Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR.
- Author:
Gen-Ji QIN
1
;
Pei-Du CHEN
;
Yao-Guang LIU
;
Yu-Da FANG
;
Da-Jun LIU
Author Information
1. Cytogenetics Institute, Nanjing Agricultural University, Nanjing 210095, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Base Sequence;
Binding Sites;
Blotting, Southern;
Chromosomes, Artificial;
Genomic Library;
Molecular Sequence Data;
Polymerase Chain Reaction;
methods;
Translocation, Genetic;
Triticum;
genetics
- From:
Chinese Journal of Biotechnology
2002;18(3):313-317
- CountryChina
- 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.
