CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS¹

Abstract

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.

Nutrients, but not genetic diversity, affect Gracilaria chilensis (Rhodophyta) farming productivity and physiological responses

“…When the individuals were not mature, sex markers published for G. chilensis (Guillemin et al. ) were used to determine ploidy. DNA extraction and PCR amplification reactions were carried out following protocols described by Cohen et al.…”

“…() and Guillemin et al. (), respectively, and the amplification products were visualized in 1.5% agarose gel (w/v) after adding 2 μL of GelRed™ (Biotium, Fremont, CA, USA).…”

“…Direct observation under stereoscope microscope (Stemi DV4; Zeiss, Jane, Germany) was used for phase determination of all individuals presenting development of reproductive structures. When the individuals were not mature, sex markers published for G. chilensis (Guillemin et al 2012) were used to determine ploidy. DNA extraction and PCR amplification reactions were carried out following protocols described by Cohen et al (2004) and Guillemin et al (2012), respectively, and the amplification products were visualized in 1.5% agarose gel (w/v) after adding 2 lL of GelRed TM (Biotium, Fremont, CA, USA).…”

AFLPandSCARmarkers associated with the sex inGracilaria lemaneiformis(Rhodophyta)

“…RAPD and SCAR methods were used to screen sex‐linked molecular markers in the haploid–diploid Gracilaria chilensis (Guillemin et al. ). In G. lemaneiformis , ISSR (inter‐simple sequence repeat) markers and RAPD markers were used to differentiate sporophytes, male gametophytes, and female gametophytes (Xiang‐feng et al.…”

“…Sixty-nine RAPD (random amplified polymorphic DNA) primers were used to identify sex-linked markers in Gracilaria gracilis and two fragments were linked to sex determination (Martinez et al 1999). RAPD and SCAR methods were used to screen sex-linked molecular markers in the haploid-diploid Gracilaria chilensis (Guillemin et al 2012). In G. lemaneiformis, ISSR (inter-simple sequence repeat) markers and RAPD markers were used to differentiate sporophytes, male gametophytes, and female gametophytes (Xiang-feng et al 1998, Xue et al 2006).…”