Figure 1.
Identification of EMS-induced mutants by NGS-assisted mapping.
For mutants identified in the reference Col-0 background, taking the green path leads to mutation identification. For mutants identified in a non-reference background, a parallel sequencing of the parental genotype (black path) is required. *Replacement of Col-0 specific SNPs with sites specific for the parental genotype, followed by use of the new constructed genome to extract the EMS-induced mutations in the mutant.
Figure 2.
Annotation of putative causal mutations (A).
Locations of EMS-induced mutations affecting the CDŚs on chromosome IV are marked with asterisks. The red asterisk indicate the mutation located in the region defined by mapping, which is flanked by the UPSC mapping markers [5]. Below, the structure of the CUL1 gene is shown, indicating positions of known recessive mutations giving viable mutants used in this study for complementation tests. The position and nature of the cul1-494 mutation, compared to wild type are highlighted on the DNA sequence. PCR amplification of the 494 and sur2-1gl1 cDNAs with primers spanning the splicing site affected by the cul1-494 mutation (B). A 463 bp fragment, corresponding to the correct spliced variant was detected in sur2-1gl1, while an additional 551 bp splicing variant was detected in 494.
Figure 3.
axr6-3 and cul1-7 mutations reduce the AR numbers produced by double mutants with sur2.
The number of adventitious roots was counted on at least 35 seedlings of each line in two replicates and the data were pooled. Error bars indicate standard errors. One-way ANOVA and Tukey’s multiple-comparison post-tests indicate that the double mutants are not significantly different from their respective wild types (P<0.05; n>70).
Figure 4.
Seedling and rosette phenotypes of the 494 and cul1-494 mutants.
Seedlings were first etiolated in the dark for four days (A) and then transferred to the light for seven days (B). For phenotypic evaluation of soil-grown plants, seeds were first germinated in vitro and the resulting seedlings were subsequently transferred into pots, which were placed in growth chambers providing short day (8 h darkness/16 h light) conditions, at 22°C/18°C (light/dark temperatures) (C). Arrowheads indicate the root–hypocotyl junction; arrows indicate adventitious roots. Bars, 1 cm.