Method S1. Positional cloning of nuc and cyb
To generate a mapping population, homozygous nuc mutants (Col-0 background expressing
35Sprom:GFP-HDEL) were crossed to Landsberg erecta. GFP-HDEL expression facilitated rapid
identification of nuc homozygotes containing ER-derived nuclear cages with a Leica MZ16FA
stereo-fluorescence microscope. Coarse mapping linked nuc to within 2cM of the g4026a CAPS
marker on chromosome 1. Fine mapping of 336 F2 nuc homozygotes with 8 SSLP and CAPS
markers located the mutation to the 20 MB region between the markers SNP69 and F14J16, a
150 kb region containing 25 genes. Sequencing analysis identified the point mutation in
At1g54030.
To identify the cyb locus, homozygous cyb mutants (Col-0 background) were crossed to the Ler
ecotype. Coarse mapping located cyb to the lower arm of chromosome 1. Fine mapping of 47 cyb
homozygotes with 23 InDel markers narrowed down the site of mutation to a 127 kb region.
Sequencing of 5 candidate genes identified the point mutation in the second exon of At1g57620.
To confirm that the identified gene encodes CYB, a T-DNA insertion line in At1g57620 –
SAIL_664_A06 - was ordered from ABRC and tested for the cyb phenotype by
immunofluorescence with the TUB-1A2 antibody.