Efficient isolation and mapping of <i>Arabidopsis thaliana</i> T‐DNA insert junctions by thermal asymmetric interlaced PCR

TLDR

TAIL‑PCR is an efficient, highly specific technique for amplifying insert ends from YAC and P1 clones without complex manipulations. The authors adapted this method to recover and map genomic sequences flanking T‑DNA insertions in Arabidopsis thaliana. The adapted protocol uses TAIL‑PCR to amplify insertion ends directly, enabling rapid mapping without additional processing steps. The method amplified insertion‑specific products from 183 of 190 lines, recovered single‑copy sequences from complex genomes, mapped 26 insertion sites with 122 RFLP probes, and identified hygromycin‑resistant lines suitable for fine‑scale mapping.

Abstract

Thermal asymmetric interlaced (TAIL‐) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T‐DNA insertions in Arabidopsis thaliana is described. Insertion‐specific products were amplified from 183 of 190 tested T‐DNA insertion lines. Reconstruction experiments indicate that the technique can recover single‐copy sequences from genomes as complex as common wheat (1.5 × 10 10 bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T‐DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T‐DNA insertions confer hygromycin resistance, can be used for fine‐scale mapping of linked phenotypic loci.