Traffic lines: new tools for genetic analysis in Arabidopsis thaliana

Abstract

Genetic analysis requires the ability to identify the genotypes of individuals in a segregating population. This task is straightforward if each genotype has a distinctive phenotype, but is difficult if these genotypes are phenotypically similar or identical. We show that Arabidopsis seeds homozygous or heterozygous for a mutation of interest can be identified in a segregating family by placing the mutation in trans to a chromosome carrying a pair of seed-expressed green and red fluorescent transgenes (a "traffic line") that flank the mutation. Nonfluorescent seeds in the self-pollinated progeny of such a heterozygous plant are usually homozygous for the mutation, whereas seeds with intermediate green and red fluorescence are typically heterozygous for the mutation. This makes it possible to identify seedlings homozygous for mutations that lack an obvious seedling phenotype, and also facilitates the analysis of lethal or sterile mutations, which must be propagated in heterozygous condition. Traffic lines can also be used to identify progeny that have undergone recombination within a defined region of the genome, facilitating genetic mapping and the production of near-isogenic lines. We produced 488 transgenic lines containing single genome-mapped insertions of NAP:dsRED and NAP:eGFP in Columbia (330 lines) and Landsberg erecta (158 lines) and generated sets of traffic lines that span most regions of the Arabidopsis genome. We demonstrated the utility of these lines for identifying seeds of a specific genotype and for generating near-isogenic lines using mutations of WUSCHEL and SHOOTMERISTEMLESS. This new resource significantly decreases the effort and cost of genotyping segregating families and increases the efficiency of experiments that rely on the ability to detect recombination in a defined chromosomal segment.

Keywords: T-DNA; mapping; recombination; stm; wus.

Figure 1

Dosage-sensitive expression of NAP:eGFP and NAP:dsRED in dry seeds. (A) Structure of the T-DNA vectors (pCAM–NAP:eGFP and pCAM–NAP:dsRED) used in this study. (B) T1 seeds transformed with NAP:eGFP. (C) T1 seeds transformed with NAP:dsRED. (D) T2 seeds from a transgenic plant segregating 3:1 for NAP:eGFP. (E) T2 seeds from a transgenic plant segregating 3:1 for NAP:dsRED. Nonfluorescent seeds are marked with an asterix.

Figure 2

Genomic locations of NAP:eGFP and NAP:dsRED insertions. (A) Transgene insertions in Col. (B) Transgene insertions in Ler. Maps were generated using the TAIR chromosome map tool (https://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp). CG and CR, NAP:eGFP and NAP:dsRED insertions in Columbia. LG and LR, NAP:eGFP and NAP:dsRED in Ler.

Figure 3

Crossing scheme used to produce TLs

Figure 4

Location of NAP:eGFP and NAP:dsRED insertions in TLs. (A) TLs in Col; (B) TLs in Ler. Numbers indicate the nucleotide position of the insertion in megabases. Vertical lines connect the red and green transgenes in a TL. TLs are identified by the ecotype, chromosome, and the order in which the line was created (e.g., CTL1.2, Columbia, chromosome 1, second line).

Figure 5

Using a TL to identify seeds homozygous for a recessive mutation. (A) The most common genotypes in the F2 progeny of a plant heterozygous for a TL and a mutation (m) of interest. (B) F2 seeds from a plant of the genotype CTL2.4/wus-5 photographed using a broadband GFP filter set (see Materials and Methods), which allows GFP and dsRED fluorescence to be visualized simultaneously. (C) F2 seeds from a CR128/wus-5 plant. nf, nonfluorescent; bf, bright fluorescent; if, intermediate fluorescent, r, red; g, green. (D) Phenotype of plants derived from nonfluorescent and bright fluorescent seeds in the F2 progeny of CTL2.4/wus-5. Plants were sprayed with Basta after flowering to determine the genotype of the phenotypically wild-type plants. All nonfluorescent seeds were resistant to Basta and had a wus-5 mutant phenotype, indicating they were wus-5/wus-5, and most of the bright fluorescent seeds were homozygous for the wild-type WUS allele, as indicated by their sensitivity to Basta.

Figure 6

Using a TL to select recombinants in a specific chromosome segment. Procedure used to introgress stm-1 into a Col genetic background. The transgenic chromosome is CTL1.13. Recombinants containing stm-1 were identified by progeny testing or by molecular genotyping. stm-1 has a much weaker phenotype in a Col background than in Ler. Col flowered 28 days after planting.