Construction And Application Of Large Insert Libraries For Zhikong Scallop (Chlamys Farreri)

Zhikong scallop(Chlamys farreri) is one of the most commercially important bivalves in China, but study on its genome is underdeveloped.To apply genome-based technologies for genetic improvements using marker-assisted selection, genome research involving large-insert genome library construction, genetic linkage mapping and physical mapping is required, and integration of genetic and physical maps would significantly enhance the capacities for genome research.In this study, the first fosmid library and another two BAC libraries of Zhikog scallop are constructed and characterized.Additionally, linkage-group specific clones from fosmid library are screened with markers on the microsatellite linkage map of Zhikong scallop. The major results are as follows:1.Construction and characterization of fosmid library in Zhikong scallopThe first Zhikong scallop fosmid library was constructed in this study. It consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents.Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between 2 and 8 positive clones, and none of those tested was absent from the library. The fosmid library will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of Zhikong scallop genome. 2. Construction and characterization of two BAC libraries in Zhikong scallopTwo Zhikong scallop BAC libraries were constructed with BamHⅠand HindⅢseparately, using only one scallop individual.The whole BAC library consists of 133,851 clones (33782 and 97680 for each) with an average insert size of about 96kb (80kb and 102kb for each),amounting to 9.8 X genome equivalents.BAC stability assays indicate that Zhikong scallop DNA was stable during propagation in the BAC system. The BAC library will serve as a useful resource for physical mapping and positional cloning, and provide a good tool for large scale sequencing of Zhikong scallop genome.3. Identification of linkage group specific clones from fosmid library of Zhikong scallop:A resource for integration of linkage and physical mapsThe first Zhikong scallop fosmid library was used to construct three-dimensional PCR screening system,110 microsatellite markers from 19 linkage groups of Zhikong scallop were used to screen a subset (2.7×)of the fosmid library. Of the 110 microsatellites,102 (92.7%) gave at least one positive clone and 8 (7.3%) failed to hit any clone. As a result,195 positive fosmid clones representing 19 linkage groups were identified, further verifying the genome coverage and utility of the library. These linkage group-specific clones provide resources essential for research of the Zhikong scallop's genome, connecting linkage group to its chromosome, physical mapping and integration of the genetic map and physical map.Another, a high-throughput PCR-based screening method was developed which combines fosmid three-dimensional PCR screening system and amplified fragment length polymorphism (AFLP) technology.We used 1 AFLP primer to screen 8 superpools of the fosmid library,and got 46 AFLP-linked positive clones. This methodology allowed us to identify fosmid clones containing AFLP genetic markers,can link DNA-based physical map to the Zhikong scallop genetic map.This combination of approaches provides a low cost,efficient way to build high-quality integrated genetic and physical genome maps.