SUPPLEMENTAL FIGURE CAPTIONS
SUPPLEMENTAL FIGURE 1. Genotypes of recombinant chromosomes. A: M39A-1-6 allele;
B: M77A-1 allele. n.d.: not determined. Vertical bars: recombination points.
SUPPLEMENTAL FIGURE 2. Typical appearance of rye seedlings from an Eriochrome
Cyanine R Al-tolerance assay. Seedlings from an Alt4-segregating family derived from the
M39A-1-6 × M77A-1 cross were stained with Eriochrome Cyanine R immediately following
exposure to toxic (150 µM) Al, and grown at non-toxic Al levels for a further 48 hours. The
distance between the middle of the purple-stained section and the toot tip represents the regrowth following relief from stress. Tolerant genotypes (Alt4/Alt4 or Alt4/alt4) show extensive
re-growth and a diffusely stained zone, whereas intolerant genotypes (alt4/alt4) show little regrowth, a more concentrated stained zone and swelling of root tips caused by Al-toxicity.
SUPPLEMENTAL FIGURE 3. Chromosome arm designation of rye PCR markers using
wheat-rye chromosome addition lines. Genomic DNA templates were Chinese Spring wheat, an
unidentified rye line (‘rye’) and Chinese Spring disomic and ditelosomic addition lines
containing individual chromosomes or chromosome arms of Imperial rye. PCR products were
separated by agarose gel electrophoresis, with or without prior digestion with the indicated
restriction enzymes. Arrows indicate rye-derived fragments. Chromosome arm designations for
markers are indicated. Sizes of molecular weight marker (m) bands are indicated.
SUPPLEMENTAL FIGURE 4. Alignment of rye ALMT1 partial genomic sequences. The
sequences span introns-1 and -2 (lowercase). Sequence differences are highlighted by shading,
and gaps due to insertion/deletion polymorphisms are indicated by dashes. Locations of selected
primers are shown. Sequences matching the 5′ ends of primers Alm-6 and -7 are not illustrated.
Primer Almt-10 spans the Intron-2 splice sites. ScALMT1-1 and ScALMT1-2 are previously
described ALMT1 genes from tolerant and non-tolerant rye lines, respectively (FONTECHA et al.
2007). The inverted repeats of the MITE element within the intron-2 insertion region in
ScALMT1-M77.1 are underlined. Accessions: EU146229-EU146235; DQ158086, DQ158087.
SUPPLEMENTAL FIGURE 5. Localization of rye ALMT1 homologues using wheat-rye
chromosome addition lines. A. Southern blot analysis of Chinese Spring wheat and Chinese
Spring disomic and ditelosomic addition lines containing individual chromosomes or
chromosome arms of Imperial rye. DNAs were cut with EcoRV and probed with a rye AMLT1
cDNA. Rye derived fragments were only detected in the 7R and 7RS addition lines. B. PCR
analysis of the same lines indicated in B and an unidentified rye line (‘rye’). Genomic DNAs
were used as templates in PCR amplifications using primers Alm-2 and Alm-3 which amplify a
section from rye ALMT1 genes. Rye products were only detected in the 7R and 7RS addition
lines. Sizes of molecular weight marker (m) bands are indicated.
SUPPLEMENTAL FIGURE 6. Alignment of rye and wheat ALMT1 cDNA sequences.
Included are rye ScALMT1-1 and ScALMT1-2 (FONTECHA et al. 2007) and wheat TaALMT1-1
and TaALMT1-2 (SASAKI et al. 2004), derived from tolerant and intolerant varieties of these
species, respectively. Translation start and stop codons are underlined. The ends of the
ScALMT1-1 and ScALMT1-2 ORF seqeunces are unknown. Polyadenylation sites in ScALMT1M39.1 and -M39.3 determined by 3′ RACE are shown in bold. The 3′ transcript ends for the
remaining rye genes, and 5′ transcript ends for all of the rye genes, were not determined.
Sequence differences are highlighted by shading, and gaps due to insertion/deletion
polymorphisms are indicated by dashes. Positions of splice junctions of individual introns,
determined by comparison to rye and wheat ALMT1 genomic sequences from this and the other
studies, are indicated by triangles. Accessions: EU146236, EU146237, EU146239, EU146241,
DQ158086, DQ158087, AB081803, AB081804.
SUPPLEMENTAL FIGURE 7. Alignment of ALMT1 protein sequences. Included are rye
ScALMT1-1 and ScALMT1-2 (FONTECHA et al. 2007) and wheat TaALMT1-1 and TaALMT1-2
(SASAKI et al. 2004), derived from tolerant and intolerant varieties of these species, respectively.
The ends of the ScALMT1-1 and ScALMT1-2 protein seqeunces are unknown. Sequence
differences are highlighted by shading, and gaps due to insertion/deletion polymorphisms are
indicated by dashes. The 50 amino acid region underlined in ScALMT1-M77.1 is deleted as a
consequence of omission of exon-2 in a proportion of the ScALMT1-M77.1 transcripts. The
amino acid in bold and underlined in ScALMT1-M39.2 is the last amino acid before the frameshift caused by skipping of exon-4 in a proportion of ScALMT1-M39.2 transcripts.
SUPPLEMENTAL FIGURE 8. RT-PCR amplification of fragments containing full-length
ORFs of expressed rye ALMT1 genes. RT-PCRs were performed using primers Alm-23 and
Alm-27 and root-tip RNAs from the M39A-1-6 and M77A-1 parents and 859 and 1135
homozygous recombinants. Arrow indicates transcript variant lacking exon-2 from the
ScALMT1-M77.1 gene present in M77A-1 and 859. Sizes of molecular weight marker (m) bands
are indicated.