Plant & Animal Genome VI Conference

P61

USE OF DATABASE SEQUENCES TO RAPIDLY DEVELOP SIMPLE SEQUENCE REPEATS (SSRs) MARKERS ON RAPESEED (Brassica napus L)

Cargill Genetic Europe, B.P. 17, F-28313 TOURY FRANCE

Microsatellites or simple sequence repeats (SSRs) are composed of di-, tri- or tetra-tandemly-repeated nucleotides. SSRs are an excellent source of molecular markers because they are highly polymorphic (due to the difference in repeats number) and abundant in the DNA of most eucaryotic organisms. Development of microsatellite markers requires a prior characterization of the SSRs. Normally, SSRs are identified and sequenced from small inserts of genomic DNA libraries and oligonucleotide primers are designed in the flanking regions of the repeats (SSRs) for PCR amplification. In our attempt to develop microsatellite markers on rapeseed (Brassica napus L), we have used sequence information available in the databases to search for repeated sequences and identify putative SSRs in 980 sequences from different Brassica species and Arabidopsis. Flanking primers for a total of 102 putative SSRs (34 from Brassica species and 68 from Arabidopsis) were designed and tested on 16 European rapeseed cultivars. For the 34 SSRs from different sequences of the Brassica species, 23 were successfully amplified and 7 of them were polymorphic among the 16 rapeseed cultivars. With a first set of 34 SSRs from Arabidopsis sequences, only 8 were successfully amplified in the 16 rapeseed cultivars and 3 of them were polymorphic. Careful observations show that all of the 8 successfully amplified SSRs were identified in the coding regions of Arabidopsis sequences. In a second experiment, flanking primers for 34 new SSRs, this time designed in the coding regions of Arabidopsis sequences, were tested on the 16 rapeseed cultivars. This leads to successful amplification of 17 SSRs and 6 of them were polymorphic. This study shows that it is possible to rapidly develop microsatelite markers in related plant species and genera by using sequence information available in the databases. We are now using this strategy to identify more microsatelite markers for our breeding and mapping programs.