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Background  The green alga  Chlamydomonas reinhardtii , although a premier model organism in biology, still lacks extensive insertion mutant libraries with well-identified Flanking Sequence Tags (FSTs). Rapid and efficient methods are needed for FST retrieval.    Results  Here, we present a novel method to identify FSTs in insertional mutants of  Chlamydomonas . Transformants can be obtained with a resistance cassette lacking a 3’ untranslated region (UTR), suggesting that the RNA that is produced from the resistance marker terminates in the flanking genome when it encounters a cleavage/polyadenylation signal. We have used a robust 3’-RACE method to specifically amplify such chimeric cDNAs. Out of 38 randomly chosen transformants, 27 (71%) yielded valid FSTs, of which 23 could be unambiguously mapped to the genome. Eighteen of the mutants lie within a predicted gene. All but two of the intragenic insertions occur in the sense orientation with respect to transcription, suggesting a bias against situations of convergent transcription. Among the 14 insertion sites tested by genomic PCR, 12 could be confirmed. Among these are insertions in genes coding for  PSBS3  (possibly involved in non-photochemical quenching), the NimA-related protein kinase  CNK2 , the mono-dehydroascorbate reductase  MDAR1 , the phosphoglycerate mutase  PGM5  etc..    Conclusion  We propose that our 3’-RACE FST method can be used to build large scale FST libraries in  Chlamydomonas  and other transformable organisms.

A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE