Supplementary Figure S1. Molecular characterization of abph1 alleles.
a. Map of the abph1 locus, showing the position of the transposon insertion
(Mu7). Southern blot of abph1 alleles (b). In the abph1-0 parental line, the Mu-flanking
probe hybridizes to a doublet at around 12kb (panel b, lane1), and in the Mutator (“Mu”)
transposon parental line to two bands of 2kb and 7kb (panel b, lane 2, a, a*). In the
abph1-mum181 insertion allele, both of these bands are increased in size by
approximately 2.2 kb, which is the size of the Mu7 transposon 1(panel b, lane 4; b,
~4.2kb; b* ~9.2kb). The probe fragment itself is not digested by BamHI (even if it is
amplified by PCR from the Mu parental line), therefore the presence of the two bands is
1
most easily explained by a BamHI site that undergoes partial digestion. This could
happen if it is sometimes methylated, for example 2. The map accounting for these bands
is shown in panel a, with the probe fragment marked in red, and the partially digesting
BamHI site marked by an “*”. If this site cuts, the hybridizing band should be 2kb in the
Mu parental line and 4.2kb in the abph1-mum181 allele carrying the Mu7 insertion. When
the site does not cut the bands should be 7 and 9.2kb, respectively, as is observed (panel
b, compare lanes 3, 4).
In the three other transposon alleles, the fragment recognized by the probe
appears to be missing rather than altered in size (Fig. S1A, lanes 6, 8, 9, position of
“missing” bands marked by upward arrows; “^”), compared to segregating normal sibs in
lanes 5 and 7. Mu transposons are known to give rise to deletions 3, so these alleles may
be caused by a deletion of the probe region. The probe that we used is single copy in
standard maize inbred lines (panel b, lanes 10,11). The alleles and genotypes are listed
below panel b.
c. The 2kb of cloned Mu7 flanking sequence contained the complete coding
region of a small gene with 5 predicted exons, E1 to E5. The position of primers (P1 to
P6) used to amplify the predicted coding region from the abph1-0 allele DNA is shown.
We were able to amplify the entire coding region from wild type (B73) DNA (panel d,
lanes 2-6). However, we could not amplify it from the abph1-0 allele DNA, though
shorter fragments, such as the first three exons or the fifth exon each could be amplified
(panel d, lanes 7, 11). This suggests that the abph1-0 allele contains a rearrangement or
insertion between intron 3 and intron 4. We were never able to PCR amplify this region
from the abph1-0 allele DNA, even when using long extension times and Extaq DNA
polymerase (Takara) which routinely can amplify more than 15kb.
Therefore the
insertion may be recalcitrant to amplification, as is common for example for transposons
(R.A. Martienssen, pers. comm.). Southern blotting of abph1-0 DNA suggests that the
two fragments of the gene are still contained on a relatively small DNA fragment (not
shown), arguing against a major rearrangement.
We also amplified and sequenced cDNA and genomic clones from the abph1-sal
allele (panel e). A single nucleotide change was detected in the second nucleotide after
the 5’ junction of the first intron (Fig. 1e). This nucleotide (“T”, or “U” in the RNA) is
2
extremely highly conserved in plant introns 4, and the change to a “C” (“^”, Fig. 1e) in
the abph1-sal allele correlates with altered transcript processing that we observed using
reverse transcriptase-PCR amplification of RNA from abph1-sal seedlings. We detected
two major transcripts (panel e, lane 3), whereas wild type plants had only one transcript
(lane 2). DNA sequencing showed that in the larger abph1-sal RT-PCR product the first
intron was unspliced, whereas in the smaller one there was a 10 base pair insertion caused
by apparent cryptic splicing 10bp downstream of the normal exon-intron boundary (**,
Fig. 1e, splice junction denoted “// ”). Both mis-splicing events lead to a frame shift and
introduce a premature stop codon after approximately 40 amino acids.
In summary, we found molecular lesions in six independent abph1 alleles. abph1mum181 has a novel Mu7 insertion just after the predicted ATG, abph1-mum43, abph1mum77 and abph1-spm799 appear to be deleted for most of the coding region, and
abph1-0 likely has an insertion between exon 3 and exon 5. This allele also does not
produce any detectable transcript (Fig. 2a). The abph1-sal allele has a mutation at the 5’
end of the first intron that leads to non-splicing or mis-splicing of this intron.
REFERENCES.
1. Schnable, P. S., Peterson , P. A. & Saedler, H. The bz-rcy allele of the Cy transposable
element system of Zea mays contains a Mu-like element insertion. Mol Gen Genet 217,
459-63 (1989).
2. Kouzminova, E., Selker, E. dim-2 encodes a DNA methyltransferase responsible for all
known cytosine methylation in Neurospora. The EMBO journal. 20(15), 4309-23 (2001).
3. Robertson, D., Stinard, P. & Maguire, M. Genetic evidence of Mutator-induced
deletions in the short arm of chromosome 9 of maize. II. wd deletions. Genetics. 136,
1143-9 (1994).
4. Brendel, V., Kleffe, J., Carle-Urioste, J. C. & Walbot, V. Prediction of splice sites in
plant pre-mRNA from sequence properties. Journal of molecular biology. 276(1), 85-104
(1998).
3
Supplementary Figure S2. in situ hybridization analysis of ABPH1 expression.
(A) Expression of ABPH1 in a transverse section of a 2-week old vegetative seedling
shoot apex (similar stage to the one in Fig. 2g). Expression is visible as pale blue
staining at the periphery of cells in the meristem, and occupies a segment of 1/3 to 1/2 of
the meristem diameter, opposite to the plastochron 1 (P1) leaf. The plastochron 2 (P2)
leaf and the shoot apical meristem (SAM) are also labeled.
A transverse section of the ear inflorescence meristem is shown in (B) and is also stained
in situ for ABPH1 mRNA. At this developmental stage ABPH1 is expressed evenly
throughout the meristem. Bar in A, B = 50m.
4