Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has u niquely small sex chromosomes, with X chromosomes present only in female ga metophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-deri ved artificial chromosome (pCYPAC2). With an average insert size of approxi mately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened f or by differential hybridization using male and female genomic DNAs as sepa rate probes. Seventy male-specific PAC clones were identified. The male spe cificity of one of the clones, pMM4G7, was verified by Southern hybridizati on and PCR analysis. This clone was indeed located on the Y chromosome as v erified by fluorescence in situ hybridization (FISH). This result shows tha t the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and fe male libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of s ex determination.