Rapid identification of Arabidopsis insertion mutants by non‐radioactive detection of T‐DNA tagged genes

Abstract

SummaryTo assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M 2 family segregating a characterized gene mutation can be identified within 4 weeks.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…Although subsequent studies did not confirm in vivo formation of a CRK5-PRL1 complex under normal growth conditions, we found that inactivation of the CRK5 gene by a T-DNA insertion in the MPIZ mutant line 38225 (crk5-1; Ríos et al, 2002; see Supplemental Figure 1A online) resulted in reduced primary root elongation ( Figure 1A), analogously to the prl1 mutation (Németh et al, 1998). By contrast, a second T-DNA insertion in the CRK5 gene (Salk_003774, crk5-2) obtained from the SIGnAL collection (Alonso et al, 2003) failed to confer a root elongation detect.…”

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

“…The accessions Columbia (Col‐0) and Nossen (No‐0) were used as the wild‐type control. cycb1;1 and cycb1;2 T‐DNA insertion lines were isolated from the Koncz collection (Rios et al , 2002). T‐DNA insertion line cycb1;3 (pst15850) was obtained from the RIKEN collection (Yokohama, Japan) and cycb1;4 from the GABI‐KAT collection (Bielefeld, Germany) (Kleinboelting et al , 2012).…”

Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes

“…The atswi3c-1 (Koncz_27320) and brm-1 (SALK_030046) mutant alleles were previously characterised by Sarnowski et al (2005) and Hurtado et al (2006), respectively. The brm-6 (Koncz_77269) mutant allele was identiWed by PCR screening of our T-DNA mutant collection (Ríos et al 2002). The NMD pathway mutants upf1-5 (SALK_112922) and upf3-1 (SALK_025175) were also described previously (Hori and Watanabe 2005;ArcigaReyes et al 2006).…”

“…In a previously described mutant screen (Ríos et al 2002), we identiWed a new BRM T-DNA insertion mutant allele designated brm-6 (Fig. 1a).…”