Sex identification and genetic variation of Saccharina (Phaeophyta) gametophytes as revealed by inter-simple sequence repeat (ISSR) markers

Inter-simple sequence repeat (ISSR) polymerase chain reaction (PCR) markers were utilized to investigate the genetic variation between male and female gametophyte populations of strains Rongfu and 901 of Saccharina. In total, 11 ISSR primers were able to generate 135 satisfactory and reproducible loci, of which 134 were polymorphic with 99.26 % polymorphism. The percentages of polymorphism of female gametophyte populations (60 and 62 % for their respective strains) were higher than those of the males (53 %), and the Nei's genetic diversity and Shannon's information index showed a similar tendency. The clustering of gametophytes of the same sex from each strain was well resolved by both an unweighted paired group method using the arithmetic average and a principal component analysis, suggesting that any male/female gametophyte pair could represent each strain. However, a single pair was not adequate for germplasm maintenance because the genetic variance among individuals within a population accounted for 57.45 % of the total (P < 0.0001), as shown by the analysis of molecular variance. The gametophyte sex could be identified by amplification with primer UBC809 because of a differential band present in the females. According to the sequence of this band, a pair of ISSR-derived sequence-characterized amplified region (SCAR) primers was designed. With the primers, one female-specific fragment was detected using PCR and Southern blot hybridization. This converted SCAR marker was localized on one unique chromosome of the female gametophytes of these two strains by use of fluorescence in situ hybridization, confirming that it was a female chromosome-specific marker.