Isolation And Application Of Microsatellite Markers For Turbot (Scophthalmus Maximus L.)

A microsatellite-enriched genomic DNA library of turbot (Scophthalmus maximus)was generated by the FIASCO (Fast Isolation by AFLP of Sequences ContainingRepeats) method. Firstly, using MseI enzyme and PCR amplification, establish smallfragments genomic library. Secondly, using the single nucletide probes with biotin sign,(CA)12，(AC)17，(AG)17，(AT)17，(AAT)10，(AAC)10，(GACA)10，(GATA)10with tandemrepeat sequence hybridization, constructs the microsatellite-enriched genomic DNAlibrary of turbot. Thirdly, being connected with the carrier, transformed into competentcells by the ampicillin-containing LB medium, to screen2027positive colonies.Fourthly, after three sets of PCR reaction, secondary screen655cloned, enrichmentefficiency is32.3%. Of the597sequenced clones,76clones (12.73%) are lack of clearmicrosatellite sequences, and65cloned sequence (10.89%) for the same by theanalysis. So this experiment received containing469microsatellite loci of a singlesequence, and the efficiency of the FIASCO method enrichment is78.56%.The screening of microsatellite sequences were signature analyzed. Of the469sequenced clones,597microsatellites DNA were identifidaad. Among them,309were perfect (51.76%),207were imperfect (34.67%) and81were compound(13.57%). The core sequence repetitions of micro-satellite DNA Obtained in therange of3to96times, the average number of repetitions is13.39times. And therepetitions is mainly concentrated in the3to19times, accounting for78.02%. Theselected turbot microsatellite DNA focuses on dinucleotide repeat type,accounting for85.25%. The central tapes of microsatellite DNA are (CA/GT)ntypes and (AG/CT)ntypes, accounting for77.6%and11.50%separately. Thelength of microsatellite sequence is mainly concentrated in150bp and349bp,accounting for81.3%. In addition, there is a clone containing repeating units ofeight nucleotide (AGACGGAC)ntandem repeats, and the other two clonescontaining (AC)6(GC(AC)3)6and (TG)14(AGCGCA(TG)5)4repeat sequences. The microsatellite primers of turbot were to develop.469microsatellite lociflanking sequences had been analyzed, and384sites (81.88%) had the rightflanking sequence for primer development.413pairs of primers were designed,and360pairs of primers can get stable expression product. By microsatellitemarkers primary screening test,168pairs of primers (46.7%) had a singleexpression product, and183pairs of primers (50.8%) were represented forpolymorphism,110microsatellite loci (30.6%) of which were conformed to themapping standards of the genetic linkage map.To obtain the mapping marker of building genetic linkage map of turbot,turbot microsatellite markers screening experiments were performed. Thepreliminary screening of748pairs of microsatellite primers (360pairs of primersfrom this experiment and388pairs of primers from literature published) will beused for mapping the family of two parents and six individuals on behalf of Fl.There are721pairs of primers being obtained clear amplification products,254pairs of which were epresented for a single purpose strip products, and467pairswere represented for polymorphism,294microsatellite loci of which wereconformed to the mapping standards of the genetic linkage map.The microsatellite markers were represented for the separation mode in F1generation of the mapping pedigrees.120pairs of microsatellite selectingrandomly from the294microsatellite loci to study the separation of two parents ofthe mapping pedigrees and92individuals of F1generation,115pairs of primersof which were able to obtain clear amplification product. Obtaining five separationmode in all：ab×cd、ef×eg、hk×hk、lm×ll、nn×np. The separation of each markgot2to4allele, a total of254alleles, and the average number of alleles was2.21.And2microsatellite markers (FF0919, Sam-USC138) are abnormal genotypeseparation. The remaining113microsatellite markers mean expectedheterozygosity was0.57, and the mean observed heterozygosity was0.55. Throughthe chi-square test,16markers (14.2%) are deviated from Mendelian separationseriously (P <0.01), and87microsatellite markers (77.0%) are in accordancewith Mendelian law (P>0.05). And the latter could be used to construct geneticlinkage map of turbot.