Construction of a bacterial artificial chromosome library of Villosiclava virens UV-2 genome

Abstract:

[Objective] Rice false smut caused by Villosiclava virens (Cooke) Tak. is a serious fungal disease of rice. This study aimed to construct a high molecular weight bacterial artificial chromosome (BAC) library of UV-2 genome. The BAC library will help identify patho-genicity-associated genes and provide genome resource for map-based cloning and compara-tive genomic analysis. [Methods] High molecular weight genomic DNA was isolated from young mycelia of UV-2 strain, digested with restriction enzyme Hind III and size selected by PFGE. Large genomic DNA fragments were recovered and ligated to the pIndigoBAC536-S vector. The ligation product was used to transform the Escherichia coli strain DH10B T1 Phage-Resistant cells and the transformants were selected on a blue-white selection medium. White colonies were individually picked into 384-well microtiter plates and stored at ?80 °C. [Results] We constructed a high quality deep coverage BAC library of UV-2 strain. The BAC library consisted of 10 368 clones with an average insert size of 124.4 kb and an empty clone rate of lower than 1%, covering 36.8-fold of the UV-2 genome. [Conclusion] We established a method for preparation of fungal high molecular weight genomic DNA, and constructed the first BAC library of V. virens genome successfully. The BAC library has been opened to re-searchers as a public genomic resource (http://GResource.hzau.edu.cn).