtained.
Technical Abstract: Researchers working with many fruit and vegetable crops manipulate important traits through traditional breeding and without the aid of molecular markers. Simple sequence repeat (SSR) molecular markers are desirable but development from genomic clones is time-consuming and expensive. Some researchers have tried to reduce costs by using SSR markers developed from closely and even distantly related species. Others have designed SSR primers from genomic and expressed sequence tag (EST) sequences archived in GenBank. Our objectives were to use these multiple approaches to develop SSRs for two closely related target genera, strawberry (Fragaria L.) and brambles (Rubus L.), including blackberry (Rubus hybrid), red raspberry (Rubus idaeus L.) and black raspberry (Rubus occidentalis L.), and to evaluate the results, especially to determine if sequences from congeners, related genera, or related family members could be used for SSR development. Two different methods, BLAST- and SSRIT- based, were used to identify SSR-containing sequences in GenBank in addition to developing SSRs from genomic clones. The resulting polymerase chain reaction (PCR) primers were used to amplify genomic DNA of 31 genotypes of strawberry, blackberry and raspberry to evaluate their ability to amplify products and detect polymorphisms. Our results indicate that use of SSRs across genera within the Rosaceae family is possible but limited. SSRs derived from P. persica did not amplify a product in Fragaria or Rubus, and amplified only P. persica DNA. Eight SSRs designed from Rubus alceifolius Poir. were not useful with Fragaria and were no more useful with blackberry or raspberry than were Fragaria-derived SSRs. Approximately 19% to 27% of Fragaria-derived SSRs amplified a product from Rubus template, and an average of 40% of these detected polymorphisms. SSRs designed from any Fragaria could be used equally well with any other Fragaria with about 80% polymorphism detected among tested genotypes. SSRIT was superior to BLAST for identifying GenBank sequences containing repeats. SSRs developed from repeats found in either the 5'UTR (80% polymorphic) or 3'UTR (85% polymorphic) were most likely to detect polymorphisms, compared with those developed from coding regions (30%) or introns (33%). SSRs developed from genomic clones were only slightly better able to detect polymorphisms than SSRs from GenBank sequences; this may be partly due to repeat size. Use of GenBank to develop SSRs is, however, limited by the number of archived sequences from a crop species of interest. This limitation can be prohibitive if large numbers of SSRs are required for genetic mapping of a trait of interest.