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Genes encoding aluminum-activated malate transporter II and their association with fruit 1

acidity in apple 2

Baiquan Ma1,3

, Liao Liao1, Hongyu Zheng

1,3, Jie Chen

2,3, Benhong Wu

2, Collins Ogutu

1,3, Shaohua Li

1, 3

Schuyler S. Korban4, Yuepeng Han

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1Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden of 7

the Chinese Academy of Sciences, Wuhan, 430074, P.R. China 8

2Beijing Key Laboratory of Grape Sciences and Enology, and CAS Key Laboratory of Plant Resources, 9

Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 10

3Graduate University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing, 100049, P.R. China 11

4Department of Biology, University of Massachusetts Boston, Boston, MA 02184 USA 12

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�Corresponding author: [email protected] 15

Tel/Fax: 86-27-8751-0872 16

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of 43The Plant Genome Accepted paper, posted 06/23/2015. doi:10.3835/plantgenome2015.03.0016

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Abstract: A gene encoding aluminum-activated malate transporter (ALMT) was previously reported as a 1

candidate for the Ma locus controlling fruit acidity in apple. In this study, we found that apple ALMT genes 2

can be divided into three families and the Ma1 gene belongs to the ALMTII family. Duplication of ALMTII 3

genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has 4

occurred between the Ma1 gene and its homologues in the ALMTII family and only the Ma1 gene is 5

significantly associated with malic acid content. The Ma locus consists of two alleles Ma1 and ma1. Ma1 6

resides in the tonoplast, and its ectopic expression in yeast was found to significantly increase the influx of 7

malic acid into yeast cells, suggesting it may function as a vacuolar malate channel. In contrast, ma1 8

encodes a truncated protein due to a single nucleotide substitution of G with A in the last exon. As this 9

truncated protein resides within the cell membrane, it is deemed non-functional as a vacuolar malate 10

channel. The frequency of the Ma1Ma1 genotype is very low in apple cultivars, but high in wild relatives, 11

which suggests that apple domestication may be accompanied by selection at the Ma1 gene. In addition, 12

variations in malic acid content of mature fruits were also observed between accessions with the same 13

genotype in the Ma locus. This suggests that the Ma gene is not the only genetic determinant of fruit 14

acidity in apple. 15

Key words: Apple, malic acid, domestication, association mapping, gene duplication 16

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Introduction 1

Apple (Malus × domestica Borkh.) is one of the most important fruit trees in temperate regions, and 2

displays a juvenile period of 6 to 10 years or even longer. The apple genome is highly heterozygous due 3

to self-incompatibility. Genetic improvement of apple using conventional breeding methods is hampered 4

by this long juvenility phase. To improve efficiency of apple breeding efforts, it is critical to accurately 5

identify young seedlings possessing desirable horticultural traits early using molecular-marker assisted 6

selection. In the past few decades, several studies have been conducted to identify molecular markers 7

that are closely linked to either major genes or quantitative trait loci (QTL) controlling economically 8

important traits in apple, including plant architecture (Kenis and Keulemans 2007; Segura et al. 2008), 9

disease resistance (Xu and Korban 2002; Belfanti et al. 2004; Le Roux et al. 2010), bud dormancy (Brunel et 10

al. 2002; Dyk et al. 2010), and fruit quality (Espley et al. 2009; Mellidou et al. 2012; Longhi et al. 2012 , 11

2013; Khan et al. 2012, Zhang et al 2012). 12

Genetic mapping in apple, as well as in other fruit trees, is usually undertaken using experimental 13

populations derived from bi-parental crosses. However, establishing and maintaining bi-parental crosses 14

and progenies of fruit trees are very difficult and costly. In addition, genetic resolution of QTL maps is 15

often limited due to occurrence of only limited numbers of meiotic events within a bi-parental mapping 16

population (Flint-Garcia et al. 2003). These negative factors together with the long juvenility phase 17

significantly contribute to low efficiency of linkage mapping, particularly for fruit quality traits. As a 18

result, despite numerous linkages mapping studies conducted for a variety of fruit tree species, only few 19

genes have been cloned using linkage-based mapping (Khan and Korban 2012). 20

Recently, an alternative approach, known as association mapping, has become increasingly popular 21

in pursuing genetic mapping studies in plants as it has demonstrated promise in overcoming the 22

limitations of linkage mapping (Rafalski, 2010). Association mapping, also referred to as linkage 23

disequilibrium (LD) mapping, identifies genetic polymorphisms that are significantly associated with 24

phenotypic variation. Compared to QTL mapping, association mapping offers several advantages, 25

including: (1) reduced time frame due to the use of an existing natural population instead of a progeny 26

resulting from a bi-parental cross; (2) availability of greater allele numbers; and (3) higher mapping 27

resolution as it exploits historical and evolutionary recombination events at the population level 28

(Flint-Garcia et al. 2005; Yu and Buckler 2006). Association analysis is divided into genome wide 29

association study (GWAS) and candidate gene association analysis. In GWAS, a high density of single 30


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nucleotide polymorphisms (SNPs) across the genome is often used to test potential loci associated with 1

phenotypic variation. Candidate gene association analysis relies on developing molecular tags of potential 2

candidate genes for detecting their association with phenotypic variation. 3

To date, association mapping has been successfully used to identify candidate genes responsible for 4

complex traits of interest in a variety of plants such as maize, pine, and Arabidopsis (Thornsberry et al. 5

2001; Gonzalez-Martinez et al. 2007; Ehrenreich et al. 2009; Li et al. 2013). With the release of whole 6

genome sequences of several fruit species including grape (Jaillon et al. 2007), apple (Velasco et al. 2010), 7

peach (Verde et al. 2013), pear (Wu et al. 2013), and citrus (Xu et al. 2013), several commercial SNP arrays 8

have been developed (Chagné et al. 2012; Khan et al. 2012; Myles et al. 2010; Verde et al. 2012), paving 9

the way for pursuing association mapping studies. For example, association mapping has recently been 10

reported in apple (Kumar et al. 2012, 2013). In addition, association mapping combined with QTL 11

mapping has also been conducted in both apple and grapevine (Fournier-Level et al. 2010; Dunemann at al. 12

2012; Khan et al. 2013; Longhi et al. 2013). 13

Fruit quality is a highly desirable trait in fruit breeding efforts. Organic acids together with sugars 14

and aromas contribute to the taste of fresh apple fruits. In mature apple fruits, malic acid is the 15

predominant organic acid, accounting for about 90% of the total organic acid content (Wu et al. 2007; 16

Zhang et al 2012). To date, several studies have been carried out to identify QTLs responsible for apple 17

fruit acidity, and a major QTL (designated Ma) has been mapped onto the top of linkage group (LG) 16 18

(Maliepaard et al. 1998; Liebhard et al. 2003; Kenis et al. 2008). More recently, a gene encoding 19

aluminum-activated malate transporter (ALMT) was identified as a potential candidate for the Ma locus 20

(Bai et al. 2012; Khan et al. 2013). Apple is a diploid, but has a polyploidy origin (Velasco et al. 2010). It 21

is unclear whether the homologues of the Ma1 gene are also responsible for fruit acidity although several 22

QTLs other than the Ma locus have been reported in apple (Liebhard et al. 2003; Kenis et al. 2008). The 23

Overall knowledge on genetic basis of the biosynthesis and accumulation of malic acid in apple fruits is still 24

limited. 25

Recently, we initiated the measurement of fruit quality characteristics in apple germplasm, and 26

found that fruit acidity is likely to have undergone selection during apple domestication (Ma et al. 2015). 27

The malic acid database of apple germplasm also provides an opportunity to investigate genetic basis of 28

fruit acidity using association mapping method. Since duplicated genes often share redundant functions 29

(Qian et al. 2010), we investigated the effect of homologues of the Ma1 gene on fruit acidity using 30


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candidate gene-based association mapping. The subcellular localization and functionality of the Ma1 gene 1

were also investigated. This study not only provide an insight into the role of the Ma1 gene in 2

determining fruit acidity in apple, but it also demonstrates that candidate gene-based association mapping 3

is an efficient strategy for identifying genes involved in complex traits in apple, and perhaps in other fruit 4

species as well. 5

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Materials and Methods 7

Plant material 8

The apple collection used in this study is maintained at Xingcheng Institute of Pomology of the 9

Chinese Academy of Agricultural Sciences, Xingcheng, Liaoning, China. The collection consists of 352 10

cultivars and/or accessions that were reported in our previous study (Ma et al. 2015) and one Chinese wild 11

accession Lenghaitang (Additional data 1). 12

For the accession Lenghaitang, leaf sample was collected in the spring season and fruit sample was 13

collected at mature stage in 2010. The maturity was assessed by checking the colour of the peel and a 14

confirmation of the seed colour changing to brown. Each accession had three replicates, consisting of ten 15

fruits. Fruit skins were peeled off and then the flesh was cut into small sections. Both fruit and leaf 16

samples of ‘Lenghaitang’ were immediately frozen in liquid nitrogen, and then stored at -75 °C until use. 17

Leaf samples of the rest 352 accessions were the same as described in our previous study (Ma et al. 2015). 18

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Measurements of malic acid contents in apple fruits 20

The measurement of malic acid content was only conducted for the accession Lenghaitang according 21

to our previous protocol (Ma et al. 2015). Briefly, five grams of frozen fruit were ground into powder in 22

liquid nitrogen, homogenized with 20 mL ddH2O, and incubated at room temperature for 5 min. The 23

mixture was then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was filtered successively 24

through solid phase extraction (Waters Spe-pak C18 Vac cartridges) and 0.22 μM millipore membrane. The 25

filtered fluid was used for measurement of malic acid content using high performance liquid 26

chromatography (HPLC), and the standard malic acid was purchased from Sigma (St. Louis, MO, USA). The 27

malic acid contents of the rest 352 accessions were measured in our previous study (Ma et al. 2015). 28

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Development of molecular tags of ALMT genes and their utility in genotyping of individuals in an apple 30


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collection 1

Genomic DNA sequences of ALMT genes were retrieved from the draft genome of cv. Golden 2

Delicious (Velasco et al. 2010) to design and develop SSR and SNP markers. SSRs were identified using 3

the program of Perfect Microsatellite Repeat Finder 4

(http://sgdp.iop.kcl.ac.uk/nikammar/repeatfinder.html), and primers flanking SSR loci were designed 5

using Primer 3.0. In addition, a SNP in the coding region of Ma1 was used to develop a cleaved amplified 6

polymorphic sequence (CAPS) marker, and the corresponding primer sequences are as follows: 7

5’-AGAGTTGGTGTGGAATGTGC-3’ and 5’-TTGCCTTCTCACTCAGCTCT-3’. Amplification was performed using 8

the following program: 95 °C 5 min, 35 cycles of 30 s at 95 °C, 30 s at 60 °C, 45 s at 72 °C, along with a final 9

extension of 10 min at 72 °C. 10

A total of 5 μL of PCR products was mixed with an equal volume of loading buffer (98% formamide, 11

0.05% bromophenol blue, 0.05% xylene cyanol, and 10 mM NaOH), denatured at 95 °C for 5 min, and then 12

immediately chilled on ice. Denatured PCR products were resolved on 8% denaturing polyacrylamide gel 13

in 1X Tris-acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 20 mM acetic acid, and 1 mM EDTA). 14

Electrophoresis was carried out at 1200 V for 40 min. Bands were visualized using silver staining, and 15

their sizes were estimated using a 25 bp DNA ladder standard (Promega, Madison, WI). 16

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Analysis of population structure and marker-trait association 18

Seventeen SSR markers distributed along different chromosomes (Table S1) were randomly selected 19

to genotype individuals using polyacrylamide gel electrophoresis, as described above. Genotyping data 20

were collected to estimate the most likely K value (Q matrix) using STRUCTURE 2.2 software (Pritchard et 21

al. 2000). Genetic stratification was analyzed using a variant of the Markov chain Monte Carlo (MCMC) 22

algorithm. Patterns of genetic structure were calculated in multiple runs, wherein each run consisted of 23

500,000 iterations with a burn-in period of 50,000. The parameter K was set from 1 to 15, and 10 24

replicates were performed for each value of K. A modal value of ΔK was used to assess the most likely K 25

corresponding to Q matrix (Evanno et al. 2005). An individual was classified into a cluster when the 26

assignment probability was greater than 0.75. In addition, a microsatellite analyzer (MSA) software was 27

used to estimate relative kinship (K matrix) (Dieringer and Schlštterer 2003). 28

A total of 353 individuals were used to detect association between molecular markers and malic acid 29

content. Association analysis was performed with the software package TASSEL 3.0 using a mixed linear 30


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model (MLM) (Yu et al. 2006; Bradbury et al. 2007). This method utilized both the population structure Q 1

matrix and a K matrix. The critical p value for analyzing the significance of molecular markers was 2

calculated based on the false discovery rate (FDR) for malic acid content (Benjamini and Hochberg 1995; 3

Benjamini and Yekutieli 2001), which was found to be highly stringent. The criterion for marker-trait 4

association was set at P ≤0.005. In addition, the haplotype association test was performed using the PLINK 5

software, as described by Purcell et al. (2007), and the criterion for haplotype-trait association was set at 6

P ≤0.01. 7

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RNA isolation and quantitative real-time PCR (qRT-PCR) 9

Eight cultivars, i.e. Aifeng, Belle de Boskoop, Zhumary, Jincui, Xinnongjin, McIntosh Red, Annuo, and 10

Yanhongmei, were selected for qRT-PCR assay and their fruit samples collected at 30, 60, and 90 days after 11

full bloom (DAFB). Each accession had three replicates, consisting of five fruits. Fruit flesh tissues of each 12

replicate were cut into small sections, mixed, and then subjected to total RNA extraction. Total RNA 13

extraction was performed using RNAprep pure Plant kit (Tiangen, Beijing, China) according to the 14

manufacturer’s instructions, and then adjusted to 500 ng/μL using NanoDrop™ Lite Spectrophotometer. 15

RNA extractions were treated with DNase I (Takara, Dalian, China) to remove any contamination of 16

genomic DNA. Approximately, 3 μg total RNA per sample was used for cDNA synthesis using TransScript 17

One-Step gDNA Renoval and cDNA Synthesis SuperMix (TRANS, Beijing, China) according to 18

manufacturer's instructions. A pair of primers 5’-GACTTGGGCTTCAACAGCTC-3’/5’-TTTTCGAGGATCCGAATGAC-3’ 19

was designed to investigate expression profiles of Ma1, a candidate gene of the Ma locus on LG16 of 20

apple. qRT-PCR was carried out in a total volume of 20 µL reaction mixture containing 10.0 µL of 2× SYBR 21

Green I Master Mix (Takara, Dalian, China), 0.2 µM of each primer, and 100 ng of template cDNA. 22

Amplifications were performed using Applied Biosystems® 7500 Real-Time PCR Systems (Applied 23

Biosystems, USA). The amplification program consisted of 1 cycle of 95 °C for 3 min, followed by 40 cycles 24

of 95 °C for 30 sec, and 60 °C for 34 sec. The fluorescent product was detected at the second step of each 25

cycle. Melting curve analysis was performed at the end of 40 cycles to ensure proper amplification of 26

target fragments. An actin gene was used as a constitutive control along with the following primer 27

sequences: 5’-TGACCGAATGAGCAAGGAAATTACT-3’ and 5’-TACTCAGCTTTGGCAATCCACATC-3’. All analyses were 28

repeated three times using biological replicates. 29

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Subcellular localization using a green fluorescent protein (GFP) 1

Subcellular localization of two alleles Ma1 and ma1 were conducted. The allele ma1 contains an 2

early stop codon due to a single nucleotide mutation, and its coding region is 252 bp shorter in length 3

compared to that of the allele Ma1. Therefore, two pairs of primers, 4

5’-GCGTCATGAATGGCGGCCAAAATCGGGTCC-3’/5’-CGTTCTTCAACCGCAAACTC-3’ and 5

5’-GCGTCATGAATGGCGGCCAAAATCGGGTCC-3’/5’-CTGATATTGGTCGTTTTAAAAGAC-3’, were designed to amplify the 6

coding regions of Ma1 and ma1, respectively, using cDNA templates from leaf tissues of apple cv. Golden 7

Delicious. 8

PCR products were purified, inserted into the TA cloning vector pEASY-T1, and inserts were validated 9

by direct sequencing. Plasmid DNAs were extracted and digested with two restriction enzymes, BspHI 10

and XbaI. DNA fragments were recovered and inserted into the expression vector pCAMBIA1302::GFP 11

under the control of cauliflower mosaic virus (CaMV) 35S promoter, thus generating constitutively 12

expressed fusion proteins Ma1-GFP and ma1-GFP. The two constructs were individually transformed 13

into epidermal cells of onion (Allium cepa) using a modified Agrobacterium infection method as described 14

by Sun et al. (2007). GFP-dependent fluorescence was detected at 12-36 h following transfection using a 15

D-FL EPI-Fluoresecence Attachment Instructions (Nikon, Japan). 16

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Construction of yeast expression vectors 18

A pair of primers, 5’-CTCTTGTACGCTGTTCCGCT-3’/5’-ATGAGATCCAACCTTGGCC-3’, was designed to amplify 19

cDNA fragments of Ma1 using cDNA templates from fruits of cv. Golden Delicious. cDNA fragments were 20

cloned into the pEASY-T1 vector, and plasmid DNAs were used as PCR templates to generate full coding 21

sequences of Ma1 allele using a pair of primers 22

5’-GCGTGATCAATGGCGGCCAAAATCGGGTCC-3’/5’-GCGGCGGCCGCTTAGTTCTTCAACCGCAAAC-3’. The full coding 23

sequences were also cloned into the pEASY-T1 vector. Plasmid DNAs were then extracted, digested with 24

BclI and NotI, and ligated into a BamHI/NotI-digested mediated vector. DNA fragments covering the 25

TDH3 promoter, full coding regions of either Ma1 or ma1 alleles, and terminator sequences were then 26

amplified with a pair of primers 27

5’-GGTACCCTGCCATTTCAAAGAATACGTAAATAA-3’/5’-GTCGACATCATGTAATTAGTTATGTCACGCT-3’, and subsequently 28

inserted into the pEASY-T1 vector. 29

The cloned vector was digested with two enzymes, KpnI and XhoI. DNA fragments were retrieved, 30


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and introduced into the KpnI/SalI site of an integrative yeast expression vector pRS406, generating a 1

construct pRS406-Ma1. The integration vectors pRS406 and pRS406-Ma1 were linearized with the 2

restriction enzyme StuI, and then integrated into the ura3-52 locus of Saccharomyces cerevisiae WAT11 3

strain to generate yeast strains WAT11/pRS406 and WAT11/pRS406-Ma1, based on the PEG/LiAc method 4

(Gietz and Woods 2002). 5

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Yeast collection and malic acid content measurements 7

Yeast strains were inoculated into 250 mL YPDA liquid medium, and incubated overnight at 30°C with 8

shaking at 250 rpm. Yeast cells were collected by centrifugation, and resuspended in 5 mL ddH2O. One mL 9

yeast solution was transferred into a 1.5 mL eppendorf tube, and 1.0 g of acid washed glass beads 10

(425-600 µm, Sigma) was added to mechanically disrupted yeast cells using grinder mill Scientz-48 11

(Zhejiang, China). Cellular fragments were sedimented by centrifugation at 12,000 rpm for 30 min at 4°C, 12

and the supernatant was collected and transferred to a new 1.5 mL Eppendorf tube. The supernatant was 13

subsequently filtered through solid phase extraction (Waters Spe-pak C18 Vac cartridges) and 0.22 μM 14

millipore membrane. The filtered fluid was used for measurement of malic acid content using HPLC. All 15

analyses were repeated three times using biological replicates. One-way analysis of variance (ANOVA) was 16

conducted using SPSS statistics 17.0 software (SPSS Inc. Released 2008. SPSS Statistics for Windows, 17

Version 17.0. Chicago: SPSS Inc). 18

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Results 20

Genes encoding aluminum-activated malate transporter in the apple genome 21

The DNA coding sequence of ALMT family in Arabidopsis (Kovermann et al. 2007) was used as a 22

query sequence to identify homologous genes by Blast Searches of the reference genome sequence of 23

apple (Velasco et al. 2010). A total of 22 homologues were identified on chromosomes 1, 2, 3, 6, 11, 13, 14, 24

15, and 16, or among the unanchored sequences (Fig. 1a). Phylogenetic analysis revealed that these 25

homologues are grouped into three gene families, designated ALMTI, ALMTII and ALMTIII (Fig. 1b). The 26

ALMTI family includes eight homologues (MDP0000160829, MDP0000553830, MDP0000226551, 27

MDP0000873233, MDP0000227375, MDP0000851070, MDP0000186135, and MDP0000269351), whereas 28

the ALMTIII family includes six homologues (MDP0000392887, MDP0000261132, MDP0000315809, 29

MDP0000255420, MDP0000144432, and MDP0000230322). The ALMTII family consists of eight 30


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homologues (MDP0000791995, MDP0000259751, MDP0000312421, MDP0000252114, MDP0000768640, 1

MDP0000244249, MDP0000299697, and MDP0000290997), one of which (MDP0000252114) was 2

designated Ma1 gene in previous report (Bai et al. 2012). Thus, the Ma1 gene and its closely related 3

homologues in the ALMTII family were chosen to investigate their association with fruit acidity in apple. 4

Additionally, five more ALMT like genes (MDP0000217912, MDP0000241893, MDP0000315808, 5

MDP0000271850, and MDP0000196725), besides the 22 ALMT genes, were also reported in the apple 6

genome (Khan et al. 2013). However, MDP0000241893, MDP0000315808, and MDP0000271850 are 7

almost identical in DNA sequence to MDP0000261132, MDP0000255420, and MDP0000791995, 8

respectively. MDP0000196725 has a very short open reading frame (468 bp in size), and are supposed to 9

be a pseudogene. MDP0000217912 has low levels of sequence identity to all the 22 ALMT genes, and was 10

separated into an outgroup (data not shown). Thus, these additional five ALMT like genes were excluded 11

from the ALMT gene family in this study. 12

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Association between ALMTII genes and fruit acidity in apple 14

Molecular tags, including 7 SSRs and 1 CAPS, were developed for the eight apple ALMTII genes (Table 15

1). These gene tags were subsequently used to screen a collection of 353 Malus accessions, and 16

genotyping data were collected for association mapping analysis (additional data 1). The malic acid 17

content in mature fruits ranged from 0.53 to 22.74 mg/g FW among the apple collection, with an average 18

of 4.85 mg/g FW. To avoid false associations between markers and traits of interest, the genetic structure 19

of the apple collection was evaluated. The STRUCTURE results indicated a peak ΔK value of K=3 (Fig. S1). 20

This suggested that there were three genetically distinct clusters present in this collection. Thus, the 21

corresponding Q and Kinship matrices were calculated, and used in the TASSEL 3.0 software. Candidate 22

gene-based association mapping was performed using a mixed linear model (MLM) method. As a result, 23

only the Ma1 gene (MDP0000252114) was significantly associated with malic acid content (P=6.07E-04), 24

accounting for ~7.58% of the observed phenotypic variation (Table 1). Molecular tag of the Ma1 gene 25

(WBG90, 1.4 kb downstream of the stop codon) revealed three genotypes, (TC)17(TC)17, (TC)20(TC)17, and 26

(TC)20(TC)20, in the apple collection (Fig. 2a, 2b-I). Mean values of malic acid contents in mature fruits 27

displayed the following pattern among the three genotypes: (TC)17(TC)17 > (TC)20(TC)17 > (TC)20(TC)20 (Fig. 3). 28

In contrast, no significant associations were observed between the homologues of the Ma1 gene and 29

malic acid contents (Table 1, P≥0.14). 30


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Since an A/G SNP 252 bp upstream of the stop codon of the Ma1 gene has been reported in previous 1

study (Bai et al. 2012), we also developed a CAPS marker to detect the A/G polymorphism (Fig. 2b-II). This 2

CAPS marker revealed three genotypes, designated AA, AG, and GG, in the apple collection. The allele ‘A’ 3

contained a GACC sequence resulting in a premature stop codon, while the ‘G’ allele containing a GGCC 4

sequence had a complete coding sequence (the same hereinafter). Mean values of malic acid contents in 5

mature fruits showed significant differences among the three genotypes and displayed the following 6

patterns: GG > AG > AA (Fig. 3). 7

The combination of a SNP locus and an SSR motif were evaluated, and four haplotypes, designated 8

HapA through HapD, were identified (Table 2). All haplotypes, except for HapC were significantly 9

associated with malic acid content. HapA and HapD showed the highest effects on malic acid content in 10

apple fruits. 11

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Expression profiling of ALMTII genes in apple fruits at different stages 13

RT-PCR was initially conducted to investigate the expression profiles of ALMTII genes in fruits of four 14

cultivars, Yanhongmei, Belle de Boskoop, Zhumary, and Aifeng (Fig. 4). These four cultivars have similar 15

ripening periods, with maturation occurring about 90 days after full bloom, but show a great variation in 16

malic acid content. Of the eight ALMTII genes, the Ma1 gene was expressed during fruit development in all 17

tested cultivars. However, six ALMTII genes (MDP0000312421, MDP0000791995, MDP0000244249, 18

MDP0000259751, MDP0000290997, and MDP0000768640) were not expressed in all tested cultivars. One 19

ALMT gene (MDP0000299697) was weakly expressed during early stage of fruit development. 20

Furthermore, we evaluated transcript levels of the Ma1 gene using qRT-PCR. As mentioned above, 21

three genotypes were detected using a CAPS marker developed from the A/G polymorphic site present in 22

the last exon of the Ma1 gene. Thus, fruits of eight cultivars representing different genotypes were 23

selected for gene expression analysis. Transcript accumulation of the Ma1 gene was detected in fruits at 24

different developmental stages and in all genotypes, but showed different patterns during fruit 25

development (Fig. 5). For example, during fruit development, increased transcript accumulation was 26

observed in cvs. Xinnongjin, Jincui, and Yanhongmei, while slight reduction in transcript levels was 27

detected in cvs. Belle de Boskoop, Annuo, and McIntosh Red. In contrast, transcript levels were relatively 28

stable during fruit development in cvs. Aifeng and Zhumary. 29

Finally, we evaluated the relationship between transcript levels of Ma1 and malic acid content. 30


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Although transcript levels for cvs. Aifeng and Zhumary were quite similar, malic acid contents were 1

significantly different. Similarly, transcript accumulation patterns were very similar between cvs. 2

Xinnongjin and Yanhongmei, while malic acid contents of cv. Yanhongmei were significantly higher than 3

those of cv. Xinnongjin. Furthermore, transcript levels of the Ma1 gene in fruits of cv. Aifeng were slightly 4

higher than those of cv. Belle de Boskoop, yet malic acid content of cv. Aifeng was lower than that of cv. 5

Belle de Boskoop. 6

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Subcellular localization of Ma1 and its functional analysis by ectopic expression in yeast 8

The TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) predicted that Ma1 9

consists of seven transmembrane domains (TMα1 to TMα7), and a single nucleotide mutation that results 10

in a premature stop codon located between TMα6 and TMα7 (Fig. 2c). To investigate effects of single 11

nucleotide mutation on gene functionality, subcellular localization analysis of two alleles of the Ma1 gene 12

was conducted. Allele containing a GACC sequence was designated as ma1, while the other containing a 13

GGCC sequence was designated as Ma1. Two constructs, 35S: Ma1-GFP and 35S: ma1-GFP, were 14

transiently transformed into onion epidermal cells using agro-infection (Fig. 6). It was found that the 15

Ma1-GFP fusion protein resided in the tonoplast, as the nucleus was surrounded with fluorescence, while 16

the ma1-GFP fusion protein was targeted to the cell membrane. This finding indicates that the C-terminal 17

amino acid sequence of Ma1 plays an important role in directing the protein to a specific subcellular site. 18

The Ma1 gene is closely related to AtALMT9 in Arabidopsis (Fig. 1b). AtALMT9 serves as a vacuolar 19

malate channel in Arabidopsis (Kovermann et al. 2007). Thus, the functionality of the Ma1 gene was 20

investigated via ectopic expression in yeast. When yeast lines were incubated in a standard YPDA liquid 21

medium, no differences in malic acid contents were observed between transgenic lines expressing Ma1 or 22

ma1 and control yeast lines carrying an empty vector (Table 3). However, in the presence of 0.02 g/mL 23

malic acid added to the YPDA liquid medium, malic acid contents increased significantly in transgenic lines 24

expressing Ma1 when compared to control lines carrying ma1 or the empty vector. However, no 25

significant difference in malic acid contents was detected between transgenic lines expressing ma1 and 26

control lines carrying the empty vector. This result suggests that Ma1, similar to AtALMT9, may also 27

function as a channel to increase the influx of malic acid, but ma1 is likely nonfunctional. In other words, 28

Ma1 is potentially associated with accumulation of malic acid in apple fruits. 29

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Discussion 1

Duplication of ALMTII genes in apple and their divergence in expression 2

Gene duplication, which can arise from polyploidization and/or segmental duplication, is a major 3

driving force for recruitment of genes in plants. Apple is a diploid, with an autopolyploidization origin (Han 4

et al., 2011; Velasco et al. 2010). In this study, eight ALMTII genes have been identified in the apple 5

genome. Of these ALMTII genes, seven are located on chromosomes 1, 6, 13, 14, and 16, and one is 6

among the unanchored sequences. Chromosomes 6 and 14 are homologous pairs (Han et al., 2011; 7

Velasco et al. 2010) and both contain an ALMTII gene on the bottom chromosome. Similarly, 8

chromosomes 13 and 16 are also homologous pairs and both contain a cluster of two ALMTII genes on the 9

top chromosome. These results suggest that duplication of ALMTII genes in apple is related to whole 10

genome duplication during the process of speciation, and the tandem duplication on chromosomes 13 11

and 16 is likely to have occurred in the ancestor of domesticated apples. Since chromosomes 1 and 7 are 12

known to be homologous pairs (Han et al., 2011; Velasco et al. 2010), the ALMTII gene among unanchored 13

sequences (MDP0000791995) is likely located on chromosome 7. 14

Most of polyploid-derived duplicated genes show a rapid divergence in expression (Blanc and Wolfe 15

2004). This phenomenon is also observed for the apple ALMTII genes. For example, two duplicate gene 16

pairs, Ma1 /MDP0000312421 and MDP0000299697/MDP0000290997, have acquired divergent expression 17

patterns. The Ma1 gene is expressed in fruit during the entire process of fruit development, while 18

MDP0000312421 is not expressed in fruit. Similarly, MDP0000299697 is weakly expressed in fruit at early 19

stage of fruit development, whereas MDP0000290997 is not expressed in fruit. However, it is unclear 20

whether or not the two duplicate gene pairs, MDP0000259751/MDP0000791995 and 21

MDP0000768640/MDP0000244249 diverge in expression although none of them showed expression in 22

fruit. 23

In summary, duplication of ALMTII genes in apple is related to the polyploid origin of the apple 24

genome. Divergence in expression has occurred between the Ma1 gene and its closely related 25

homologues, which is consistent with our findings above that only the Ma1 gene out of the ALMTII family 26

is associated with fruit acidity in apple. 27

28

Characterization of the Ma1 gene and complexity of the genetic basis of fruit acidity in apple 29

Our study indicates that the Ma1 gene is significantly associated with fruit acidity in an apple 30


14

collection, which confirms previous studies (Bai et al. 2012; Khan et al. 2013). To gain further insight into 1

the role of Ma1 in determining fruit acidity, the functionality of this gene was investigated. Subcellular 2

localization has indicated that Ma1 resides in the tonoplast, and ectopic expression in yeast has further 3

demonstrated its potential functionality as a vacuolar malate channel. Ma1 consists of a TMD that is 4

formed by seven putative transmembrane α-helices, and the last putative transmembrane α-helix at the C 5

terminus seems to be essential for directing it to a specific subcellular compartment or membrane. A 6

single nucleotide substitution of G with A in the last exon results in a premature stop codon, which 7

contributes to a truncated protein lacking the last putative transmembrane α-helix. The truncated protein 8

resides in the cell membrane instead of the tonoplast, thus, losing the functionality as a vacuolar malate 9

channel. As malic acid is stored in the vacuole, the single nucleotide mutation must have an influence on 10

accumulation of malic acid in fruit tissues. Besides the A/G SNP locus, seven other SNPs in coding regions 11

were also identified in eight cultivars, which were subjected to qRT-PCR analysis (data not shown). More 12

studies are needed to address if these SNPs have an effect on gene function. 13

Linkage mapping studies have shown that the Ma locus on LG16 can explain 22.8-36% of the 14

observed variation in fruit acidity (Kenis et al. 2008; Liebhard et al. 2003). However, our association 15

mapping study indicates that the Ma1 gene accounts for ~ 7.46% of the observed phenotypic variation for 16

malic acid content in mature fruits. This result strongly suggests that the Ma locus on LG16 is not the only 17

determinant of fruit acidity in apple. Moreover, it is revealed that apple cv. Belle de Boskoop contains two 18

homozygous alleles ma1ma1; however, it has lower pH values in fruits at different stages of development. 19

In addition to the Ma1 gene, a major QTL for fruit acidity was detected on linkage group 8 (Liebhard et al. 20

2003; Zhang et al. 2012). These results suggest the likely presence of major gene(s), other than the Ma1 21

gene, in the apple genome that are also responsible for fruit acidity. The apple accessions used in this 22

study, such as cv. Belle de Boskoop, are highly valuable for identifying these additional major genes 23

responsible for apple fruit acidity. 24

In earlier studies, it was reported that Ma1 has a strong additive effect in increasing fruit acidity, and 25

it is incompletely dominant over ma1 (Xu et al. 2012), while other studies have indicated that Ma1 is 26

completely dominant over ma1 (Iwanami et al. 2012). Based on mean values of malic acid contents in 27

mature fruits of the apple collection used in this study, it is revealed that the following allelic dominance 28

effects are observed, wherein Ma1Ma1 > Ma1ma1> ma1ma1. Thus, it seems that Ma1 is incompletely 29

dominant over ma1. 30


15

1

Variability in levels of fruit acidity within a collection and its implications to domestication of apple 2

Findings of this study not only confirms malic acid as the predominant organic acid in mature apple 3

fruits (Wu et al. 2007), but that wide variations in malic acid content of mature fruits are also observed 4

between cultivars from different geographic regions. Apple cultivars originating from the European 5

countries such as Germany, England, and France produce acidic fruits with average malic acid contents of 6

7.20, 5.80, and 5.42 mg/g FW in mature fruits, respectively. In contrast, apple cultivars from Asian 7

countries such as China, Japan, and North Korea produce sweet fruits, with average malic acid contents of 8

3.32, 3.15, and 2.95 mg/g FW in mature fruits, respectively. These observed differences may be attributed 9

to the following reasons. First, people in Asian countries prefer sweet and low-acid apples, while 10

Europeans prefer more acidic and/or tart apples. Second, cider is a popular drink in Europe and many 11

apple cultivars have been developed for cider production. Levels of malic acid are very critical in cider 12

processing as it plays an important role in maintaining a consistently sharp and crispy taste 13

(Pereira-Lorenzo et al. 2009). In general, fruits of apple cultivars used for cider processing contain high 14

levels of malic acid contents. Thus, these findings suggest that fruit acidity is an important determinant for 15

fresh consumption as well as processing in apple breeding programs (Crisosto et al 2002; Juniper and 16

Mabberley 2006; Wen et al. 2013; Khan et al. 2014). 17

As mentioned above, the Ma1 gene plays an important role in determining fruit acidity, and three 18

genotypes, AA (ma1ma1), AG (Ma1 ma1), and GG (Ma1Ma1), are identified in the apple collection based 19

on the A/G SNP locus of the Ma1 gene. Most of wild apple relatives have GG genotype (58%), followed by 20

AG (27%) and AA (15%) genotypes (Fig. 7a). In contrast, most apple cultivars have AA genotype (58%), 21

followed by AG (38%) and GG (4%) genotypes. On average, mature fruits of domesticated cultivars have 22

lower malic acid contents compared to the wild relatives (Fig. 7b). These results suggest that apple 23

domestication may be accompanied by selection of the Ma1 gene that plays an important role in 24

determining fruit acidity. In addition, it is worthy to note that the observed higher levels of fruit acidity in 25

wild species may be related to natural selection as higher levels of malic acid could contribute to fruit 26

survival by circumventing damage resulting from both biotic and abiotic stresses such as pests, pathogens, 27

heat, and drought (Fernie and Martinoia 2009). 28

Variations in malic acid content of mature fruits are also detected between accessions with the same 29

genotype in the Ma locus. For example, among accessions with the same genotype, wild relatives have 30


16

generally the highest value of malic acid content in mature fruits, followed by European cultivars and Asian 1

cultivars (Fig. 7b). Asian cultivars have higher frequency of GG genotype (8%) when compared with 2

cultivars from Europe (2%) or other countries (3%). Among accessions with the same GG genotype, 3

however, Asian cultivars have lower value of malic acid content in mature fruits than cultivars from Europe 4

and other countries (Fig. 7b). These results further confirm our findings above that gene(s) other than 5

Ma1 are also responsible for fruit acidity in apple. 6

Apple was first domesticated in the Tian Shan Mountains of central Asia, wherein the wild species M. 7

sieversii is known to be widely distributed (Dzhangaliev 2003). Following sequencing of the apple genome, 8

M. sieversii has been identified as the main contributor to the genome of the cultivated apple (Velasco et 9

al. 2010). Thus, it is reasonable to speculate that sweet apples were initially developed in Central Asia, and 10

then introduced to Europe along the famous Silk Road. Hybridization between M. sylvestris and sweet 11

apples has been undertaken to develop cultivars for cider production and/or fresh fruit consumption 12

(Wagner and Weeden 2000). 13

14

Relationship between the origin of the cultivated apple and the population structure 15

As mentioned above, the initial progenitor of the cultivated apple, M. × domestica, was initially 16

domesticated from M. sieversii (Dzhangaliev 2003). Subsequently, the introgression of wild species into 17

the cultivated apple must have occurred as a distinct event during domestication. In East Asia, interspecific 18

hybridization between M. sieversii and another wild species, M. baccata, contributed to the development 19

of a new hybrid species, M. × asiatica, which has been grown as a local landrace since ancient time 20

(Pereira-Lorenzo et al. 2009). In Europe, interspecific hybridization between the initial progenitor of M. × 21

domestica and the European crabapple M. sylvestris has been reported to be an important event in the 22

evolution of domesticated apple cultivars that are more closely related to M. sylvestris than to M. sieversii 23

(Richards et al. 2008; Cornille et al. 2012). In short, M. sieversii and M. sylvestris are the two predominant 24

contributors to the M. × domestica gene pool (Velasco et al. 2010; Cornille et al. 2012), and the 25

introgression of other wild species such as M. baccata into the domesticated apple also contributes to the 26

diversity of apple. 27

The apple accessions used in this study predominantly originated from Europe and Asia, and they are 28

clustered into three groups based on the result of population structural analysis (Fig. S2). All accessions, 29

except Hongroupingguo (Malus sieversii) and Tukumanpingguo(Malus sieversii) , in the second group 30


17

(marked with predominant green color) are cultivars. The first group (marked with predominant red color) 1

is composed of cultivars and most wild species such as M. baccata. The third group (marked with 2

predominant blue color) consists of cultivars and several wild species such as Malus sylvestris and Malus 3

prunifolia (Willd.) Borkh. Thus, our finding suggests that these apple collections are divided into three 4

groups based on different origin and evolution of the domesticated apple. 5

6

7

Acknowledgements 8

This work was supported by the National Natural Science Foundation of China (Grant Nos. 31420103914 9

and 31372048) and the National Basic Research Program of China (Grant No. 2011CB100600). 10

11

12


18

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WW, Kuang H, Zhang HY, Roose ML, Nagarajan N, Deng XX, Ruan Y (2013) The draft genome of sweet 2

orange (Citrus sinensis). Nat Genet 45: 59-66 3

Yu J, Pressoir G, Briggs WH, Vroh BiI, Yamasaki M, Doebley JF, McMullen MD, Gaut BS, Nielsen DM, Holland 4

JB, Kresovich S, Buckler ES (2006) A unified mixed-model method for association mapping that accounts 5

for multiple levels of relatedness. Nat Genet 38: 203-208 6

Yu JM, Buckler ES (2006) Genetic association mapping and genome organization of maize. Curr Opin 7

Biotech 17: 155-160 8

Zhang J, Baetz U, Krügel U, Martinoia E, De Angeli A. (2013) Identification of a probable pore-forming 9

domain in the multimeric vacuolar anion channel AtALMT9. Plant Physiol 163:830-843 10

Zhang Q, Ma BQ, Li H, Chang YS, Han YY, Li J, Wei GC, Zhao S, Khan MA, Zhou Y, Gu C, Zhao XZ, Han ZH, 11

Korban SS, Li SH, Han YP.(2002) Identification, characterization, and utilization of genome-wide simple 12

sequence repeats to identify a QTL for acidity in apple. BMC Genomics 13:537. 13

14


25

Table legends 1

Table 1 Candidate gene-based association analysis of Ma1 homologues and malic acid content in apple 2

mature fruits. *: Negative and positive numbers indicate upstream or downstream, respectively, of start or 3

stop codons. The names with prefix ‘MDP0000’ represent accession numbers of apple genes deposited in 4

Genome Database for Rosaceae (https://www.rosaceae.org). 5

6

Table 2 Correlations between haplotypes in the Ma1 gene and malic acid contents in mature fruits of an 7

apple population. 8

9

Table 3 Functional analysis of the Ma1 gene based on expression in yeast lines. *Ma1 containing a GGCC 10

sequence at the A/G polymorphic site and has a complete coding sequence. N/D, Not determined. 11

Means with different letters within the same column are significantly different at the 0.01 level of 12

probability. 13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

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26

Figure legends 1

Figure 1. Figure 1. Genes encoding aluminum-activated malate transporter in the apple genome. (a) 2

Chromosomal distribution of the apple ALMT genes, whose accession numbers are retrieved from the 3

Genome Database for Rosaceae (GDR) database (http://www.rosaceae.org/). The dashed line indicates 4

anchored sequences. Genes belonging to the ALMTII family are highlighted in black background. Accession 5

numbers in brackets are identical in coding DNA sequences to their neighbours. (b) Phylogentic tree 6

derived from amino acid sequences of ALMT genes in apple and Arabidopsis. The numbers near branches 7

indicate bootstrap values calculated from 1,000 replicate analyses. 8

9

Figure 2. Characterization of the Ma1 gene responsible for fruit acidity in apple. (a) Genomic structure of 10

the Ma1 gene. SNP and WBG90 represent A/G polymorphic and microsatellite (TC)n loci, respectively. (b) 11

An example of genotyping of the apple population using two molecular tags of the Ma1 gene. I, a 12

polyacrylamide gel of SSR marker WBG90, and the polymorphic bands are indicated with arrows. II, an 13

agarose gel electrophoresis of PCR products digested with HaeIII enzyme. PCR products containing GGCC 14

sequences can be digested, while PCR products with GACC sequences cannot be digested. (c) The TMD of 15

the Ma1 gene is predicted to be formed by six putative transmembrane a-helices (TMa1–TMa6) with the N 16

terminus being located in the vacuole. The star indicates the A/G polymorphic locus, with the allele ‘A’ 17

corresponding to a premature stop codon. 18

19

Figure 3. Mean values of malic acid contents in mature fruits of different genotypes that are classified 20

based on either the A/G SNP or the microsatellite (TC)n locus of the Ma1 gene in apple. Different 21

lowercase letters indicate significant differences among genotypes (T-test, least significant difference test 22

at P < 0.01). Error bars show the SE of the mean. 23

24

Figure 4. Analysis of expression profiles of the eight apple ALMT genes using RT-PCR. S1, S2, and S3 25

indicate 30, 60, and 90 days after full bloom, respectively. 1, 2, 3, and 4 represent cv. Yanhongmei, Bell de 26

Boskoop, Zhumary, and Aifeng, respectively. No RT-PCR products of the expected sizes were obtained for 27

five ALMT genes (MDP0000312421, MDP0000791995, MDP0000244249, MDP0000259751, 28

MDP0000290997, and MDP0000768640). 29

30

Figure 5. Expression profiles of the Ma1 gene in fruits of different apple varieties. The genotypes at the 31


27

A/G SNP locus are as follows: AA in cv. Aifeng and Belld de Boskoop; AG in cv. Zhumary, Jincui and 1

Xinnongjin; GG in cv. McIntosh Red, Annuo and Yanhongmei. All the eight cultivars show similar ripening 2

periods, with maturation occurring at approximately 90 days after full bloom. 3

4

Figure 6. Subcellular localization of Ma1 -GFP by transient expression in epidermal cells of onion. A, 5

Fluorescence of the GFP protein is detected throughout the whole cell, including both cytoplasm and 6

nucleus. B, Cells stained with the membrane dye Dil. C, Fluorescence of the ma1-GFP fusion protein is 7

targeted to the cell membrane. D, Fluorescence of the Ma1-GFP is targeted to the tonoplast, which 8

surrounds the nucleus (marked by arrows). E, Fluorescence of the AtALMT12-GFP fusion protein that was 9

previously localized at the cell membrane (Meyer et al. 2010). F, Fluorescence of the AtMT9-GFP fusion 10

protein that was previously localized at the tonoplast (Zhang et al. 2013). Ma1 and ma1 represent two 11

different alleles harboring GGCC and GACC sequences, respectively, at the A/G polymorphic site 252 bp 12

upstream of the stop codon. 13

14

Figure 7. Characterization of the apple collection used in this study. (a) Distribution of three genotypes (AA, 15

AG, and AG) among cultivars from different geographic regions and their wild relatives. Others include 16

United States, New Zealand, and Australia. The genotypes are classified based on the A/G SNP locus of the 17

Ma1 gene. (b) Mean values of malic acid contents in mature fruits of three genotypes (AA, AG, and AG) 18

among cultivars from different geographic regions and their wild relatives. Different lowercase letters 19

indicate significant differences among genotypes (T-test, least significant difference test at P < 0.01). 20

21

22

23

24

25

26

27

28

29

30


28

Supplemental data 1

Table S1. SSR markers used for assessment of the structure of an apple population. *WBGCAS is a newly 2

developed marker in this study. E-SSR and G-SSR correspond to expressed and genomic microsatellites, 3

respectively. 4

5

Table S2. RT-PCR primers of ALMTII genes in apple. 6

7

Figure S1. Determination of the optimal value of K and inferred population structure of the apple 8

collection. The blue dot represents the ad hoc procedure described in Pritchard et al. (2000), and the 9

blue star indicates the second order of statistics (ΔK) based on Evanno et al. (2005). 10

11

Figure S2. Bayesian population assignment test based on 17 microsatellite loci with the software 12

STRUCTURE 2.2. Then numbers represent the apple accessions as listed in additional data 1. 13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31


29

Additional Data 1

Additional data 1. Genotype of apple germplasm based on the screening of two molecular markers of the 2

Ma1 gene 3

4

Additional data 2. Additional data 2: Genotyping data of SSR markers used for analysis of pulation 5

structure of apple germplasm. Note: the missing data are indicated with "-9". The numbers represent 6

genotypic values. 7

8

9


1

1

2

Figure 1. Genes encoding aluminum-activated malate transporter in the apple genome. (a) Chromosomal 3

distribution of the apple ALMT genes, whose accession numbers are retrieved from the Genome Database 4

for Rosaceae (GDR) database (http://www.rosaceae.org/). The dashed line indicates anchored sequences. 5

Genes belonging to the ALMTII family are highlighted in black background. Accession numbers in brackets 6

are identical in coding DNA sequences to their neighbours. (b) Phylogentic tree derived from amino acid 7

sequences of ALMT genes in apple and Arabidopsis. The numbers near branches indicate bootstrap values 8

calculated from 1,000 replicate analyses. 9

10

11

12

13

14

15

16

17

18

19

20


2

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Figure 2. Characterization of the Ma1 gene responsible for fruit acidity in apple. (a) Genomic structure of 15

the Ma1 gene. SNP and WBG90 represent A/G polymorphic and microsatellite (TC)n loci, respectively. (b) 16

An example of genotyping of the apple population using two molecular tags of the Ma1 gene. I, a 17

polyacrylamide gel of SSR marker WBG90, and the polymorphic bands are indicated with arrows. II, an 18

agarose gel electrophoresis of PCR products digested with HaeIII enzyme. PCR products containing GGCC 19

sequences can be digested, while PCR products with GACC sequences cannot be digested. (c) The TMD of 20

the Ma1 gene is predicted to be formed by six putative transmembrane a-helices (TMa1–TMa6) with the N 21

terminus being located in the vacuole. The star indicates the A/G polymorphic locus, with the allele ‘A’ 22

corresponding to a premature stop codon. 23

24

25

26

27

28

29

30


3

1

2

3

4

5

6

7

8

9

10

11

12

13

Figure 3. Mean values of malic acid contents in mature fruits of different genotypes that are classified 14

based on either the A/G SNP or the microsatellite (TC)n locus of the Ma1 gene in apple. Different 15

lowercase letters indicate significant differences among genotypes (T-test, least significant difference test 16

at P < 0.01). Error bars show the SE of the mean. 17

18

19

20

21

22

23

24

25

26

27

28

29

30

31


4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Figure 4. Analysis of expression profiles of the eight apple ALMT genes using RT-PCR. S1, S2, and S3 21

indicate 30, 60, and 90 days after full bloom, respectively. 1, 2, 3, and 4 represent cv. Yanhongmei, Bell de 22

Boskoop, Zhumary, and Aifeng, respectively. No RT-PCR products of the expected sizes were obtained for 23

five ALMT genes (MDP0000312421, MDP0000791995, MDP0000244249, MDP0000259751, 24

MDP0000290997, and MDP0000768640). 25

26

27

28

29

30

31

32

33

34

35


5

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Figure 5. Expression profiles of the Ma1 gene in fruits of different apple varieties. The genotypes at the 26

A/G SNP locus are as follows: AA in cv. Aifeng and Belld de Boskoop; AG in cv. Zhumary, Jincui and 27

Xinnongjin; GG in cv. McIntosh Red, Annuo and Yanhongmei. All the eight cultivars show similar ripening 28

periods, with maturation occurring at approximately 90 days after full bloom. 29

30


6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Figure 6. Subcellular localization of Ma1 -GFP by transient expression in epidermal cells of onion. A, 15

Fluorescence of the GFP protein is detected throughout the whole cell, including both cytoplasm and 16

nucleus. B, Cells stained with the membrane dye Dil. C, Fluorescence of the ma1-GFP fusion protein is 17

targeted to the cell membrane. D, Fluorescence of the Ma1-GFP is targeted to the tonoplast, which 18

surrounds the nucleus (marked by arrows). E, Fluorescence of the AtALMT12-GFP fusion protein that was 19

previously localized at the cell membrane (Meyer et al. 2010). F, Fluorescence of the AtMT9-GFP fusion 20

protein that was previously localized at the tonoplast (Zhang et al. 2013). Ma1 and ma1 represent two 21

different alleles harboring GGCC and GACC sequences, respectively, at the A/G polymorphic site 252 bp 22

upstream of the stop codon. 23

24

25

26

27

28

29

30

31

32


7

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Figure 7. Characterization of the apple collection used in this study. (a) Distribution of three genotypes (AA, 22

AG, and AG) among cultivars from different geographic regions and their wild relatives. Others include 23

United States, New Zealand, and Australia. The genotypes are classified based on the A/G SNP locus of the 24

Ma1 gene. (b) Mean values of malic acid contents in mature fruits of three genotypes (AA, AG, and AG) 25

among cultivars from different geographic regions and their wild relatives. Different lowercase letters 26

indicate significant differences among genotypes (T-test, least significant difference test at P < 0.01). 27

28

29


1

1

Table 1 Candidate gene-based association analysis of Ma1 homologues and malic acid content in apple 2

mature fruits. 3

Homologue of Ma1

Molecular tag

LG

Primer (5’-3’)

P-value

Name Type Polymorphic site Location* Forward Reverse

MDP0000244249 WBG86 SSR (ATG)5 -566 bp 16 AAACCATTCACTTGATATGACA GACGATATCCCAAGTTAACAA 0.08

MDP0000252114 WBG90 SSR (TC)20 -1408 bp 16 GCCCACTAATGCTTTCTGTA CTCCAACAGATTGTCGAATC 0.0007

MDP0000768640 WBG106 SSR (GT)10 +1473 bp 13 CTGCTGGTGCCTACATGTGGT CAGGTAACTATCCGTGACGAG 0.21

MDP0000290997 WBG112 SSR (GGTTTT)3 +1740 bp 6 AGAAGCCAAGAGGACACTCT TCTGTATCGACCATGTGGTG 0.47

MDP0000791995 WBG113 SSR (TG)16 +665 bp U/N TTAATTTGTTTGGCGTTGGG TCATGGACGGTGATAGCTGA 0.02

MDP0000259751 WBG117 SSR (CTG)7 -252 bp 1 GTAGAGGCGGCCATGAATAA CTTCTCCGATCTCACAAGCC 0.03

MDP0000299697 WBG119 SSR (TA)9 +384 bp 14 CCTGTAACGACCCTGTATCG ACTTTTTCTTCGTCCGAATC 0.24

MDP0000312421 WBG121 CAPS GKAC +513 bp 13 AGATAACTCTGCAATCCCCAG TCCGACTTCGGTTTCAACAG 0.79

*: Negative and positive numbers indicate upstream or downstream, respectively, of start or stop codons. 4

The names with prefix ‘MDP0000’ represent accession numbers of apple genes deposited in Genome 5

Database for Rosaceae (https://www.rosaceae.org). 6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22


2

1

2

3

Table 2 Correlations between haplotypes in the Ma1 gene and malic acid contents in mature fruits of an 4

apple population. 5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

Haplotype SNP

(R=A/G)

WBG90 Frequency Correlation between haplotype and malic

acid content

Regression coefficient Probability

HapA G (TC)17 0.192 3.24 4.69E-18

HapB A (TC)17 0.296 -1.09 0.00676

HapC G (TC)20 0.115 1.32 0.01400

HapD A (TC)20 0.397 -3.12 1.42E-15


3

1

2

3

Table 3 Functional analysis of the Ma1 gene based on expression in yeast lines. 4

YPDA liquid

medium

Construct* Malic acid content (mg/g) Mean value

1 2 3

Containing

0.02g/mL

malic acid

Ma1 3.85 3.67 3.87 3.80a ±0.11

ma1 1.45 1.55 1.41 1.47b ±0.07

Empty vector 1.38 1.29 1.40 1.36b ±0.06

Without malic

acid

Ma1 N/D N/D N/D

ma1 N/D N/D N/D

Empty vector N/D N/D N/D

* Ma1 containing a GGCC sequence at the A/G polymorphic site and has a complete coding sequence. N/D, 5

Not determined. Means with different letters within the same column are significantly different at the 6

0.01 level of probability. 7

8

9

10

11

12


1

Supplemental data 1

2

Table S1. SSR markers used for assessment of the structure of an apple population 3

Marker* LG Type Motif Primer (5’-3’) Expected

size Forward Reverse

Hi20b03 8 G-SSR (ACA)7 AAACTGCAATCCACAACTGC GTTTAGTTGCTAATGGCGTGTCG 226 bp

Hi08g06 10 G-SSR (GAA)5 AATCGAACCAGCACAGGAAG GTTTAGATGGAGGTCGTGGTTACG 192 bp

Hi08h03 4 G-SSR (TTG)6 GCAATGGCGTTCTAGGATTC GGTGGTGAACCCTTAATTGG 153 bp

CN868471 9 E-SSR (TTC)6 TTCCTGTGAACCCAGTCCTC GATCGACAGGCAACATCCTT 281 bp

BACSSR20 11 G-SSR (GA)21 TGATACATGAAGTGTGCTTCCTC TGAGAGAACTGAATCGGGCT 229 bp

BACSSR51 15 G-SSR (TA)10 ACCAGCTGTTTCCAGTTTCC AAAGATTTATTGGCGGGCAT 134 bp

CN893277 5 E-SSR (AG)11 CACTGCAGGAACTGCAAAAA AGAAAGGGGTTGAATTTGGG 246 bp

CN876284 17 E-SSR (AGA)6 CAGCGAGGAGAAGGAAATTG GTTCCAGAACTTCACGCCAT 102 bp

BACSSR58 3 G-SSR (AT)10 ATGATCTGCATGGTGGTTCC GGCTTTGACTTCGTTTCAGC 245 bp

CH05h05 13 G-SSR (TC)19 ACATGTCACTCCTACGCGG GTGCAGTGATTAGCATTGCTGT 180 bp

WBGCAS84 16 G-SSR (AGG)6 CATGTTGTTTGGATATGGATATG TCTCACATCGGTTTCTCTGC 157 bp

WBGCAS130 6 G-SSR (GA)20 GGAGAGAGGATATGGCGTGA TACCAACACGCTCTCCACTC 141bp

WBGCAS131 2 G-SSR (AT)15 TGCTGGTAAGGGACATTACG TCTGGTTTGACCAAGGTTCA 274bp

WBGCAS132 14 G-SSR (CT)10 CGCGTCTTGAAACATTCCAA TGCGGTTTTTGAGATTGTGA 183bp

WBGCAS133 7 G-SSR (AT)11 CTTGGCCCCATTACGAAA GACATCCTTGATGTGCCACA 255bp

WBGCAS134 1 G-SSR (CT)30 ATGGGATACATTGGCTCCAC TGCACCACCTTCACACAGAA 220bp

WBGCAS135 12 G-SSR (CT)22 GCTGCTGCTGGAGGAAGTTT GAAGTTGGTCTCCTTGAGCA 180p

*WBGCAS is a newly developed marker in this study. E-SSR and G-SSR correspond to expressed and 4

genomic microsatellites, respectively. 5

6

7

8

9

10

11

12

13

14


2

1

2

3

4

Table S2. RT-PCR primers of ALMTII genes in apple 5

Gene Primer sequence (5’-3’) Expected

size (bp) Forward Reverse

MDP0000252114 CCACAGCTTCGCCGAGAGAA CGACGACAGTGAGAATTGCC 323

MDP0000312421 ATGCCAGCCAGTATTCCATC GTCCCAGACACCAAGACGAT 304

MDP0000244249 GGCAGTTGTGACTCGACTGG GTGGTTCCCAGACTGCAAAT 308

MDP0000791995 TCCTCACTGTTGCCATCATG AGTCAGCCACCGAGTCAAAG 444

MDP0000299697 CTGATGATCGTCTCGCTGCT TCCGATACCCAGACACCATG 355

MDP0000259751 GATTTCACCTGCAAAGCCTC CCTTAAAGGGGAAGCCTTCT 196

MDP0000768640 CCCAGGAAAATCATCTTTGC ACTTCCTCCAACTTTGCAGC 257

MDP0000290997 GCTTTGGTTGTGGCAAACTT CCACCAAGTTGTGCAGATCC 313

Actin TGACCGAATGAGCAAGGAAATTACT TACTCAGCTTTGGCAATCCACATC 245

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7

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3

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Figure S1. Determination of the optimal value of K and inferred population structure of the apple 19

collection. The blue dot represents the ad hoc procedure described in Pritchard et al. (2000), and the 20

blue star indicates the second order of statistics (ΔK) based on Evanno et al. (2005). 21

22

23

24

25

26

27

28

29

30

31

32

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Figure S2. Bayesian population assignment test based on 17 microsatellite loci with the software 23

STRUCTURE 2.2. Then numbers represent the apple accessions as listed in additional data 1. 24

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26

27

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