| Laminaria* has the longest history of artificial cultivation and the highest yield among macroalgae cultured in China. Laminaria japonica was introduced into China for cultivation in 1927, and L. longissima and other species later. China has achieved richly in cultivation method, seedling raising technique and breeding of Laminaria, however, the genetic studies (e.g., evaluation of genetic diversity and determination of genetic basis of economic traits of Laminaria) were far behind the cultivation of Laminaria. An appropriate molecular marker linkage map (markers are transferable and dense enough) will certainly facilitate the genetic study of Laminaria. Unfortunately, such a map was not available currently.Dongfang No.2 is the first interspecific hybrid Laminaria commercially cultivated in the world, which was raised through crossing a female gametophyte clone of L. japonica with a male one of L. longissima. The hybrid holds high heterozygous vigor (heterosis) in yield and yield related traits. Whether Dongfang No. 2 is a true interspecific hybrid remained an attention from both scientific community and farmers. The parentage of Dongfang No. 2 hybrid Laminaria should be determined.A group of microsatellite DNA markers were developed and used to determine the genetic diversity of gametophyte clones isolated from L. japonica and L. longissima and construct a linkage map of Laminaria in combination with amplified fragment length polymorphism markers (AFLP). In addition, AFLP markers, single nucleotide polymorphism (SNP) of internal transcribed spacers (ITS) region and two microsatellite DNA markers were used to determine the parentage of Dongfang No.2 hybrid Laminaria.The following are the major findings:A (AC)n microsatellite DNA containing fragment enriched library was constructed for L. japonica using FIASCO method. One hundred and thirty eight microsatellite DNA containing fragments were identified and sequenced, from them, 65 pairs of primers were designed. Of these pairs of primers (hereafter, microsatellite DNA markers), 22 amplified products from the genomic DNA of the gametophyte clones and young sporophytes of Laminaria.In 36 Laminaria gametophyte clones, the number of alleles each locus detected by these microsatellite DNA markers ranged from 2 to 7, and the gene diversity and Shannon's information index from 0.21 to 0.75 and from 0.37 to 1.55, respectively. The average number of alleles (1.5 vs. 3.3), average gene diversity (0.12 vs 0.45) and average Shannon's information index (0.20 vs 0.81) of L. japonica and L. longissima were significantly different. The frequencies of the majority of the two most abundant alleles of two species were also different significantly. The genetic differentiation between two species reached 35.6% (gene diversity) and 36.9% (Shannon's information index) in average.Two linkage maps of microsatellite DNA and AFLP markers were constructed using 40 gametophyte clones randomly isolated from a sporophyte of the hybrid of a male gametophyte clone of L. japonica and a female one of L. longissima. One was done using the marker alleles specific for the male gametophyte clone parent (coupling each other) of the hybrid, and the other using those specific for the female gametophyte clone parent. Actually, a single linkage map can be constructed with microsatellite DNA markers. Two maps were constructed; the recessive AFLP alleles corresponding to their dominants were not traceable.The map constructed with the female gametophyte clone specific marker alleles consisted of 100 markers(94 AFLP and 6 microsatellite DNA)arranged into 27 linkage groups. The map length was 678.4cM, covering 56.4% of the estimated genome size. The map constructed with the male gametophyte clone specific marker alleles consisted of 121 markers(115 AFLP and 6 microsatellite DNA) assigned into 30 linkage groups, which was 745.7cM in length,covering 53.6% of the estimated genome size. We are currently trying to develop a microsatellite DNA marker linkage map at the appropriate density.Dongfang No. 2 hybrid Laminaria shared 70 and 55 bands with L. japonica and L. longissima, respectively, obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No. 2 held both"T"and"C"at position 847 of the ITS region, while"T"at this position was specific for L. japonica and"C"for L. longissima, respectively. In addition, Dongfang No. 2 also held the alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No. 2 is a true hybrid of L. japonica and L. longissima. Unfortunately, the origin of its chloroplast was not determined based on the variation of RuBisCo spacer.
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