ORIGINAL PAPER

Characterization of Transcriptional DifferencesBetween Columnar and Standard Apple TreesUsing RNA-Seq

Yugang Zhang & Jun Zhu & Hongyi Dai

Published online: 17 January 2012# Springer-Verlag 2012

Abstract The columnar apple tree is a valuable resource forgenetic improvement of cultivated apples due to its specialarchitecture. To identify genes involved in the columnararchitecture, expression profiles of newly developing shootsof standard and columnar trees from a segregating popula-tion of “Fuji”דTelamon” have been analyzed using thenew generation of high-throughput RNA-Seq technology.Following Blast analysis against Nr, SwissProt, KEGG, andCOG databases, a total of 69,558 unigene annotations havebeen identified. Among them, more than 80.68% and79.87% clean reads of Apple-Co and Apple-St samples,respectively, have been mapped to reference apple genome.Interestingly, 57% of genes were covered with more than50% clean reads. Moreover, 1,252 and 1,443 unigenes fromApple-Co and Apple-St tissues, respectively, had alternativesplicing sites. Among these, 614 and 666 were alternative 3′splice sites and the most abundant, followed by alternative5′ splice sites, while exon skipping sites were the leastfrequent. In addition, 13,142 and 13,334 novel transcriptunits were identified form Apple-Co and Apple-St samples,respectively. Moreover, analysis of RPKM (reads per kilo-base per million reads) values found that expression of5,237 unigenes differed by more than twofold. Amongthese, 1,359 were enriched in 232 metabolic pathways basedon KEGG database annotation, and 2,233 were enriched inbiological regulation, cellular process, etc. pathways based

on GO functional annotation, and 287 unigenes were relatedwith apple architecture. Among the 287 unigenes, 31 unig-enes mapped to chromosome 10 of apple genome, and 25unigenes were GRAS transcription factor, which were sug-gested to play an important role in architecture formation ofcolumnar apple trees. Taking together, this study provided atheoretical basis for further enriching gene resources ofimportant agronomic traits of fruit trees and for understand-ing the formation mechanism of columnar apple trees.

Keywords Columnar apple trees .Co gene .

Transcriptional . RNA-Seq

Introduction

Plant architecture is of importance for crop yield, quality,and cultivation model. Columnar apple trees are favorableamong fruit growers because they have short internodes,fewer lateral shoots, and more spurriness. The first columnarapple variety “McIntosh Wijcik” is a mutant of “McIntosh”discovered in Canada. Its columnar gene (Co) at chromo-some 10 is linked with AFLP and SSR markers and used tobreed new columnar apple varieties (Hemmat et al. 1997;Tian et al. 2005). Whether other genes are also involved incolumnar architecture of apple trees is not clear.

Currently, studies on genes related to plant architecturehave been mainly conducted in annual plants or modelplants such as rice, Arabidopsis, maize, wheat (Kuraparthyet al. 2008), and barley (Rossini et al. 2006). Genes involvedin plant height, shoot number, and shoot angle have beencloned from wheat, barley, maize (Gallavotti et al. 2004),rice (Li et al. 2003; Takeda et al. 2003; Qiao and Zhao2011), Arabidopsis (Kim et al. 2005; Laufs et al. 2004;Greb et al. 2003; Otsuga et al. 2001; Komatsu et al. 2003;

Electronic supplementary material The online version of this article(doi:10.1007/s11105-011-0396-0) contains supplementary material,which is available to authorized users.

Y. Zhang : J. Zhu :H. Dai (*)College of Landscaping and Horticulture,Qingdao Agricultural University,Qingdao, Shandong Province 266109, Chinae-mail: hydai@qau.edu.cn

Plant Mol Biol Rep (2012) 30:957–965DOI 10.1007/s11105-011-0396-0

Booker et al. 2005; Dai et al. 2006), tomato (Schumacher etal. 1999), chrysanthemum (Jiang et al. 2010), snapdragon(Weir et al. 2004), and Pisum (Foo et al. 2005). In addition,MKK (Dai et al. 2006) and polyamines (Ge et al. 2006) werealso involved in regulation of plant branching. Lactone wasreportedly involved in shoot suppression and plant architec-ture formation of Arabidopsis and rice (Gomez-Roldan et al.2008; Umehara et al. 2008), and some genes associated withflowering can regulate plant architecture formation (He et al.2010; Flachowsky et al. 2010). However, studies on genesrelated to the architectures of apple trees and other woodyperennials are rare. In this study, we analyzed the transcrip-tion profiles of standard and columnar trees from the segre-gating progenies of “Fuji”דTelamon” utilizing the newlydeveloped high-throughput RNA-Seq technology to furtherenrich the gene resources important to the traits of fruittrees, hoping to provide a theoretical basis for understandingthe mechanisms of columnar apple tree formation and forimproving the architecture of major cultivated apple treesthrough genetic transformation in the future.

Materials and Methods

Materials

Five of each 4-year-old columnar and standard seedlings ofthe progeny of “Fuji”דTelamon” cultivated in QingdaoLaixi Fengshi Horticultural Field of Qingdao AgriculturalUniversity were used in this study. New shoots in length of1–2 cm sprouted at 1.5 m height of the southward stem ofeach columnar (Apple-Co) and standard (Apple-St) appletrees were collected three times during May to July 2010and immediately sent back to our laboratory in an icebox,snap frozen in liquid nitrogen, and stored at −80°C forfuture use.

Total RNA Isolation and mRNA Purification

Total RNA was isolated using a commercial plant RNAextraction kit (Bio Teke Corporation, Beijing) according tothe method of Chen et al. (2011) from new shoots collectedeach time and purified using an Oligotex mRNA purifica-tion kit (Qiagen, USA). After checking their integrity on anAgilent Technologies 2100 Bioanalyzer, equal amount oftotal RNA collected from same seedlings were mixed andused to prepare cDNA libraries.

Preparation of cDNA Library for RNA-Seq

The cDNA libraries were prepared using a commercial kit(Illumina, USA) based on the manufacturer's instruction and

method reported previously (Bruno et al. 2010), andsequenced on an Illumina HiSeq™ 2000.

Sequencing Assessment and Assembly

The original sequencing images were converted into rawdata (raw reads) using base calling method. After eliminat-ing those containing adaptors and with low quality, theremaining clean reads were mapped to the apple genome(http://genomics.research.iasma.it/) using SOAPaligner/soap2. The unigenes were acquired after assembling theclean reads with least Ns and inextensible 3′ and 5′ endsusing software SOAPdenovo.

Gene Coverage

After mapping clean reads to the apple genome, the cover-age of each gene was calculated by the percentage of genescovered by reads, that was, the percentage ratio of the basenumbers of the genes matched with unique mapping reads.

Alternative Splicing (AS)

The junction sequences were used to analyze seven types ofAS events in Apple-Co and Apple-St including skippedexons (SE), retained introns (RI), alternative 5′-splice sites(A5SS), alternative 3′-splice sites (A3SS), mutually exclu-sive exons (MXE), alternative first exons (AFE), and alter-native last exons (ALE) according to the method of Wang etal. (2010).

Novel Transcript Unit (Novel TU)

Gene models with length >150 bp and coverage >2 werefirst selected from potential gene models. Those located200 bp downstream of 3′ of one gene and 200 bp upstreamof 5′ of another gene were defined as novel TU.

Functional Annotation

Unigenes were functionally annotated as protein functionalannotation, pathway annotation, COG functional annota-tion, and gene ontology (GO) functional annotation usingthe method reported previously (Xue et al. 2010).

Differential Gene Expression Analysis

Analysis of differentially expressed genes was performedusing the method previously reported (Xue et al. 2010). Thedifferential expression fold of genes between columnar andstandard apple trees was calculated based on their reads perkilobase per million reads (RPKM). Differentially expressedgenes were defined using false discovery rate (FDR ≤0.001

958 Plant Mol Biol Rep (2012) 30:957–965

and differential expression fold >2). The confirmed differ-entially expressed genes were subjected to GO functionaland pathway analyses.

Screening of Genes Related to Architecture of Apple Trees

The differentially expressed unigenes related to the architectureof apple trees (plant height, internode length, shoot branchingnumber, branching angle, etc.) were screened by comparing tothe sequences of other species published in literatures andGenBank after GO functional and pathway analyses.

Results and Analysis

Summary and Assessment of RNA-Seq Data Set

A total of 1.2 Gb bases were sequenced from each Apple-Coand Apple-St samples (Table 1). These bases contain4,014,905 and 4,234,765 raw reads, respectively. After as-sembling, 340,149 and 346,286 contigs containing no Nswere identified from Apple-Co and Apple-St samples, re-spectively. By linking contigs together, 112,470 and114,259 scaffolds were obtained from Apple-Co andApple-St samples, respectively. After paired-end reads cor-rection, 56,440 and 57,695 non-redundant unigenes wereobtained from Apple-Co and Apple-St samples, respective-ly. Mapping of the clean reads to apple genome database(http://genomics.research.iasma.it/) found that 80.68% and79.87% unigenes obtained from Apple-Co and Apple-Stsamples were covered in the genome database (Table 1).Figure 1 shows the distribution of all the reads in the longest25 chromosomes of apple trees.

Assembly Quality of RNA-Seq Results of Apple Trees

The length distribution of unigenes is an important indicatorof sequencing quality. It can be seen from Table 2 thatunigenes with length <500 nt, 500–1,000, 1,000–1,500,1,500–2,000, and ≥2,000 nt accounted for 78.94%,17.37%, 2.91%, 0.59%, and 0.19%, respectively, in Apple-Co samples, and 79.08%, 17.19%, 2.89%, 0.63%, and

0.21%, respectively, in Apple-St samples. The N50 valuesfor columnar and standard apple trees were 426 and 424.The results indicated that the obtained sequences met therequirement for further analysis.

Gene Coverage Analysis

It can be seen from Fig. 2 that genes covered by more than90%, 50%, and less than 10% of clean reads accounted for21%, 57%, and 5% of total genes of Apple-Co and Apple-St, respectively. For Apple-Co samples, 4,261,955 Uniqreads covered 33,312 genes, while for Apple-St samples,4,563,276 Uniq reads covered 33,886 genes.

Alternative Splicing Events in Apple-Co and Apple-St

Seven alternative splicing (AS) events are present in organ-isms in vivo. They are exon skipping (ES), intron retention(IR), alternative 5′ splice site (A5SS), alternative 3′ splicesite (A3SS), alternative first exon (AFE), alternative lastexon (ALE), and mutually exclusive exon (MEE).Figure 3 shows the former four alternative splicings withhigh-throughput sequencing in apple tree samples.Expression level equaled Log2 (reads number). It also can

Table 1 Sequencing output of apple cDNA library

Samples Totalreads

Total nucleotides(nt)

Q20 (%) No. of rawreads

No. ofcontigs

No. ofscaffolds

No. ofunigenes

Percentage of reads mappedto apple genome

Apple-Co 6,666,667 1,200,000,060 94.24 4,014,905 340,149 112,470 56,440 80.68

Apple-St 6,666,667 1,200,000,060 94.21 4,234,765 346,286 114,259 57,695 79.87

Total nucleotides0 total reads×read size; Q20 percentage means percentage of bases whose quality is larger than 20 in clean reads; N percentagemeans percentage of Ns in clean reads; GC percentage means GC content of clean reads

Fig. 1 Distribution of reads mapped to the longest 25 chromosomes ofapple genome

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be seen from Fig. 3 that 1,252 and 1,443 AS events occurredin Apple-Co and Apple-St samples, respectively, of whichalternative 3′ splice sites were the most, reaching 614 and666 followed by alternative 5′ splice sites, and exon skip-ping occurred the least.

Prediction of Novel Transcript Units (Novel TUs)

Novel TUs can be found by analyzing reads distribution andgene annotation. It can be seen from Fig. 4 that 13,142 and13,334 novel TUs were discovered from Apple-Co andApple-St samples, respectively.

Functional Annotation

A total of 69,558 unigenes were annotated after blastingagainst nucleic acid and protein databases SwissProt,KEGG, and non-redundant protein Nr database in GenBank(E<1e−5; Table 3). Among them, 48,477 unigenes were an-notated from Nr database, accounting for 69.69%; 29,527unigenes were annotated from SwissProt database, accountingfor 42.45%; 20,527 unigenes were annotated from KEGG;and 27,620 were annotated from GO. Of unigenes, 28,850were involved in various metabolic pathways, some of whichwere involved in multiple metabolic processes. By comparing

with COG database, 12,513 unigenes were annotated intodifferent functional pathways (Fig. 5). The other unigenesequences unmatched in above protein databases were ana-lyzed using software ESTScan (Zhuang et al. 2011; Li et al.2011), and a total of 2,403 amino acid coding sequences wereobtained.

Difference in Gene Expression Level Between Columnarand Standard Apple Trees

The difference in levels of differentially expressed genesbetween columnar and standard apple trees was calculatedaccording to RPKM value of the obtained unigenes. A totalof 5,237 differentially expressed genes with FDR ≤0.001and |log2Ratio| ≥1 were found, of which 2,704 genes wereupregulated and 2,533 were downregulated (Fig. 6). Theseresults demonstrated the overall difference in transcriptionalexpression level of differentially expressed genes betweencolumnar and standard apple trees.

GO Functional Annotation of Differentially ExpressedGenes Between Columnar and Standard Apple Trees

GO functional annotation of the 2,233 differentiallyexpressed unigenes indicated that 566 unigenes were

Table 2 Length distribution of all unigenes obtained by using RNA-Seq of apple trees

Sample No./% 100–500 nt 500–1,000nt

1,000–1,500nt

1,500–2,000nt

≥2,000 nt N50 Mean All unigenes Length of allunigenes (nt)

Apple-Co No. 44,555 9,802 1,643 334 106 426 402 56,440 22,669,159

% 78.94 17.37 2.91 0.59 0.19

Apple-St No. 45,624 9,918 1,667 363 123 424 402 57,695 23,184,412

% 79.08 17.19 2.89 0.63 0.21

All No. 51,239 13,956 3,115 909 339 500 447 69,558 31,109,603

% 73.66 20.06 4.48 1.31 0.49

Fig. 2 Distribution of genes' coverage in Apple-Co and Apple-St

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enriched in molecular function, 1,006 were enriched incellular component, and 661 were enriched in biologicalprocess (Fig. 7). In the above three gene ontologies, 42differentially expressed genes were enriched in biologicalregulation, 190 were in cellular process, 52 in establishment

of localization, 194 in metabolic process, 337 in cell, 337 incell part, 245 in organelle, 250 in binding, and 227 incatalytic process.

Pathway Analysis

Blasting against KEGG database indicated that a total of1,359 differentially expressed genes were involved in 232

Fig. 3 AS events in Apple-Co and Apple-St. a Exon skipping (ES). b Intron retention (IR). c Alternative 5′ splice site (A5SS). d Alternative 3′splice site (A3SS). e Number of AS in Apple-Co. f Number of AS in Apple-St

Fig. 4 Novel transcripts in Apple-Co and Apple-St

Table 3 Results of blast against important public databases

Database 69,558 unigenes in apple tree cDNA library

Annotated (n) Percentage (%)

Nr 48,477 69.69

SwissProt 29,527 42.45

KEGG 20,527 29.51

GO 27,620 39.71

COG 12,513 17.99

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pathways. As shown in Table 4, those genes related toregulating architecture formation of apple trees weremainly involved in biosynthesis of secondary metabolites,phenylpropanoid biosynthesis, zeatin biosynthesis, metab-olism of xenobiotics by cytochrome P450, brassinosteroidbiosynthesis, metabolic pathways, indole alkaloid biosyn-thesis, etc. Of differential expressed genes, 15.23% wereinvolved in the biosynthesis of secondary metabolites,

and 23.69% were involved in metabolic pathway. Somegenes were involved in multiple metabolic processes.

Analysis of Genes Related to the Architecture Formationof Apple Trees

Compared with standard apple trees, columnar apple treeshave very short internodes, fewer long lateral spurs, andmany fruitful short spurs (Supplementary Fig. S1). Thesespecific traits of columnar apple trees are controlled by Coand related genes. According to RPKM expression, GOannotation, and KEGG pathway annotation of the differen-tially expressed genes, literatures, and sequences of otherspecies in GenBank, 287 unigenes were defined as genesrelated to the formation of apple tree's architecture includinginternodes, branching angle, branch number, apical domi-nance, etc. (Supplementary Table S1). The acquisition ofthese candidate genes provided theoretical basis for furtherunderstanding the formation mechanism of columnar appletrees.

Discussion

Analysis of Assessment of RNA-Seq, AS Events, and NovelTUs

Illumina-based RNA-Seq technology is a high-throughputsequencing platform allowing people to rapidly obtain the

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A: RNA processing and modification B: Chromatin structure and dynamics C: Energy production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H: Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and biogenesis K:Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope biogenesis N: Cell motility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion transport and metabolismQ: Secondary metabolites biosynthesis, transport and catabolismR: General function prediction onlyS: Function unknownT: Signal transduction mechanismsU: Intracellular trafficking, secretion, and vesicular transportV: Defense mechanismsW: Extracellular structuresY: Nuclear structureZ: Cytoskeleton

Fig. 5 COG function annotation of all unigene sequences

Fig. 6 Differential expression level comparison of columnar withstandard apple trees

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sequences of almost all mRNA transcripts of a specificorgan or tissue of a species under certain status. Using thistechnology, we obtained 56,440 and 57,695 non-redundantunigenes from columnar and standard apple trees, whichaccount for 81.14% and 82.95% of the total 69,558 anno-tated unigenes, respectively. From columnar and standard

apple trees, 80.68% and 79.87% clean reads were mapped tothe apple genome (Velasco et al. 2010). The results revealedthe overall transcription differences of columnar and stan-dard apple trees during stem growth and development.

A gene can be transcribed into multiple mRNAs throughalternative splicing, and consequently translated into

Fig. 7 Comparison ofdifferentially expressedunigenes with GOannotation

Table 4 List of pathway-enriched differentially expressedgenes in columnar and standardapple trees

No. Pathways Differentially expressedgenes (No./%)

P value Q value

1 Phenylpropanoid biosynthesis 68 (5) 1.244214e−13 2.886576e−11

2 Flavonoid biosynthesis 46 (3.38) 1.245754e−09 1.445075e−07

3 Stilbenoid, diarylheptanoid, andgingerol biosynthesis

39 (2.87) 2.653209e−07 1.538861e−05

4 Biosynthesis of secondary metabolites 207 (15.23) 2.150759e−06 9.979522e−05

5 Diterpenoid biosynthesis 15 (1.1) 3.781856e−06 1.462318e−04

6 Zeatin biosynthesis 18 (1.32) 0.0001340108 3.886313e−03

7 Flavone and flavonol biosynthesis 15 (1.1) 0.0001591288 4.101987e−03

8 Metabolism of xenobiotics bycytochrome P450

18 (1.32) 0.0005460577 1.151685e−02

9 Benzoxazinoid biosynthesis 12 (0.88) 0.0006627399 1.281297e−02

10 Starch and sucrose metabolism 58 (4.27) 0.001077734 1.841614e−02

11 Drug metabolism, cytochrome P450 18 (1.32) 0.001111319 1.841614e−02

12 Limonene and pinene degradation 30 (2.21) 0.001329454 1.927708e−02

13 Steroid hormone biosynthesis 9 (0.66) 0.009565441 9.246593e−02

14 Phenylalanine metabolism 12 (0.88) 0.03030184 2.393606e−01

15 Brassinosteroid biosynthesis 4 (0.29) 0.1106730 6.419034e−01

16 Biotin metabolism 2 (0.15) 0.2362574 9.356458e−01

17 Metabolic pathways 322 (23.69) 0.4025811 9.999446e−01

18 MAPK signaling pathway 14 (1.03) 0.7973493 9.999446e−01

19 Indole alkaloid biosynthesis 1 (0.07) 0.8630132 9.999446e−01

20 Ubiquitin mediated proteolysis 27 (1.99) 0.940312 9.999446e−01

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different proteins to exert different functions, thus greatlyincreasing protein diversity. In Apple-Co and Apple-St sam-ples, although the absolute AS event numbers were differ-ent, they presented the same trend, which was the number ofalternative 3′ splice sites was the most frequent, followed byalternative 5′ splice sites, and the number of exon skippingwas the least frequent.

In this study, 13,142 and 13,334 novel TUs werediscovered from Apple-Co and Apple-St samples, re-spectively. RNA-Seq technology can detect transcriptswith low abundance, identify novel transcript units,and reveal their differential expression between differentsamples based on RPKM values. Therefore, it has beenwidely applied in different species (Bruno et al. 2010;Xue et al. 2010).

Difference in Gene Expression Level Between Columnarand Standard Apple Trees

In this study, we obtained a total of 5,237 unigeneswith twofold difference in transcription level, indicatingthat the transcription level of columnar apple trees wasconsiderably different from standard apple trees. GOfunctional annotation and pathway analysis in KEGGdatabase also revealed similar results. Some of thepathway-enriched unigenes encode the key enzymesregulating gibberellin, indole-3-acetic acid, and brassi-nosteroid biosynthesis, indicating that the architectureformation of apple trees may be related to hormonemetabolic regulation. This was consistent with a previ-ous study (Watanabe et al. 2006).

Of differentially expressed genes, 15.23% were involvedin the biosynthesis of secondary metabolites, and 23.69%were involved in metabolic pathway, implying there weresignificant differences between columnar and standard appletrees in metabolic level.

Analysis of Genes Related to the Architecture Formationof Apple Trees

Compared with standard apple trees, columnar apple treesshowed significant differences in internode length, numberof branches, branch angle, and apical dominance. Thesedifferences in phenotype were controlled by genes.Classical genetic studies showed that the growth habit ofcolumnar apple trees was a quality trait controlled by thesingle dominant Co gene, which was mapped to the chro-mosome 10 of apple genome using linked DNA markers(Hemmat et al. 1997; Tian et al. 2005). The expression ofCo gene was affected by one or more other genes. However,considering the complexity and quantity of apple genome,the genetic distance is still far from these DNA markers to

Co gene; thus, it is not clear how these genes affect thearchitecture formation of columnar apple.

Transcriptome analysis based on high-throughput Illuminapaired-end sequencing is a powerful tool for discovery ofdifferences in gene expression between columnar and stan-dard apple trees. According to GO functional annotationand KEGG pathway database, 287 unigenes among theobtained 5,237 ones were related to plant architectureformation, among which 60% unigenes were involved inregulating branch formation, 20% were involved in regu-lating plant height, and 15% were involved in plant archi-tecture formation.

Blasting against apple genome database indicated that,among the 287 unigenes (of which 106 unigenes are GRAStranscription factor), 31 unigenes were mapped to chromo-some 10 of apple genome, and 25 unigenes were GRAStranscription factor, which included the DELLA, LS, SCR,and other gene families. DELLA proteins are gibberellinresponse-inhibiting GRAS transcription factors found inmany plants. Plants with DELLA mutations have dwarfinheritance and are not sensitive to gibberellin. In this study,we found that DELLA family genes including GAI, RGA,and RGL were expressed differentially between columnarand standard apple trees, which further suggested thatgenes regulating gibberellin biosynthesis might be in-volved in architecture of apple trees. Branches of woodyplants and tiller of annual grasses are also importantcomponents of plant architecture. Obtained from the tran-scriptome sequencing, 14 apple LAS unigenes of GRASgene family showed a higher homology in proteinagainst the grasses tiller genes, such as Arabidopsis'sLAS (Greb et al. 2003), tomato's LS (Schumacher et al.1999), and rice's MOC1 gene (Li et al. 2003). Aboveresults suggest that GRAS transcription factors play animportant role in regulating the architecture formation ofthe columnar apple trees.

Currently, the relationship between 25 GRAS tran-scription factor genes and Co gene is not clear.Agrobacterium-mediated transfer of some of the genesto Gala apple trees using the binary expression vectorwill help to elucidate the functions of these GRAS tran-scription factor genes in the architecture formation ofapple trees. On the other hand, further studies on thesegenes' network will be an effective way to determinewhether the columnar phenotype of apple trees is regu-lated by a major gene or by multiple genes.

In conclusion, this study revealed differentiallyexpressed genes at transcription level between columnarand standard apple trees by utilizing RNA-Seq technolo-gy and analyzed their enriched pathways, thus providinga scientific basis for further investigating the molecularmechanisms underlying the architecture formation of co-lumnar apple trees.

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Acknowledgements This work was supported by China AgricultureResearch System Foundation (No. CARS-28-01-07), Shandong Pro-vincial Young Scientist Foundation (No. BS2009NY023), ShandongProvincial Improved Variety Engineering System Foundation (No.620902), Qingdao Scientific Research Foundation (No. 11-2-4-5-6-jch), and Qingdao Agricultural University Doctoral Foundation (No.630732).

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