CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID-DIPLOID RED ALGA GRACILARIA CHILENSIS(1).

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex-linked molecular markers in the haploid-diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male-specific bands; and one RAPD primer (OPD12), a female-specific band. The sequences of the cloned putative sex-specific PCR fragments were used to design specific primers for the female marker SCAR-D12-386 and the male marker SCAR-G16-486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex-specific association, we were able to amplify SCAR-D12-386 and SCAR-G16-486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%-9% nucleotide divergence for SCAR-D12-386 and SCAR-G16-486, respectively. SCAR-D12-386 and SCAR-G16-486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.