Short-interval traffic lines: versatile tools for genetic analysis in Arabidopsis thaliana

Abstract

Traffic lines are transgenic stocks of Arabidopsis thaliana that contain a pair of linked seed-specific eGFP and DsRed markers. These stocks were originally developed for the purpose of studying recombination, but can also be used to follow the inheritance of unmarked chromosomes placed in trans to the marked chromosome. They are particularly useful for this latter purpose if the distance between markers is short, making double recombination within this interval relatively rare. We generated 163 traffic lines that cover the Arabidopsis genome in overlapping intervals of approximately 1.2 Mb (6.9 cM). These stocks make it possible to predict the genotype of a plant based on its seed fluorescence (or lack thereof) and facilitate many experiments in genetic analysis that are difficult, tedious, or expensive to perform using current techniques. Here, we show how these lines enable a phenotypic analysis of alleles with weak or variable phenotypes, genetic mapping of novel mutations, introducing transgenes into a lethal or sterile genetic background, and separating closely linked mutations.

Keywords: fie; myb75; val1; Plant Genetics and Genomics; balancer chromosome; fluorescent-tagged lines; recombination.

Fig. 1.

Location of short-interval (right) and long-interval (left) TLs on the chromosomes of the Columbia accession of Arabidopsis thaliana.

Fig. 2.

Self-progeny of a ch42-4sd/TL4.22 plant, planted according to the seed fluorescence phenotype. Nonfluorescent seeds are expected to be predominantly ch42-4sd/ch42-4sd and albino, moderately fluorescent seeds are expected to be predominantly ch42-4sd/TL4.22 and yellow-green, and strongly fluorescent seeds are expected to be predominantly TL4.22/TL4.22 and dark green. Seedlings that do not conform to the expected phenotype are circled.

Fig. 3.

The first leaf with abaxial trichomes in the self-progeny of a myb75-1/TL1.55 plant, planted according to seed fluorescence phenotype. Nonfluorescent seeds are expected to be predominantly myb75-1/myb75-1 and moderately fluorescent seeds are expected to be predominantly myb75-1/TL1.55. Error bars = SEM; ** = P < 0.01, 2-tailed Student’s t-test, n > 45.

Fig. 4.

Rescuing lethal or sterile mutations with a transgene. a) Moderately fluorescent seeds from a fie-11/TL3.31 plant were selected and the resulting plants were transformed with a pFIE-HA BaR T-DNA construct by floral dipping. The expected genotypes of the nonfluorescent seeds from these plants are shown. Fully formed (i.e. viable) seeds will either be homozygous for fie-11 and hemizygous for pFIE-HA BaR, or be heterozygous for fie-11 as a result of double recombination within the interval marked by TL3.31. Given the relatively low frequency of transformation, this latter class of seeds will probably lack pFIE-HA BaR. b) Using a TL to rescue a seedling lethal or sterile mutant with a transgene. This approach is useful if the mutation does not affect seed morphology or viability, making it impossible to distinguish transgenic from nonransgenic seeds prior to planting.

Fig. 5.

Using a TL to separate closely linked mutations. F2 seeds from a val1 bmi1a/TL2.31 plant were screened for seeds that displayed strong red and moderate green fluorescence, or strong green and moderate red fluorescence. The genotype of these seeds was determined by examining the molecular genotype of their F2 progeny, using allele-specific PCR primers.