The Plant Journal (2015) 84, 417–427
doi: 10.1111/tpj.13021
A microRNA allele that emerged prior to apple domestication
may underlie fruit size evolution
Jia-Long Yao1,*, Juan Xu1,2, Amandine Cornille3,4, Sumathi Tomes1, Sakuntala Karunairetnam1, Zhiwei Luo1,
Heather Bassett5, Claire Whitworth6, Jonathan Rees-George1, Chandra Ranatunga6, Alodie Snirc3,4, Ross Crowhurst1,
Nihal de Silva1,†, Ben Warren1, Cecilia Deng1, Satish Kumar6, David Chagne5, Vincent G. M. Bus6, Richard K.Volz6,
Erik H. A. Rikkerink1, Susan E. Gardiner5, Tatiana Giraud3,4, Robin MacDiarmid1,7 and Andrew P. Gleave1
1
The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand,
2
Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, Wuhan, 430070,
Hubei, China,
3
Ecologie, Syste matique et Evolution, Universite Paris-Sud, Ba^ timent 360, F-91405 Orsay, France,
4
CNRS, F-91405 Orsay, France,
5
The New Zealand Institute for Plant & Food Research Limited, Palmerston North 4442, New Zealand,
6
The New Zealand Institute for Plant & Food Research Limited, Havelock North 4157, New Zealand, and
7
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Received 2 June 2015; revised 25 August 2015; accepted 28 August 2015; published online 10 September 2015.
*For correspondence (e-mail [email protected]).
†
Deceased.
SUMMARY
The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops,
despite size being an important agronomic trait. Here we show that the expression level of a microRNA
gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing
reduced expression associates with large fruit in an apple breeding population, whereas over-expression of
miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to
be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple
fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently
strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective
sweeps are normally weaker in perennial crops than in annual crops.
Keywords: APETALA2, domestication, fruit size, Malus 3 domestica, microRNA.
INTRODUCTION
The cultivated apple (Malus 9 domestica) has both cultural
and economic significance, being second only to banana
in terms of worldwide fruit crop production. Although
most wild apple species produce bitter, small fruits (<1 cm
in diameter) termed ‘crab apples’ (Cornille et al., 2014), the
species that have contributed to the genome of the cultivated apple (M. sieversii, M. sylvestris and M. orientalis)
(Velasco et al., 2010; Cornille et al., 2012) produce relatively large fruits (>1 cm). Malus sieversii, the primary progenitor of the cultivated apple (Velasco et al., 2010), has
fruit up to 8 cm in diameter, which approaches the average
fruit size of cultivated apples. It has been suggested that
large apples were first selected for in the Tian Shan Mountains on the China-Kyrgyzstan border, where the Malus
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd
fruit-dispersing vector changed from birds to large mammals, prior to domestication by humans (Juniper and Mabberley, 2006). Selection for even larger fruit would have
occurred after the Tian Shan Mountain apples were
brought to Europe over the Silk Road, between 4000 and
6000 years ago (Juniper and Mabberley, 2006).
Fruit crop domestication is typically associated with a
dramatic increase in fruit size (Gross and Olsen, 2010; Miller
and Gross, 2011). Despite its fundamental and applied
importance, the molecular genetics underlying this important agronomic trait are poorly understood in perennial
species. In the annual species tomato, three genes have
been identified that control quantitative trait loci (QTLs) that
influence the differences in fruit size between wild and
417
418 Jia-Long Yao et al.
cultivated plants (Frary et al., 2000; Cong et al., 2008;
Munos et al., 2011). Our goals were to investigate the
molecular basis and evolutionary trajectory of apple fruit
size, by examining both wild and cultivated apple species.
MicroRNA172 (miRNA172) inhibits the translation of a
subfamily of APETALA2 (AP2) genes (Chen, 2004) that govern floral organ development (Yant et al., 2010) and organ
size (Jofuku et al., 2005) in Arabidopsis, and a recent study
has shown that miRNA172 positively regulates Arabidopsis
silique size (Ripoll et al., 2015). As apple fruit develop from
more than one type of floral organs (Pratt, 1988), miRNA172
was an attractive candidate to study in relation to its potential role in the regulation of apple fruit size. Although 16
miRNA172 genes have been predicted, 15 (miRNA172a-o)
from the M. 9 domestica genome sequence (Xia et al.,
2012) and one (miRNA172p) from expressed sequence tag
(EST) sequences (Gleave et al., 2008), we focused our study
on the only member of the miRNA172 family where expression has been confirmed (miRNA172p).
RESULTS
Expression of miRNA172p and its target genes
Bioinformatics analysis showed that miRNA172p potentially targets 10 apple genes of which eight are AP2-like
genes (Table S1). Phylogenetic analysis shows that two of
these eight (MDP0000137561 and MDP0000204900) are
most closely related to AP2 while the other six are more
closely related to an AP2 sister-clade containing TOE1 (Figure S1). RNA sequence data showed that these two AP2
genes have a high expression level in flowers and fruit
while the six genes of the AP2 sister-clade have lower (or
no) expression in these tissues (Figure S1). There are five
distinct mature miRNA sequences from 16 predicted
miRNA172 genes in the apple genome. The mature miRNA
sequence for miRNA172p, is shared with miRNA172d-h,
and present at a relatively high level in flower and fruit tissues as shown by analysis of small RNA sequences
(Table S2). Another mature miRNA172 sequence present at
high level in flowers and fruit matches miRNA172 m-o.
The overlapping expression patterns of miRNA172p and
AP2 genes in flowers and fruit and the presence of miRNA172p target site in AP2 genes indicate that miRNA172p
could influence apple fruit development by modulating
levels of AP2 proteins.
Over-expression of miRNA172p reduces fruit size and
alters floral organ development
Over-expression of miRNA172p resulted in the reversion of
cultivated apple fruit to crab apple size, in addition to causing other altered phenotypes in transgenic ‘Royal Gala’
(RG) plants (Table S3). The transgenic plants TRG1 and
TGR2 did not show over-expression of miRNA172 or an
altered phenotype. The transgenic plant TRG3, which over-
expressed miRNA172p 15-fold, exhibited significantly
smaller fruit and seeds than the RG control (Figure 1a,b).
In addition, TRG3 had some flowers with sectors of sepals
showing petal-colour, indicating a conversion of sepal to
petal identity (Figure S2). TRG3 was similar in height to RG
control (Figure S3b). Plants TRG4, TRG5 and TRG6, with
20- to 24-fold over-expression of miRNA172p, exhibited
more dramatic changes in phenotype, including flowers
consisting entirely of carpel tissues, with no sepals, petals
or stamens (Figure 2). The carpels showed a wide range of
development changes from leaf-like tissue carrying ectopic
ovules to fully fused carpels consisting of ovary, style and
stigma (Figure 2b–d). These carpels were apocarpic (occurred separately) rather than syncarpic (fused together) as
in normal apples. All the carpels of the TRG4, TRG5 and
TRG6 plants failed to produce any fruit after hand pollination irrespective of the degree of developmental defects. In
addition, TRG5 was a semi-dwarfed plant (Figure S3d) and
TRG6 was not only dwarf (Figure S3e), but also flowered
2 years later than RG control and other transgenic plants.
Key developmental differences between the large fruit of
domesticated apples and the smaller crab apples are
reported to be reductions in fruit cell number and fruit cell
size in the latter (Harada et al., 2005). TRG3 fruit displayed
a significantly thinner hypanthium at full bloom and thinner fruit cortex tissue at 2 weeks post full bloom when
compared with fruit of RG; however, they both exhibited
similar cell sizes (Figure S4a,b), suggesting that TRG3 fruit
had fewer cells than RG fruit in both the hypanthium and in
the cortex of 2-week post full bloom fruit. From 5 weeks
post full bloom through to maturity TRG3 fruit cortex tissues displayed a reduced cell size compared with RG (Figures 1c and S4c). These developmental data indicate that
the elevated miRNA172p expression inhibited cell division
and cell expansion at the early and late stages of fruit
development, respectively. The crab apple M. sieboldii
‘Aotea,’ known to bear small fruit (Table S4), exhibited a
similar reduction in fruit cell number and size, to TRG3 (Figures 1c and S4b,c). Although yellow granules were also
observed in ‘Aotea’ fruit tissue sections (Figure S4b,c),
these are not likely related to fruit size, but rather are vacuoles accumulating phenolic compounds, such as condensed tannins, and are specific to the yellow fruit flesh
variety ‘Aotea’. Given the similarity in fruit size and fruit cell
number and size between TRG3 and crab apples, we postulated that a modified allele of miRNA172p with reduced
expression might be responsible for the increase in fruit
size in domesticated apple and its large-fruited progenitors.
Identification of a miRNA172p mutant allele
To identify mutant alleles of miRNA172p, we sequenced
DNA amplicons (up to 3957 bp) of miRNA172p from 64
accessions of four apple species with relatively large
fruit (M. 9 domestica, M. sieversii, M. orientalis and
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
Apple fruit size evolution 419
(a)
180
160
Fruit weight (g)
140
120
100
80
60
40
1 cm
20
0
(b)
Seed weight
(mg/10 seed)
500
400
300
200
1 cm
100
0
(c)
35 000
100 μm
Cell area (µm²)
30 000
25 000
20 000
15 000
10 000
5000
RG
TRG3
‘Aotea’
0
RG
TRG3 ‘Aotea’
Figure 1. Over-expression of miRNA172p in transgenic Malus 9 domestica ‘Royal Gala’ (RG) TRG3 apple reduces the size of fruit, seeds and fruit cells.The photographs show (a) mature fruit, (b) mature seeds, and (c) thin (10 lm) sections of mature fruit cortex tissues of RG, TRG3 and crab apple Malus sieboldii ‘Aotea’
from left to right. The graphs on the far right panel show mean fruit weight (n = 20), mean weight of 10 seeds (n = 10) and mean fruit cortex cell area (n = 20)
for the fruit from the three plants. The error bars in the graphs represent standard deviation.
M. sylvestris) and 12 accessions of a crabapple species
(M. baccata) that bears very small fruit (Figure 3a and
Table S4). These accessions were representative of the
genetic diversity of the wild apples (Cornille et al., 2013a,
b). A phylogenetic tree derived from these sequences
showed that all 12 M. baccata accessions clustered
together, and that the accessions of the other four species
formed a separate clade, with no further phylogenetic
structure according to species (Figure S5). The two-clade
structure was due to six small indels (1–5 bp) and 38 sin-
gle-nucleotide polymorphisms (SNPs) (Figure 3b) between
M. baccata and the four large-fruited species. In addition,
the four large-fruited species exhibited a transposable element (TE) insertion in the 30 end of pri-miRNA172p (Figures 3b, S6 and S7) that was absent in the sequences from
M. baccata. The location of the TE insertion was identical
for all accessions tested indicating that all these accessions
most likely have the same insertion event. However, there
are five SNPs in the TE (Figure S7) indicating genetic diversity appeared after the insertion event.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
420 Jia-Long Yao et al.
1 mm
style
5 mm
1 cm
lct
Stigma
Stigma
style
style
ovary
(a)
ovules
(c)
Figure 2. Flowers consists of carpel tissues in
TRG5.(a) Floral inflorescences on stem of TRG5
show defective flowers consisting of leaf-like carpel
tissues (lct), style and stigma.(b) Defective carpels
removed from the inflorescences of (a) show a series of developmental aberrations from a leaf-like
structure carrying ectopic ovules (eo) to an almost
fully closed carpel.(c) A florescence of (a) with leaflike carpels removed reveals more advanced carpels consisting of ovary, style and stigma.(d) A single carpel removed from the inflorescence in (c)
reveals ovules after cutting the ovary open.
(d)
eo
(b)
1 cm
The 154-bp TE belongs to a miniature inverted-repeat
transposable elements (MITEs) family (Figure S8). As the
TE can form stem-loop structures and alter gene expression (Han and Korban, 2007), we suggest that the presence
of the TE might reduce the expression level of miRNA172p
although we cannot rule out the possibility that other SNPs
and indels at the locus may change the expression of miRNA172p. We named the miRNA172p locus CrabApple Fruit
Size and its wild-type and transposon insertion alleles
CAFS and cafs, respectively.
To confirm the role of the cafs allele in apple fruit size
evolution, we allelotyped the miRNA172p locus of 153
accessions, belonging to 36 different Malus species as
classified by Phipps et al. (1990) (Table S4). The 153 accessions show a fairly well representative diversity of the
genus Malus. They were classified as 77 cafs homozygous,
58 CAFS homozygous and 18 heterozygous (Table S4). All
77 cafs homozygous accessions were from large-fruited
species. They include 73 accessions from the cultivated
species M. 9 domestica and its closest wild relatives
within the Malus series of the genus, and four accessions
from M. 9 robusta and M. platycarpa that are outside the
Malus series but are considered to be hybrids with a parent from the Malus series (Table S4). All 58 CAFS homozygous accessions are outside the Malus series and 55 of
them produce small fruit. The 18 heterozygous accessions
were from species with different fruit size and may be generated by genetic admixture, which has been shown to be
common in Malus (Cornille et al., 2012, 2013b). The above
results confirm that the cafs allele is strongly associated
with large fruit size. A slight discrepancy between the cafs
allele and fruit size may be explained by the presence of
other major fruit-size QTLs in Malus (Devoghalaere et al.,
2012; Chang et al., 2014) and genetic admixture (Cornille
et al., 2012, 2013b).
The CAFS locus underlies a major fruit-size QTL
The association between the cafs allele and a large fruit
size was confirmed by analysing a segregating progeny
from a RG (cafs/cafs) 9 A689-24 (CAFS/cafs) cross
(Table S5). Ninety-one cafs/cafs and 68 CAFS/cafs plants
displayed significantly different (P = 4.3 9 106) 3-year
average fruit weights of 207 and 176 g, respectively, with
the CAFS locus explaining 21% of the fruit weight variation
(Figure 3c and Table S5).
Using the same RG 9 A689-24 segregating population,
four significant fruit size QTLs were located on linkage
group 2 (LG2), LG10 (two QTLs), and LG11 (Table S6). The
QTL on LG11 explained 13.9% of the fruit weight variation
and the QTL confidence interval overlapped with the confidence interval for the cafs locus location (Figure 4), indicating that the CAFS locus was co-located with the QTLs on
LG11.
Quantitative PCR (polymerase chain reaction) analyses
of cDNA from RNA of two CAFS/cafs and four cafs/cafs
plants showed that the pri-miRNA172p level was reduced
approximately twofold in cafs/cafs plants (Figure 3d). This
twofold reduction in expression level is associated with
increasing fruit size although it is much smaller than the
16-fold change of miRNA172p level in the transgenic apple
plant TRG3. The difference in levels of expression
change detected here may be explained by the distinct
possibility of tissue-specific expression of native
miRNA172p (Table S2) compared with the constitutive and
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
Apple fruit size evolution 421
(a)
Dom
Sie
Syl
Ori
Bac
1 cm
(b)
C>A
CCAAT del
A>G
G>A
A del
G>A
C>T
AAG ins
T>C
C>T
C>T
C>T
G>C
1
C>T
C>A
GTCGT ins A>G
A>C
A>C
G>T T>A
T>G
T>C
A>G
G>A
Fruit weight (g)
300
n = 68
n = 91
200
150
100
50
A>G
C>T
A>C G>A
cafs/cafs
C>T
A>T
C>T
TT ins
C>A
pri-miRNA172p
TE ins
NS
4.5 kb
4
3
2
1
0
CAFS/cafs
T>C
miRNA172p
250
0
TT ins
A>G
(d)
G>A
A>T
T>C
C>T
Relative primiR172p level
350
A>T
C>T
Promoter
(c)
C>A
1
2
CAFS/cafs
3
4
5
6
cafs/cafs
Figure 3. Determination of the relationship between the cafs allele of miRNA172p and Malus fruit size.(a) Fruit of Malus 9 domestica (Dom), Malus sieversii
(Sie), Malus orientalis (Ori), Malus sylvestris (Syl) and Malus baccata (Bac).(b) The sequences specific to the 2 kb promoter region and 2 kb pri-miRNA for 12
accessions of M. baccata defined as the CAFS allele are shown in black, and 64 accessions of M. 9 domestica, M. sieversii, M. orientalis and M. sylvestris
defined as the cafs allele are shown in red, ins: insertion, del: deletion, TE ins: transposable element insertion, NS: not sequenced. (c) Box plot of fruit weight
distribution of 91 cafs/cafs and 68 CAFS/cafs progeny plants of RG 9 A689-24.(d) The pri-miRNA172p expression levels were reduced in four cafs/cafs plants
compared with the levels in two CAFS/cafs plants (error bars represent the standard deviation of three independent PCR reactions).
Figure 4. Genetic and fruit size QTL map of A68924 linkage group 11.LOD profiles for multiple QTL
mapping (MQM) and interval mapping (IM) models
show a fruit size QTL co-located with the CAFS
locus within their 95% confidence intervals (CI).
over-expression of the transgene driven by a CaMV35S
promoter. Similarly, a two-fold change of the mRNA level
of the maize domestication gene teosinte branched1 (tb1)
caused by a TE insertion has significantly changed apical
dominance (Doebley et al., 1997; Studer et al., 2011). Our
data demonstrate that the CAFS locus underlies a major
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
422 Jia-Long Yao et al.
Species
Sequence
M. 9 domestica
cafse
M. sieversii
cafs
M. orientalis
cafs
M. sylvestris
cafs
M. baccata
CAFSe
M. 9 domestica
neutralf
M. sieversii
neutral
M. orientalis
neutral
M. sylvestris
neutral
M. baccata
neutral
Wilcoxon rank sum testg
Na
Sb
hWc
pd
19
15
15
15
12
8
9
9
10
9
27
33
23
29
46
69
58
43
96
73
0.00197
0.00266
0.00180
0.00228
0.00406
0.00426
0.00323
0.00245
0.00541
0.00454
w = 23, P = 0.010
0.00158
0.00242
0.00207
0.00162
0.00331
0.00453
0.00306
0.00284
0.00502
0.00562
w = 24, P = 0.005
Table 1 Nucleotide polymorphism for
Malus species at miRNA172p and at 13
neutral genes
a
Number of accessions sequenced (See Tables S8 and S9 for details).
Number of polymorphic sites.
c
Theta of Watterson (Watterson, 1975).
d
The average number of nucleotide differences per site between sequences (Tajima, 1983).
e
4 kb sequences of the cafs or CAFS allele of miRNA172p.
f
5.409 kb concatenated sequences of 13 neutral genes (Table S9).
g
Two-sided Wilcoxon rank sum test between the group of four cafs and the group of CAFS
and neutral gene sequences.
b
QTL for apple fruit size and strongly indicates that the presence of the homozygous cafs allele results in large fruit as
a consequence of a reduction in miRNA172p transcript
accumulation. CAFS, however, cannot account for all fruit
size variation and must act in association with other fruit
size QTLs in M. 9 domestica (Devoghalaere et al., 2012;
Chang et al., 2014).
DNA sequence diversity at the miRNA172p locus
We postulated that the cafs allele was selected when large
fruit was selected. We therefore carried out standard neutrality tests using the DNA sequence data generated for
Figure 3(b). We also sequenced 13 neutral genes for
nucleotide diversity comparison. We found that nucleotide
diversities (hW and p value) of the cafs allele in the four
species with large fruit was significantly lower than those
of the CAFS allele in the small-fruited species M. baccata
and of the 13 neutral genes (5.4 kb) in the above five species (Table 1). The lower DNA sequence diversity together
with the fixation of cafs allele in large-fruited Malus species suggests the cafs allele was under selection. However,
Tajima’s D (Tajima, 1989), Fu and Li’s D (Fu and Li, 1993),
Fu and Li’s F (Fu and Li, 1993), and Hudson–Kreitman–
Aguade (HKA) (Hudson et al., 1987) tests did not detect significant departure from neutrality for the CAFS locus
(Table S7). The insignificant results of neutrality tests may
be explained by restoration of genetic diversity after an
ancient selective sweep (see the Discussion section for
more details). As the causal locus here is not a protein
encoding gene we cannot use other selection tests, such
as the MacDonald–Kreitman test or the dN/dS test, that are
able to detect footprints of selection for significantly longer
periods after a selective sweep has occurred because these
tests rely on the counting of non-synonymous substitutions compared to synonymous substitutions.
DISCUSSION
We have demonstrated that miRNA172p has negative influence on fruit growth in apple as its over-expression
reduces fruit size in transgenic apple. This negative influence is supported by the results of analysing a TE insertional allele of miRNA172p, cafs, that has reduced
expression of this miRNA and is associated with an
increase in fruit size. In the progeny of a controlled cross
between parents homozygous and heterozygote for cafs,
we found that the presence of the homozygous cafs allele
was associated with large fruit and a concomitant reduction in miRNA172p transcript level. In the same cross, the
CAFS locus was co-located with one of the four fruit size
QTLs identified. Results of the present study strongly suggest that miRNA172p underlies apple fruit size evolution
although further experiments may be required to confirm
this suggestion. These experiments may include fine QTL
mapping using a larger segregating set of progeny to more
accurately locate the fruit-size QTL with respect to the cafs
alleles, complementation of cafs allele in cultivated apple
with the CAFS allele from wild apple to show reduction of
fruit size, and suppression of the CAFS allele in wild apple
using an RNAi gene construct to show increase of fruit
size. Due to the long-generation time of apple trees, generation of an enlarged fruiting progeny set and transgenic
apple plants would take several years to complete.
It is intriguing that miRNA172 promotes fruit growth in
Arabidopsis (Ripoll et al., 2015), yet inhibits fruit growth in
apple. Superficially, our result with apple is contradictory
to that with Arabidopsis. However, the two results are in
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
Apple fruit size evolution 423
Figure 5. Model for the regulation of fruit growth
in apple and Arabidopsis by the miRNA172–dependent regulatory module.MiRNA172 negatively regulates AP2 by inhibiting its translation (Chen, 2004).
AP2 is required for sepal development (Weigel and
Meyerowitz, 1994) which contributes to apple fruit
flesh cortex development (Yao et al., 2001). AP2
directly binds to the promoter and represses the
expression of both FUL and AG (Yant et al., 2010)
that are important for carpel and Arabidopsis silique development (Yant et al., 2010; Ripoll et al.,
2015). Finally, FUL activates miRNA172 (e.g. miRNA172C) expression through promoter binding
(Ripoll et al., 2015).(a) An elevated level of
miRNA172 inhibits apple fruit growth but promotes
Arabidopsis silique growth.(b) A reduced level of
miRNA172 promotes apple fruit growth but inhibits
Arabidopsis silique growth.
fact consistent when we consider the fruit structure differences between Arabidopsis and apple. Whereas Arabidopsis fruit (siliques) derive from ovary tissues (Ripoll et al.,
2015), the major part of apple fruit, the flesh cortex, develops from the hypanthium that is hypothesized to consist of
the fused bases of the sepals, petals and stamens (Pratt,
1988), while the inferior ovary becomes the core of the
fruit. Previous molecular studies (Yao et al., 1999, 2001;
Kotoda et al., 2000) have further indicated that sepals contribute to apple flesh cortex formation, but petals and stamens contribute no or little tissue to fruit development.
The apple A class floral homeotic gene MdAP1 is exclusively expressed in sepals during flower development (Kotoda et al., 2000) and in the flesh cortex during fruit
development (Yao et al., 1999). This indicates that the base
of sepals contributes to apple flesh cortex formation. The
apple B class floral homeotic gene MdPI is strongly
expressed in petals and stamens but not in developing
fruit (Yao et al., 2001). This indicates that petals and stamens contribute no or little tissue for fruit development. A
loss-of-function mutation in MdPI completely converts
petals to sepals and confers parthenocarpic fruit growth
(Yao et al., 2001).
AP2 is another class A floral homeotic gene working
together with AP1 to regulate sepal development (Weigel
and Meyerowitz, 1994). The apple genome has two AP2
homologs, MDP0000137561 and MDP0000204900, that are
expressed strongly during flower and fruit developmental
stages and weakly in leaf and root tissues, and a further
six AP2-related genes that are expressed weakly during
flower and fruit development as demonstrated by RNA
sequencing (Figure S1). The transcripts of all eight of these
genes contain a target sequence for miRNA172p
(Table S1).
MiRNA172p shares the same mature miRNA sequence
with miRNA172d-h. This mature miRNA sequence is pre-
sent at a relatively high level in apple flower and fruit tissues as shown by sequence analysis of small RNA
samples [Table S2, (Xia et al., 2012)]. Although this analysis cannot distinguish the expression of miRNA172p from
miRNA172d-h, analysis of EST data can distinguish their
pri-miRNA sequences. Analyses of an extensive collection
of apple EST data have identified three fruit ESTs for primiRNA172p, but none for pri-miRNA172d-h (Gleave et al.,
2008). This indicates that miRNA172p is the predominantly
expressed gene although expression of miRNA172d-h cannot be ruled out. It is well known that miRNA172 inhibits
AP2 translation in Arabidopsis (Chen, 2004). Although, due
to lack of AP2 antibody, the present study did not carry out
experiments to directly show that miRNA172p inhibits
translation of apple AP2 homologs, the overlapped expression domain of miRNA172p and AP2 mRNA in flower and
fruit tissues indicates that miRNA172p has the potential to
inhibit AP2 translation during flower and fruit development
so as to modify fruit size.
The opposite impact of miRNA172 on Arabidopsis and
apple fruit growth may be explained using a model (Figure 5) developed based results of this study and previous
studies. MiRNA172 promotes Arabidopsis silique growth
through negative regulation of AP2 that would otherwise
inhibit the AGAMOUS (AG) and FRUITFUL (FUL) (Yant
et al., 2010) that are required for ovary and silique growth
(Weigel and Meyerowitz, 1994; Yant et al., 2010; Ripoll
et al., 2015). Whereas, miRNA172 inhibits apple fruit
growth through negative regulation of AP2 that is required
for sepal and fruit flesh cortex development (Figure 5).
TRG3 over-expression miRNA172p had fewer cells in
hypanthium than RG before pollination and fewer and
smaller cells than RG after pollination. This indicates that
the effect of miRNA172 on fruit growth may begin by influencing the size of floral organs that contribute tissues to
fruit development and then modulating cell division and
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
424 Jia-Long Yao et al.
expansion in these tissues in response to fertilisation-induced signals and signal transduction.
Floral structures of TRG3 also showed a conversion of
some sepals or sepal segments to petal identity on some
flowers. This partial sepal to petal conversion is similar to
that reported following miRNA172 over-expression in
tobacco plants (Mlotshwa et al., 2006). Although change of
floral organ identity may influence fruit development (Yao
et al., 2001), this non-uniform partial conversion is unlikely
to be the direct cause for the reduction of fruit size since
the small fruit phenotype is very consistent. When the
expression level of miRNA172p was further increased in
TRG4, 5 and 6, their flowers consist of carpel tissues only.
Similar floral defects including stigmatic papillae on cauline margins and solitary carpels forming from the axil of
cauline leaves (Aukerman and Sakai, 2003) have been
reported in Arabidopsis plants over-expressing miRNA172.
In our study, a continuous series of carpel development
levels from leaf-like structures to fully developed carpels
was observed in three independent transgenic plants, supporting the theory of carpels being homologous to leaves
(Honma and Goto, 2001). Early flowering is a common
phenotypic change for over-expression of miRNA172 in
annual plant species (Aukerman and Sakai, 2003; Chen,
2004; Mlotshwa et al., 2006), however, this change was not
observed in apple. One explanation for this may be that
our apple transgenic plants were produced from the vegetative tissues of mature trees that had already passed the
juvenile phase.
We found the cafs allele of miRNA172p fixed in the cultivated apple and in its wild progenitors with relatively large
fruits, but not in phylogenetically more distant crab apples
with smaller fruits, also supporting the role of cafs in regulating apple fruit size. Crabapple species are an important
source of beneficial traits for apple breeding and our findings allow for the development and application of genetic
markers for the cafs allele to accelerate selection for fruit
size in breeding programmes during the introgression of
genes from distant crabapple accessions into commercial
breeding lines (Palmgren et al., 2015).
Our results thus indicate that the cafs allele has evolved
and has been fixed in several Malus species prior to
domestication. The fixation of the cafs allele in M. 9 domestica and its three closest wild species (M. sieversii,
M. orientalis and M. sylvestris) suggests selection on this
allele prior to the split of M. 9 domestica from the other
three species. The timing of the split between the four
large-fruited species is estimated between 20 000 and
80 000 years ago based on nuclear DNA analysis (Cornille
et al., 2012), which is earlier than the estimated onset of
apple domestication by humans, approximately 5000 years
ago (Juniper and Mabberley, 2006). The timing of this
selection is also supported by significantly lower nucleotide diversities of the cafs allele in these four species com-
pared to those of the CAFS allele in M. baccata and of
neutral genes in the other five species. Nevertheless, standard tests used for statistically analysing selection on
domestication loci did not detect significant departure
from neutrality for the CAFS locus. These statistics, relying
on patterns of nucleotide variation, are however known to
lose power to detect patterns shaped by ancient selection,
as the diversity is progressively restored (Przeworski,
2002). Therefore, the time elapsed between cafs fixation
and present time allowed restoration to a large extent of
the genetic diversity in the genomic cafs region, thereby
limiting statistical support to detect footprints of this
ancient selective sweep (Przeworski, 2002; Doebley et al.,
2006). Our results are consistent with the concept that large
mammals (such as the bears and horses that co-evolved
with Malus in the Tian Shan region), or even perhaps
humans, selected for large apple fruits prior to domestication (Juniper and Mabberley, 2006).
It is well known that the domestication genetic bottleneck is weaker in perennial fruit crops than in annual crops
(Miller and Gross, 2011; Cornille et al., 2012). Lower numbers of generations and high genetic heterozygosity for
perennial crops are considered as confounding factors to
explain the differences in genetic bottlenecks between
annuals and perennials (Miller and Gross, 2011). We have
shown that the long passage of time after the fixation of
an allele, which was most likely selected prior to apple
domestication has weakened the evidence for the genetic
bottleneck. Our study suggests that the evolution of agronomic traits before domestication may be another explanation for the weaker domestication genetic bottleneck in
perennial fruit crops than in annual crops.
EXPERIMENTAL PROCEDURES
Identification of miRNA172p target genes
The gene model sequences of M. 9 domestica ‘Golden Delicious’(Velasco et al., 2010) were aligned with the reverse complement sequences of miRNA172p using the Needle alignment
program (Rice et al., 2000) and the alignment results were filtered
using the miRNA alignment score rules (Allen et al., 2005). Genes
with a score of ≤4 were selected as strong target candidates.
Sequence analysis of mRNA and small RNA samples
For RNA-seq, total RNA was isolated using a method developed
for extraction of pine tree RNA (Chang et al., 1993) and small RNA
was extracted using the NucleoSpin miRNA kit (Macherey-Nagel,
http://www.mn-net.com/) from different apple tissue. RNA
sequencing and data processing followed methods previously
described (Xia et al., 2012; Foster et al., 2014).
Production and molecular analysis of apple transgenic
plants
To over-express miRNA172 in apple, a plant transformation vector
was constructed by transferring the cDNA of the primary
transcript of miRNA172p (pri-miRNA172p) (Gleave et al., 2008)
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
Apple fruit size evolution 425
(GenBank Accession No. EG999280) in Bluescript SK into the
BamH1/XhoI sites in pART7 (Gleave, 1992) between the CaMV35S
promoter and ocs terminator in sense orientation, and then moving the CaMV35S-promoter-miRNA172-cDNA-ocs-terminator fragment from pART7 into the NotI site in pART27 (Gleave, 1992) that
also contains the plant selection marker gene NPTII conferring
kanamycin resistance. Using this vector, RG apple transgenic
plants were produced employing Agrobacterium-mediated plant
transformation and kanamycin selection as previously described
(Yao et al., 1995, 2013). The transgenic plants were grown alongside non-transgenic RG plants in a containment glasshouse. Flowers were pollinated with ‘Granny Smith’ pollen.
The transgenic status of the plants was confirmed by PCR analysis of genomic DNA using two primers binding to the NPTII gene
(Yao et al., 2013). The presence of a transgenic copy of
miRNA172p was ascertained by PCR employing primer 35SF2
(50 -GCACAGTTGCTCCTCTCAGA-30 ) which binds to the CaMV35Spromoter, and primer R4 (Figure S6) which binds to the miRNA172p cDNA.
Small RNA was extracted from young expanding leaves and
opening flowers using the NucleoSpin miRNA kit (MachereyNagel). The process included an on-column removal of genomic
DNA using DNase. Small RNA was quantified using a NanoDrop
ND-1000 spectrophotometer (Thermo Fisher Scientific, http://
www.thermofisher.co.nz/). The relative levels of miRNA172 were
analysed using a stem-loop RT-PCR miRNA assay (Varkonyi-Gasic
et al., 2007) with primers designed against miRNA172 and two reference control genes miRNA156 and miRNA159 as previously
described (Varkonyi-Gasic et al., 2010). The primers used would
detect miRNA172 expressed from all miRNA172 genes (miRNA172a-p).
Tissue preparation, staining and image analysis
To analyse the hypanthium and fruit cortex tissue width and cell
size, tissue sections (10-lm thickness) of ovaries at full bloom and
fruit at 2 and 5 weeks post full bloom and at mature stage were
prepared from RG, TRG3 and ‘Aotea’ using the method described
previously (Jackson, 1992). The sections were dewaxed in xylene,
stained in 0.05% (w/v) toluidine blue (pH 4.5) and photographed
using a Vanox AHT3 light microscope (Olympus, Tokyo, http://
www.olympus-global.com/en/). Hypanthium and cortex tissue
width and cell area were measured using the IMAGEJ software
(http://imagej.nih.gov/ij/).
DNA sequence analyses
To determine the DNA sequence diversity at the miRNA172p
locus, DNA fragments (up to 3957 bp) were PCR amplified using
primers F1 (50 -GTACGCAGTAGAAAGGCCACATGA-30 ) located in
the promoter of miRNA172p and primer R3 (Figure S6) located in
the 30 end of pri-miRNA172p from 76 accessions of five Malus species (Table S8). Primer design was based on the ‘Golden Delicious’ apple genome sequence (Velasco et al., 2010). These Malus
accessions were collected from different regions of the world to
ensure a good representation of each species and were used in
previous studies to determine the genetic contributions of wild
species to the cultivated apple (Cornille et al., 2012). To determine
the sequence diversity at neutral genetic loci, DNA fragments
were amplified from 45 accessions of five Malus species
(Table S9) with primer pairs aligning to 13 genes (Table S10) as
previously described (Velasco et al., 2010). Platinum Taq DNA
Polymerase High Fidelity (Invitrogen, https://www.thermofisher.
com/nz/en/home/brands/invitrogen.html) was used in PCR to
minimize DNA synthesis errors. The amplicon was treated with
Exonuclease I and Shrimp Alkaline Phosphatase (New England
BioLabs, http://www.neb.uk.com/) before dispatch to Macrogen
(Korea) for sequencing. Sequence assembly and alignment and
genetic tree construction were performed using Geneious v6.1.6
(www.geneious.com/). DNA nucleotide diversity and selection
tests were performed using DnaSP v5.10.01 (http://www.ub.edu/
dnasp/). HKA tests were performed using the 2010 HKA software
of Jody Hey (https://bio.cst.temple.edu/~hey/program_files/HKA/
HKA_Documentation.htm).
Allelotyping of the miRNA172p locus in Malus accessions
To genotype the miRNA172p locus in 153 Malus accessions
(Table S8), PCR amplification was performed using primers F6
and R4 (Figure S6), located upstream and downstream of the TE
insertion respectively. The amplification resulted in a 331-bp DNA
fragment from the CAFS allele of miRNA172p containing no TE
insertion and a 494 = bp DNA fragment from the cafs allele containing a 154-bp TE and a 9-bp duplication of the insertion site.
Association of the cafs allele with QTL for fruit size
The association between miRNA172p alleles and mature fruit
weight was analysed using a progeny family of the cross RG
(cafs/cafs) X A689-24 (CAFS/cafs). A689-24 is a fourth generation
descendant from a cross between M. 9 domestica and M. zumi.
Of the progeny, 159 were scored for the cafs marker and measured for fruit weight (Table S11). A partially overlapping sample
of 173 seedlings from the family had been genotyped for 153 DNA
markers. Fruit size measurements of these trees were available in
each of three seasons, 2006–2008. A genetic map of A689-24 was
available (Chagne et al., 2008) for QTL mapping and for mapping
the cafs locus. Joinmap v3.0 was used to construct the genetic
map with a LOD (logarithm of the odds) score of 5 for grouping
and the Kosambi mapping function to calculate the genetic map
distances (see Method S1 for more details).
Quantitative RT-PCR
To determine whether the cafs allele induces a lower miRNA172p
expression than the CAFS allele, quantitative RT-PCR analyses
were performed using primers F5b and R7 (Figure S6), which
bind specifically to pri-miRNA172p, thereby avoiding any possible interference from miR172a-o. Total RNA was isolated from
pooled 1-week-old fruit (n > 5) of two CAFS/cafs accessions and
four cafs/cafs accessions using a method developed for extraction of pine tree RNA (Chang et al., 1993), and analysed using an
Agilent 2100 bioanalyzer (Agilent Co., Ltd, USA) to determine
RNA concentration and integrity, then treated with DNase. For
each RNA sample, 1 lg RNA was used for cDNA synthesis using
the Quantitectâ Reverse Transcription Kit (Qiagen, https://
www.qiagen.com/) according to the instructions of the manufacturer. Using the cDNA as template, qRT-PCR reactions were carried out using Actin and EF-1a as reference control genes in a
LightCyclerâ 480 (Roche Diagnostics) following previously
described procedures (Drummond et al., 2009).
ACKNOWLEDGEMENTS
This work was supported by the New Zealand Foundation for
Research, Science and Technology (FRST) (Contract C06X0207),
Plant & Food Research internal investment, the Region Ile-deFrance (PICRI) and Reseau de Recherche sur le Developpement
Soutenable (R2DS) Ile-de-France. J.X. was supported in part by
the China Scholarship Council. The authors acknowledge Plant &
Food Research colleagues Ian Hallett and Paul Sutherland for
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
426 Jia-Long Yao et al.
advice on light microscopy and discussions regarding yellow vacuoles in ‘Aotea’ apple, Tim Holmes for assistance with photography, Richard Newcomb for advice on neutrality analysis, and
Toshi Foster and David Greenwood for commenting on the
manuscript, as well as all colleagues who helped with wild Malus
accession sampling. This paper is dedicated to the memory of Dr
Nihal de Silva.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article.
Figure S1. Phylogenetic and expression analysis of AP2 genes.
Figure S2. Conversion of sepal tissue to petal identify in TRG3.
Figure S3. Plant height of transgenic ‘Royal Gala’ over-expressing
miRNA172p.
Figure S4. Over-expression of miRNA172p reduces hypanthium
and fruit cortex width and fruit cell size.
Figure S5. Phylogenetic analysis of the 4 kb genomic region of
miRNA172p.
Figure S6. The 30 region of the pri-miRNA172p sequence contains
a transposable element (TE).
Figure S7. The TE insertions in microRNA172p of accessions of
four Malus species are at the same site.
Figure S8. The TE in pri-miRNA172p belongs to a MITE-type transposon family.
Table S1. Potential target genes of miRNA17p in M. 9 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) 9 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.
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.
Method S1. Association of the cafs allele with quantitative trait
locus (QTL) for fruit size.
REFERENCES
Allen, E., Xie, Z.X., Gustafson, A.M. and Carrington, J.C. (2005) microRNAdirected phasing during trans-acting siRNA biogenesis in plants. Cell,
121, 207–221.
Aukerman, M.J. and Sakai, H. (2003) Regulation of flowering time and floral
organ identity by a microRNA and its APETALA2-like target genes. Plant
Cell, 15, 2730–2741.
Chagne, D., Gasic, K., Crowhurst, R.N., Han, Y., Bassett, H.C., Bowatte,
D.R., Lawrence, T.J., Rikkerink, E.H.A., Gardiner, S.E. and Korban, S.S.
(2008) Development of a set of SNP markers present in expressed genes
of the apple. Genomics, 92, 353–358.
Chang, S.J., Puryear, J. and Cairney, J. (1993) A simple and efficient method
for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116.
Chang, Y., Sun, R., Sun, H., Zhao, Y., Han, Y., Chen, D., Wang, Y., Zhang, X.
and Han, Z. (2014) Mapping of quantitative trait loci corroborates independent genetic control of apple size and shape. Sci. Hortic. 174, 126–132.
Chen, X.M. (2004) A microRNA as a translational repressor of APETALA2 in
Arabidopsis flower development. Science (Wash), 303, 2022–2025.
Cong, B., Barrero, L.S. and Tanksley, S.D. (2008) Regulatory change in
YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat. Genet. 40, 800–804.
Cornille, A., Gladieux, P., Smulders, M.J.M. et al. (2012) New insight into
the history of domesticated apple: secondary contribution of the European wild apple to the genome of cultivated varieties. PLoS Genet. 8,
e1002703.
Cornille, A., Giraud, T., Bellard, C., Tellier, A., Le Cam, B., Smulders, M.J.M.,
Kleinschmit, J., Roldan-Ruiz, I. and Gladieux, P. (2013a) Postglacial recolonization history of the European crabapple (Malus sylvestris Mill.), a
wild contributor to the domesticated apple. Mol. Ecol. 22, 2249–2263.
Cornille, A., Gladieux, P. and Giraud, T. (2013b) Crop-to-wild gene flow and
spatial genetic structure in the closest wild relatives of the cultivated
apple. Evol. Appl. 6, 737–748.
Cornille, A., Giraud, T., Smulders, M.J.M., Roldan-Ruiz, I. and Gladieux, P.
(2014) The domestication and evolutionary ecology of apples. Trends
Genet. 30, 57–65.
Devoghalaere, F., Doucen, T., Guitton, B. et al. (2012) A genomics approach
to understanding the role of auxin in apple (Malus x domestica) fruit size
control. BMC Plant Biol. 12, doi: 10.1186/1471-2229-12-7.
Doebley, J., Stec, A. and Hubbard, L. (1997) The evolution of apical dominance in maize. Nature (London), 386, 485–488.
Doebley, J.F., Gaut, B.S. and Smith, B.D. (2006) The molecular genetics of
crop domestication. Cell (Cambridge), 127, 1309–1321.
Drummond, R.S.M., Martinez-Sanchez, N.M., Janssen, B.J., Templeton,
K.R., Simons, J.L., Quinn, B.D., Karunairetnam, S. and Snowden, K.C.
(2009) Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 is
involved in the production of negative and positive branching signals in
petunia. Plant Physiol. 151, 1867–1877.
Foster, T.M., Watson, A.E., van Hooijdonk, B.M. and Schaffer, R.J. (2014)
Key flowering genes including FT-like genes are upregulated in the vasculature of apple dwarfing rootstocks. Tree Genet. Genom. 10, 189–202.
Frary, A., Nesbitt, T.C., Grandillo, S., Knaap, E.v.d., Cong, B., Liu, J., Meller,
J., Elber, R., Alpert, K.B. and Tanksley, S.D. (2000) fw2.2: a quantitative
trait locus key to the evolution of tomato fruit size. Science (Wash), 289,
85–88.
Fu, Y.X. and Li, W.H. (1993) Statistical tests of neutrality of mutations. Genetics, 133, 693–709.
Gleave, A.P. (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the
plant genome. Plant Mol. Biol. 20, 1203–1207.
Gleave, A.P., Ampomah-Dwamena, C., Berthold, S., Supinya, D., Karunairetnam, S., Bhawana, N., Wang, Y., Crowhurst, R.N. and MacDiarmid, R.M.
(2008) Identification and characterisation of primary microRNAs from
apple (Malus domestica cv. Royal Gala) expressed sequence tags. Tree
Genet. Genomes, 4, 343–358.
Gross, B.L. and Olsen, K.M. (2010) Genetic perspectives on crop domestication. Trends Plant Sci. 15, 529–537.
Han, Y.P. and Korban, S.S. (2007) Spring: a novel family of miniature
inverted-repeat transposable elements is associated with genes in apple.
Genomics (San Diego), 90, 195–200.
Harada, T., Kurahashi, W., Yanai, M., Wakasa, Y. and Satoh, T. (2005) Involvement of cell proliferation and cell enlargement in increasing the
fruit size of Malus species. Sci. Hortic. 105, 447–456.
Honma, T. and Goto, K. (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 409, 525–529.
Hudson, R.R., Kreitman, M. and Aguade, M. (1987) A test of neutral molecular evolution based on nucleotide data. Genetics, 116, 153–159.
Jackson, D. (1992) In situ hybridisation in plants. In Molecular Plant Pathology: A Practical Approach (Gurr, S., McPherson, M. and Bowles, D., eds).
Oxford: IRL Press, pp. 163–174.
Jofuku, K.D., Omidyar, P.K., Gee, Z. and Okamuro, J.K. (2005) Control of
seed mass and seed yield by the floral homeotic gene APETALA2. Proc.
Natl Acad. Sci. USA, 102, 3117–3122.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427
Apple fruit size evolution 427
Juniper, B.E. and Mabberley, D.J. (2006) The Story of the Apple. Portland:
Timber Press, Inc.
Kotoda, N., Wada, M., Komori, S., Kidou, S., Abe, K., Masuda, T. and Soejima, J. (2000) Expression pattern of homologues of floral meristem identity genes LFY and AP1 during flower development in apple. J. Am. Soc.
Hort. Sci. 125, 398–403.
Miller, A.J. and Gross, B.L. (2011) From forest to field: perennial fruit crop
domestication. Am. J. Bot. 98, 1389–1414.
Mlotshwa, S., Yang, Z., Kim, Y. and Chen, X. (2006) Floral patterning defects
induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana. Plant Mol. Biol. 61, 781–793.
Munos, S., Ranc, N., Botton, E. et al. (2011) Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near
WUSCHEL. Plant Physiol. 156, 2244–2254.
Palmgren, M.G., Edenbrandt, A.K., Vedel, S.E. et al. (2015) Are we ready for
back-to-nature crop breeding? Trends Plant Sci. 20, 155–164.
Phipps, J.B., Robertson, K.R., Smith, P.G. and Rohrer, J.R. (1990) A checklist
of the subfamily Maloideae (Rosaceae). Can. J. Bot. 68, 2209–2269.
Pratt, C. (1988) Apple flower and fruit: morphology and anatomy. Hortic.
Rev. 10, 273–308.
Przeworski, M. (2002) The signature of positive selection at randomly chosen loci. Genetics, 160, 1179–1189.
Rice, P., Longden, I. and Bleasby, A. (2000) EMBOSS: the European molecular biology open software suite. Trends Genet. 16, 276–277.
Ripoll, J.J., Bailey, L.J., Mai, Q.-A., Wu, S.L., Hon, C.T., Chapman, E.J., Ditta,
G.S., Estelle, M. and Yanofsky, M.F. (2015) microRNA regulation of fruit
growth. Nat. Plants, 1, doi:10.1038/nplants.2015.36.
Studer, A., Zhao, Q., Ross-Ibarra, J. and Doebley, J. (2011) Identification of
a functional transposon insertion in the maize domestication gene tb1.
Nat. Genet. 43, 1160–1163.
Tajima, F. (1983) Evolutionary relationship of dna-sequences in finite populations. Genetics, 105, 437–460.
Tajima, F. (1989) Statistical-method for testing the neutral mutation hypothesis by dna polymorphism. Genetics, 123, 585–595.
Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E.F. and Hellens, R.P. (2007)
Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods, 3, doi:10.1186/1746-4811-3-12.
Varkonyi-Gasic, E., Gould, N., Sandanayaka, M., Sutherland, P. and MacDiarmid, R.M. (2010) Characterisation of microRNAs from apple (Malus
domestica ‘Royal Gala’) vascular tissue and phloem sap. BMC Plant Biol.
10, doi: 10.1186/1471-2229-10-159.
Velasco, R., Zharkikh, A., Affourtit, J. et al. (2010) The genome of the
domesticated apple (Malus x domestica Borkh.). Nat. Genet. 42, 833–839.
Watterson, G.A. (1975) Number of segregating sites in genetic models without recombination. Theor. Popul. Biol. 7, 256–276.
Weigel, D. and Meyerowitz, E.M. (1994) The ABCs of floral homeotic genes.
Cell (Cambridge), 78, 203–209.
Xia, R., Zhu, H., An, Y.Q., Beers, E.P. and Liu, Z.R. (2012) Apple miRNAs and
tasiRNAs with novel regulatory networks. Genome Biol. 13, R47.
Yant, L., Mathieu, J., Dinh, T.T., Ott, F., Lanz, C., Wollmann, H., Chen, X.M.
and Schmid, M. (2010) Orchestration of the floral transition and floral
development in Arabidopsis by the bifunctional transcription factor APETALA2. Plant Cell, 22, 2156–2170.
Yao, J.-L., Cohen, D., Atkinson, R., Richardson, K. and Morris, B. (1995) Regeneration of transgenic plants from the commercial apple cultivar Royal
Gala. Plant Cell Rep. 14, 407–412.
Yao, J.-L., Dong, Y.H., Kvarnheden, A. and Morris, B. (1999) Seven MADSbox genes in apple are expressed in different parts of the fruit. J. Am.
Soc. Hort. Sci. 124, 8–13.
Yao, J.-L., Dong, Y.-H. and Morris, B.A. (2001) Parthenocarpic apple fruit
production conferred by transposon insertion mutations in a MADS-box
transcription factor. Proc. Natl Acad. Sci. USA, 98, 1306–1311.
Yao, J.-L., Tomes, S. and Gleave, A.P. (2013) Transformation of apple
(Malus 9 domestica) using mutants of apple acetolactate synthase as a
selectable marker and analysis of the T-DNA integration sites. Plant Cell
Rep. 32, 703–714.
© 2015 The Authors
The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 417–427