Screening Of Cold Tolerance Markers Which Based On DArT Technology Of Larimichthys Crocea

Large yellow croaker (Larimichthys crocea) belongs to Osteichthyes, Perciformes, Sciaenidae,Pseudosciaena, which also known as fish, big, fresh, dragon, red mouth, etc. Lots of large yellow croaker fromFujian were frozen to death at8oC every year, since it was breeded in Ningbo introduced breeding from Fujian in1997. After selectedly breeding, we obtained the new breed, called “Dong hai NO.1” which is cold tolerant. Weidentified markers associated with cold tolerance by Diversity Arrays Technology (DArT), which providestheoretical basis for breeding of “Dong hai NO.1”. The results are as follows:Following low temperature screening test, we got7fish death over9oC and7fishes still healthy survivalunder6oC, and use them as experimental material.Mixing equal amounts of genomic DNA of sensitive and resistance, we selected seven kinds method (PstI/Alu I, Pst I/Ban II, Pst I/Bsp1286I, Pst I/BstN I, Pst I/Hae III, Pst I/Rsa I, Pst I/Taq I) to reduce genomic DNAcomplexity. We constructed DArT library respectively, and each library size was larger than105. The averagelength of insert fragment was500bp.We made7chips using probe amplified from monoclonal in from7DArT librarylibraries. The Preparetargent DNAs were prepared using genomic DNA of cold sensitive group and cold tolerance group, by Pst I/AluI, Pst I/Ban II, Pst I/Bsp1286I, Pst I/BstN I, Pst I/Hae III, Pst I/Rsa I, Pst I/Taq I complexity reducing methods.Then their polymorphism levels of each method were6.52%,9.66%,8.00%,10.14%,7.12%,17.49%,12.80%respectively.From the Pst I/Rsa I representation DNA library, we amplified the insertion as probe to prepare theMadescreening-type chips, select Pst I/Rsa I whose polymorphism level was highest (17.49%) screening probes. Weobtained0/1array, scatter plot and cluster dendrogram. Clustering result is consistent with the separation ofpopulations. In all we got20DArTcold tolerance candidate markers of DArT associated with cold tolerance.