Genomic DNA prepared from the Nile tilapia testis was used for Fosmid library construction.It was randomly sheared to -40 kb fragments.End-repair and recovery of the size-fractionated DNA were manipulated according to the manufacturer’s protocols.Ligation of the end-repaired fragments into the Fosmid vector pCC2FOS(Epicentre,USA)was performed by using T4 DNA ligase for 16 h at 4 ℃.Fosmid clones were packaged using MaxPlaxi Lambda packaging extract provided by the kit.Infected EPI300^TM-T1^R cells were grown at 37 ℃ in solid medium overnight. Well-separated colonies were picked out and transferred into individual wells of 384-well plates,each well with 300 L culture medium.After overnight incubation at 37 ℃,100 L medium was transferred from each of the 24 wells of every row of the 384-well plates into one well of the 96-well plate to construct rowpools.Totally 16 row-pools were constructed for each 384-well plate.Similarly,100 L medium was transferred from each of the 16 wells of every column of the 384 well plates into one well of the 96-well plate to construct column-pools.Totally 24 column-pools were constructed for each 384-well plate.Plate-pools were constructed by pooling the 16 column-pools and 24 row-pools(200 L from each column and rowpool)from the same 384-well plate.Totally 4 800 column-pools,7 200 row-pools and 300 plate-pools were constructed and arrayed.The library was further arranged into 25 super-pools,each including a mixture of culture medium from 12 plate-pools(500 L from each plate-pool).Finally,All 384-well plates and constructed pools were replicated,one copy was kept at -80 ℃ for longterm storage,another copy was kept at -20 ℃ for routine use.The constructed tilapia Fosmid library encompasses 115200 clones.Any gene of interest can be screened only by 3 rounds of minimally 77(25 12 40)colony PCR due to the array of super-,plate-,row- and columnpools.Fosmid stability assays were performed for the constructed library with 9 randomly picked clones by serial culture for 100 generations over a period of 6 days.The electrophoretic patterns of each clone digested with Hind Ⅲ and EcoRⅠ were identical from day 1 to day 6.No loss or rearrangement of the inserted DNA fragment was found during the continuous passage.Ten clones were randomly selected from the library and plasmids of these clones were prepared, digested with NotⅠ and visualized by electrophoresis.All the 10 clones contained insert with an average size of around 40 kb,ranging from 38 to 42 kb.Therefore,the coverage rate of this library is about 3 genome equivalents,leading to a 98.7% probability of recovery of any specific sequences of interest.Eighteen genes related to tilapia sex differentiation and growth were screened to test the quality of the library.All of them were successfully isolated from this library,with each having 2-5 positive clones,confirming the 3 genome equivalents coverage rate and the availability of the library.In summary,a high-quality Fosmid library of the Nile tilapia with large insert(40 kb)was successfully constructed,microarrayed and characterized using the vector pCC2FOS.