Apple fruit size evolution, Yao et al.
Method (Supporting Information).
Association of the cafs allele with quantitative trait locus (QTL) for fruit size.
Supporting Figure Legends
Figure S1. Phylogenetic and expression analysis of AP2 genes. Rooted Neighbour-joining
phylogenetic tree was constructed using protein sequence of AP2 genes from Arabidopsis (AP2,
TOE1, TOE2, TOE3, SMZ and SNZ) and apple (eight MDP numbers) and of ANT servicing
as an outgroup (left panel). Arabidopsis gene IDs, AP2, At4g36920; TOE1, At2g28550; TOE2,
At5g60120; TOE3, at5g67180; SMZ, At3g54990; SNZ, At2g39250; ANT, AT4G37750.
Relative expression levels (RPKM, Reads Per Kilobase of transcript per Million mapped reads)
of eight apple AP2 genes in seven apple tissues were determined using RNA sequence analysis
(right panel). DAFB: days after full bloom.
Figure S2. Conversion of sepal tissue to petal identify in TRG3. A flower of wild-type
‘Royal Gala’ showing five petals (a), and of transgenic ‘Royal Gala’ TRG3 showing extra
petals (b). Petal conversion in TGR3 flowers was revealed by removing normal petals as shown
in (c) extra petal converted from a sepal and half a sepal converted to petal and (d) several
sepals with sectors of them converted to petals.
Figure S3. Plant height of transgenic ‘Royal Gala’ over-expressing miRNA172p. Shown
are the same aged (two-year-old) trees of wild-type ‘Royal Gala’ (a), TRG3 (b), TRG4 (c),
TRG5 (d) and TRG6 (e) grown under the same conditions.
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Apple fruit size evolution, Yao et al.
Figure S4. Over-expression of miRNA172p reduces hypanthium and fruit cortex width
and fruit cell size. The images show thin (10 μm) sections of hypanthium at full-bloom stage
(a), fruit cortex at 2 weeks (b) and 5 weeks (c) post full bloom of wild-type ‘Royal Gala’ (RG),
transgenic ‘Royal Gala’ TRG3 and crabapple Malus sieboldii ‘Aotea’. Graphs on the right hand
side panels show mean hypanthium and cortex tissue width and mean cell area (n = 20). The
error bars represent standard deviations.
Figure S5. Phylogenetic analysis of the 4 kb genomic region of miRNA172p. Rooted
Neighbour-joining phylogenetic tree constructed using genomic sequence of miRNA172p from
12 accessions of Malus baccata (Bac) and 64 accessions of M. x domestica (Dom), M. sieversii
(Sie), M. orientalis (Ori) and M. sylvestris (Syl). The number for each sequence corresponds
to the sequence number given in Table S8. Sequences from two pear species, Pyrus communis
(Pc) and P. bretschneideri (Pb) were used as an outgroup.
Figure S6. The 3’ region of the pri-miRNA172p sequence contains a transposable element
(TE). The TE is shown in red, its 18 bp imperfect inverted terminal repeats are indicated by
red arrows, and its target site duplicated direct repeats are underlined in blue. The position of
the mature miRNA172 and PCR primers used in this study are also indicated. The sequence
GenBank Accession No. is EG999280.
Figure S7. The TE insertions in microRNA172p of accessions of four Malus species are at
the same site. DNA sequence alignment shows the TE (underlined in red) and its flanking
sequence. The insertion target site and its duplicated direct repeats are underlined in blue. The
SNP sites in the TE are indicated by arrows above the sequences.
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Apple fruit size evolution, Yao et al.
Figure S8. The TE in pri-miRNA172p belongs to a MITE-type transposon family. The TE
sequences and their duplicated target site sequences from five apple genes are aligned. The
duplicated target site sequences are underlined and imperfect inverted terminal repeats are
indicated by arrows. GenBank Accession Nos: MdmiRNA172p, EG999280; MdOmt2,
DQ886019; MdAGL1, GU56825; MsS46-RNase, EU419860; MdRfa2, AB073704.
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Apple fruit size evolution, Yao et al.
Supporting Tables
Table S1. Potential target genes of miRNA17p in M. x domestica
Table S2. Relative level of five mature sequences of miRNA172 in five apple tissues as
determined by analysis of small RNA sequences
Table S3. Descriptions of ‘Royal Gala’ apple transgenic plants developed using a
CaMV35S-pri-miRNA172p gene construct
Table S4. Distribution of CAFS and cafs alleles in the genus Malus
Table S5. Association analysis of cafs allele and fruit weight in progeny of ‘Royal Gala’
(cafs/cafs) × A689-24 (CAFS/cafs).
Table S6. QTLs for fruit size detected in a whole genome scan of 17 linkage groups
using interval and multiple QTL mapping.
Table S7. Standard neutral model tests.
Table S8. Description of 153 accessions from 36 Malus species sequenced and allelotyped
at CAFS locus tested in this study.
Table S9. Descriptions of the accessions of the five Malus species used in DNA sequence
diversity analysis utilizing 13 neutral genes.
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Apple fruit size evolution, Yao et al.
Table S10. List of primers used to amplify DNA fragments of 13 neutral genes for
sequence diversity analysis.
Table S11. Description of 159 progeny plants of RG X A689-24 tested in this study.
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