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Background  Sexual development in  Flammulina velutipes  is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear.    Methods  To examine the function of the HD-a sublocus, genome sequences of a series of  F. velutipes  strains were analyzed and tested through series of amplification by specific primer sets. Furthermore, activity of di-allelic HD-a locus was confirmed by crossing strains with different combinations of HD-a and HD-b subloci.    Results  Sublocus HD-b contained a large variety of fixed Hd1/Hd2 gene pairs, while the HD-a sublocus either contained a conserved Hd2 gene or, a newly discovered Hd1 gene that was also conserved. Identification of whole HD loci, that is, the contents of HD-a and HD-b subloci in a strain, revealed that strains with similar HD-b subloci could still form normal dikaryons if the two genes at the HD-a sublocus differed. At least di-allelic HD-a sublocus, is thus indicated to be actively involved in mating type compatibility.    Conclusions  HD-a sublocus is active and di-allelic. Using the new information on the HD subloci, primers sets were developed that specifically amplify HD-a or HD-b subloci in the majority of  F. velutipes  strains. In this way, unknown HD mating types of  F. velutipes  can now be quickly identified, and HD mating type compatibility conferred by HD-a or HD-b can be confirmed by PCR.

A second HD mating type sublocus of Flammulina velutipes is at least di-allelic and active: new primers for identification of HD-a and HD-b subloci