Chinese Journal of Biotechnology 2002;18(3):313-317

Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR.

Gen-Ji QIN 1 ; Pei-Du CHEN ; Yao-Guang LIU ; Yu-Da FANG ; Da-Jun LIU

Affiliations

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Country

China

Language

Chinese

Abstract

A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.