鳙鱼微卫星分子标记的筛选

Isolation of microsatellite markers in bighead carp Aristichthys nobilis

采用常规方法从鳙鱼(Aristichthysnobilis)血液中提取基因组DNA,经限制性内切酶Sau3AI酶切后,选取250～750bp大小的片段构建鳙鱼基因组文库。采用人工合成的重复序列(CA)15、(AG)12、(AAG)8用同位素标记作为探针,通过原位杂交筛选基因组文库,获得鳙鱼的微卫星序列。进一步用引物设计软件PrimerPremier5 0设计引物。通过杂交获得99个阳性克隆,经测序筛选出82个微卫星序列,设计引物65对。

The bighead carp Aristichthys nobilis play an important role in balancing the freshwater ecology by filtering the animal planktons.In recent decades,however,degeneration occurred in the good breed from the farms due to the polluted environment and the bad management,meanwhile,the same saturation occurred in the wild populations.In order to protect this precious species,many related researches involved in biochemistry,genetics and development were implemented by using a lot of biological techniques.Microsatellites,as a kind of molecular markers,have many advantages in construction of genetic linkage map,QTLs analysis,kinship identification and so on.In this experiment,genomic DNA of bighead was extracted from the blood by normal methods.After the genomic DNA was digested by the restriction enzyme (Sau3AI),we selected and purified 2500-750bp fragments.A genomic DNA pool of bighead carp was constructed by adopting the conventional method.Three isotopic probes of (CA)_15,(AG)_12 and (AAG)_8 were used to screen the pool to identify the positive clones contained the microsatellite sequences.As a result,99 positive clones were found out and sequenced,which truly are of 75 clones containing 82 different microsatellite loci.Then,using the software package Primer Premier 5.0,we designed 65 pairs of primers to test the polymorphism in bighead carp.