Analysis On The Genetic Variation Of Mariculture Populations And Preliminary Localization Of Lucus Of Gametophyte Sex Trait In Laminaria

Laminaria has been cultivated in China for a long time.Currently,the culturingarea of Laminaria has reached more than 40 thousands hectares,yielding more than0.84 million tons of air-dried Laminaria each year.With the fast development ofLaminaria use in chemical industry and the implementation of Laminaria processingfacilities,yield-oriented Laminaria cultivation is evolving into a cultivation industrywith diverse purposes.However,Laminaria cultivation industry has to face thedisadvantages such as limited varieties,depletion of culturing performance,heavyheterozygosity and weak stress tolerance.In addition,global warming has caused thegradual increase of surface seawater of Laminaria culturing area and strengthened thefluctuation of seawater temperature.This has caused less tolerant Laminaria varietiesin use to lose heavily blade tissue and yield.The limitation in both labor andair-drying ground also need to develop varieties with different maturing dates.It is clear that the genetic resource of Laminaria will facilitate the solution ofthese problems.However,the genetic resource available must be effectively evaluated,maintained and accordingly exploited.An appropriate set of molecular markers willhelp us to analyze the genetic diversity of the collected and natural Laminaria geneticresource.The basic research of Laminaria is far behind the breeding and cultivation.Thebiological study of Laminaria (e.g.,taxonomy,life history,gametophyte cloning) hassignificantly contributed to the development of Laminaria cultivation industry ofChina.In contrast,the research of the genetic basis of important economic and stress resistant characteristics of both Laminaria gametophyte and sporophyte is scarce.Themajor reason for this is the unavailability of molecular tools (e.g.,the linkage map ofmolecular markers).It was found in Laminaria breeding that the female gametophyte clone carriesmore control genes of the desirable characteristics of the sporophyte from which theclone is isolated,and accordingly contributing unequally to the performance of thehybrid.Therefore,it is very important to decipher the determining mechanism ofgametophyte sex for both biology and breeding practice of Laminaria.The following studies were carried out:(1) tentatively develop SSR markers anduse these markers to analyze the genetic diversity of collected gametophyte clones;and (2) tentatively construct an AFLP-SSR linkage map and locate gametophyte sexcharacterictics,providing the basis of isolating gametophyte sex determining gene(s).Eighteen polymorphic microsatellite DNA markers were developed and used todetermine the diversity of 90 gametophyte clones isolated from introduced L.japonica and L.longissima of China,the varieties of L.japonica and the varietiesderived from interspecific hybrids (female gametophyte clone of L.longissima x malegarnetophyte clone of L.japonica),respectively.In total sample,the number of allelesper locus ranged from 2 to 7 and the Nei's gene diversity ranged from 0.369 to 0.753.The allelic diversity and Nei's gene diversity of L.japonicawere were significantlyhigher than those of L.longissima (2.9 vs.1.8 and 0.414 vs.0.161),respectively.Bothallelic diversity and Nei's gene diversity of the varieties derived from interspecifichybrids were lower than those of L.japonica,indicating that only a portion ofvariation of L.japonica were incorporated into the varieties of L.japonica.Significant genetic differentiation was detected between 4 groups of materials andbetween female and male gametophyte clones each group.The variation amonggroups accounted for 39.95%,while that among populations accounted for 21.65% ofthe total.The genetic distances between L.japonica and L.longissima and between L. lonngissima and the varieties of L.japonica reached 1.142 and 1.036,respectively,while that between L.japonica and the varieties of L.japonica was only 0.278.Thegenetic distance between L.japonica and hybrids of two species was obviously longerthan that between L.lonngissima and the hybrids (0.686 vs.0.291),indicating thatmaternal gametophyte clone contributed more variation to the hybrids than thepaternal gametophyte clone did.In addition,recent population size reduction(bottleneck effect) was detected in all 4 groups of materials,reflecting the influence ofspecies introduction and variety breeding on genetic diversity.These observations willfacilitate the conservation and exploitation of Laminaria gametophyte clones.A tentative AFLP-SSR linkage map of Laminaria was constructed using ahaploid population of 40 gametophyte clones isolated from an individual of DongfangNo.2,the first commercially cultured hybrid of a female gametophyte clone of L.japonica Areschoug and a male one of L.longissima Miyabe.To the map,263markers (255 AFLP,7 SSR and the gametophyte sex) were assigned.The map isconsisted of 25 LGs with≥4 markers,5 triplets and 15 doublets,which is 1629.0cMin length,covering 66% of Laminaria genome.The maximum space between loci is24.63cM.A putative sex-determining region was identified in LG₂,which wascharacterized by a dense marker distribution around gametophyte sex locus.Thelinkage map itself and the methodology associating with its construction will facilitatethe genetic study and the further trials of the linkage map construction of Laminaria.A female gametophyte clone of L.japonica and a male gametophyte clone of L.longissima were hybridized,generating Dongfang No.2 hybrid Laminaria.Theheterozygosity of this hybrid Laminaria was determined using SNP of the ITS regionof ribosomal RNA transcription unit and microsatellite DNA variation at two loci.Dongfang No.2 held both'T'and'C'at position 847 of the ITS region,while'T'atthis position was specific for L.japonica and'C'for L.longissima,respectively.Dongfang No.2 also held the microsatellite DNA alleles of two parents together attwo microsatellite DNA marker loci.These observations clearly proved that Dongfang No.2 is a true hybrid of L.japonica and L.longissiuma.