Draft - University of Toronto T-Space · Draft Genome-wide analysis of the SBP ... Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology ... 2006), DPTF database (Zhu

Draft

Genome-wide analysis of the SBP-box gene family in

Chinese cabbage (Brassica rapa ssp. pekinensis)

Journal: Genome

Manuscript ID gen-2015-0074.R2

Manuscript Type: Article

Date Submitted by the Author: 10-Sep-2015

Complete List of Authors: Tan, Hua-Wei; Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture Song, Xiao-Ming; Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology

and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture; North China University of Science and Technology, Center of Genomics and Computational Biology, College of Life Sciences Duan, Wei-Ke; Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture Wang, Yan; Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture

Hou, Xilin; Nanjing Agricultural University, State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture

Keyword: Genome-wide analysis, SBP-box gene family, expression profile, <i>Brassica rapa</i>

https://mc06.manuscriptcentral.com/genome-pubs

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Genome-wide analysis of the SBP-box gene family in Chinese cabbage (Brassica rapa

ssp. pekinensis)

Hua-Wei Tan1, Xiao-Ming Song

1,2, Wei-Ke Duan

1, Yan Wang

1, Xi-Lin Hou

1*

1. State Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Germplasm

Enhancement of Horticultural Crops in East China, Ministry of Agriculture / College of Horticulture, Nanjing

Agricultural University, Nanjing, 210095, China

2. Center of Genomics and Computational Biology, College of Life Sciences, North China University of Science and

Technology, Tangshan, 063000, China.

*Please address all correspondence to: Xi-Lin Hou ([email protected])

__________________

Dr. Xi-Lin Hou

Professor

State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm

Enhancement of Horticultural Crops in East China, Ministry of Agriculture

Nanjing Agricultural University, Nanjing, 210095, China

Tel: +86 25 84395332

Email: [email protected]

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Highlights

Twenty-nine SBP-box genes were identified in the Chinese cabbage genome.

Phylogenetic and evolutionary relationships of the SBP gene family were illustrated.

MiR156/157 members may regulate the coding regions or 3′-UTR regions of SBP-box genes.

The expression pattern of Chinese cabbage SBP genes was analyzed.

Abstract

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene family contains highly conserved plant-specific

transcription factors that play an important role in plant development, especially in flowering. Chinese cabbage (Brassica

rapa ssp. pekinensis) is a leafy vegetable grown worldwide, and is used as a model crop for research in genome duplication.

The present study aimed to characterize the SBP-box transcription factor genes in Chinese cabbage. Twenty-nine SBP-box

genes were identified in the Chinese cabbage genome and classified into six groups. We identified twenty-three orthologous

and five co-orthologous SBP-box gene pairs between Chines cabbage and Arabidopsis, and constructed an interaction

network among these genes. Sixteen SBP-box genes were found more abundant in flowers than in other tissues, suggesting

their involvement in flowering. We show that the MiR156/157 family members may regulate the coding regions or 3′-UTR

regions of Chinese cabbage SBP-box genes. As SBP-box genes were found to potentially participate in some plant

development pathways, quantitative real-time PCR analysis was performed and showed that Chinese cabbage SBP-box

genes were also sensitive to exogenous hormone methyl jasmonic acid and salicylic acid. The SBP-box genes have

undergone gene duplication and loss, evolving a more refined regulation for diverse stimulation in plant tissues. Our

comprehensive genome-wide analysis provides insights into the SBP-box gene family of Chinese cabbage.

Keywords

Brassica rapa; Genome-wide analysis; SBP-box gene family; expression profile

Abbreviation List

SBP, SQUAMOSA PROMOTER BINDING PROTEIN; SPL, SQUAMOSA PROMOTER BINDING PROTEIN; bp, base

pair(s); UTR, untranslated regions; HMMs, hidden Markov models; PCR, polymerase chain reaction; LF, least fractionated

blocks; MF1, the medium fractionated blocks; MF2, the most fractionated blocks; BRAD, Brassica rapa Database;

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1. Introduction

Transcription factors regulate plant development and stress responses by temporarily and spatially regulating the

transcription of their target genes. Transcription factors are usually classified into different families and subfamilies based

on their DNA-binding domain sequences, the evolutionary components of sequence-specific transcription factors that

mediate the specificity of its interaction (Charoensawan et al., 2010; Jin et al., 2014). A genome-wide analysis of plant

transcription factors was first performed in Arabidopsis thaliana soon after the completion of its genome sequencing in

2000 (Initiative, 2000). SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a highly conserved

family of plant-specific transcription factors, which were first identified as nuclear proteins interacting within the promoter

region of the Antirrhinum majus floral meristem gene SQUAMOSA (Klein et al., 1996). SBP-box genes encode proteins

sharing a conserved DNA-binding domain of 79 amino acids. SBP proteins contain a zinc finger motif with two

zinc-binding sites (Cys-Cys-His-Cys and Cys-Cys-Cys-His) and most of them have a three-stranded antiparallel beta-sheet

(Yamasaki et al., 2004). SBP proteins play important roles in leaf development, vegetative phase change, flower and fruit

development, plant architecture, sporogenesis, gibberelic acid signaling and toxin response (Chen et al., 2010). In

Arabidopsis, AtSPL3 is involved in the floral transition, and is under the regulation of miR156 during the vegetative

development stage (Cardon et al., 1997). SBP-box genes are also responsible for defense against biotic and abiotic stresses

in grape (Hou et al., 2013). Given that SBP-box proteins play important roles in plant development, their further

characterization in other plant species is warranted.

In plants, miRNAs form a near-ubiquitous class of short (21–24 nt), endogenous single-strand RNA molecules that mainly

perform post-transcriptional gene regulation (Zhang et al., 2013). The SBP-box gene family exhibits different regulatory

patterns among different species: some members are only regulated by miR156 and others by miR156 and miR529 (Ling

and Zhang, 2012). Nineteen rice SPL (the SBP-box genes of rice) (OsSPL) genes and 12 rice miRNA156 (OsmiR156)

precursors were identified in the rice genome. Eleven of these OsSPL genes were putative targets of OsmiR156, which

indicated OsmiR156 and OsSPL target genes are involved in various developmental processes, especially flower

development in rice (Xie et al., 2006).

Chinese cabbage (Brassica rapa ssp. pekinensis) is a leafy vegetable grown worldwide. The genome of Chinese cabbage

was sequenced and assembled in 2011, and valuable genomics and bioinformatics information is available at BRAD

database (http://brassicadb.org/brad/) (Cheng et al., 2011; Wang et al., 2011). Genome-wide analyses have been performed

for transcription factors such as AP2 (Li et al., 2013; Liu et al., 2013; Song et al., 2013), GRAS (Song et al., 2014b),

HSF(Ma et al., 2014b; Song et al., 2014b), Aux/IAA (Mun et al., 2012), NAC (Liu et al., 2014; Ma et al., 2014a), WAKY

(Tang et al., 2014) and bHLH (Song et al., 2014a), providing a systematic view of post-transcriptional regulation in Chinese

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cabbage. SBP-box genes have been identified through whole-genome analysis in several plants, including Arabidopsis (Guo

et al., 2008; Yang et al., 2008), rice (Xie et al., 2006; Yang et al., 2008), grape (Hou et al., 2013) and castor bean (Zhang and

Ling, 2014). However, the specific biological and functional roles of each SBP-box transcription factor gene in Chinese

cabbage remain unknown. Here, we performed a genome-wide identification of SBP-box genes in the Chinese cabbage

genome, and then analyzed gene classification through an examination for exon-intron structure, gene phylogeny and

synteny, and their interaction with microRNAs.

2. Materials and methods

2.1. Identification, location and chemical characteristics of Chinese cabbage SBP-box genes

Characterization of sequence similarity within a conserved domain has usually been used to identify transcription factors.

Based on the sequence similarity, a large collection of transcription factor families have been collected in the Pfam protein

family database, and each of the families represented by multiple sequence alignments with hidden Markov models (HMMs)

(Finn et al., 2014). The HMMER3 software package has been recommended for searching transcription factors based on

HMM models (Eddy, 2011). In addition to search tools, several databases have been developed that focus on the

identification and annotation of plant transcription factors. The PlantTFDB (http://planttfdb.cbi.pku.edu.cn) is a

comprehensive resource for functional and evolutionary research of 58 plant transcription factor families. There are 129,288

transcription factors from 83 plant species deposited in this database, of which 67 species have whole-genome sequences,

providing detailed annotation for several lineages of green plants (Jin et al., 2014). Moreover, several databases contain

comprehensive data on transcription factors from a single species, such as the DATF database (Guo et al., 2005), DRTF

database (Gao et al., 2006), DPTF database (Zhu et al., 2007), TOBFAC database (Rushton et al., 2008) and SoyDB

database (Wang et al., 2010), providing solid support for research on Arabidopsis, rice, poplar, tobacco, grape and soybean,

respectively.

The genome, gene and protein sequences of Chinese cabbage were downloaded from the BRAD database (version 1.5).

PF03110.9 from the Pfam database was used as an SBP-domain search model in SBP-box gene searching against all 41019

Chinese cabbage genes using HMMER, with a cut-off E-value of 10-5

. To confirm the HMMER results, all 29 candidates

were checked against the proteomes in the SMART database (http://smart.embl-heidelberg.de/). The position of each

SBP-box gene on Chinese cabbage chromosomes or scaffolds was obtained according to information from BRAD database.

A Perl script was developed to mark 29 genes on the chromosome and construct a diagram. The boundaries of the

untranslated regions (UTRs) were obtained from an RNA-seq transcriptome research, and transformed into sequences using

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a Perl script. Protparam (http://web.expasy.org/protparam/) was employed in the physical and chemical characteristics

analysis of the SBP proteins. To estimate the divergence of duplicated Chinese cabbage SBP-box genes, MUSCLE was used

in protein sequence alignment of duplicated pairs of SBP proteins. Subsequently, synonymous rate (Ks), nonsynonymous

rate (Ka), and evolutionary constraint (Ka/Ks) between potential pairs of genes were calculated with KaKs_calculator tool.

2.2. Phylogenetic analyses construction

SBP-domain sequences for phylogenetic analyses were gathered from seven plant species (Table S1). Chlamydomonas

reinhardtii, Physcomitrella paten and Vitis vinifera SBP-box genes were gathered from a previous report (Hou et al., 2013).

A. thaliana, Populus trichocarpa and Oryza sativa SBP-box genes were downloaded from the DATF, DPTF and TIGR

databases, respectively (Table S2). Only the longest transcript was used if alternative splicing existed. For the phylogenetic

analysis, only the regions aligned to the SBP-domain were considered, and sequences consistent with a previous report

(Hou et al., 2013) were retained. ClustalX2 was used to generate the multiple sequence alignment of the SBP domains with

default settings according to previously report (Liu et al., 2014b). MEGA (version 6.06) was used to construct

maximum-parsimony phylogenetic trees with 2000 bootstrap replicates.

2.3. Gene structure and motif analysis

The Gene Structure Display Server tool (http://gsds1.cbi.pku.edu.cn/) was used to analyze the exon-intron structures.

MEME software (http://meme.nbcr.net/meme/) was used to search for motifs in all 29 SBP-box genes, with the motif length

set as at least 20 bp up to a maximum of 200 bp. The number of motifs were set to 16 and motifs were reported in this study.

2.4. Identification of orthologous and paralogous genes

OrthoMCL (version 2.0.3) was used to search for orthologous and paralogous genes in Chinese cabbage, Arabidopsis and

rice using the entire protein sequences of SBP. We used an E-value of 1e-5

for BLASTP and OrthoMCL analyses, gathered

the orthologous and paralogous relationships and displayed them using the Circos software (http://circos.ca/).

2.5. Identification miRNAs related to SBP-box genes

A collection of 157 Chinese cabbage miRNAs from the public miRBase database (version 21) were used to identify

SBP-box gene-related miRNAs. TargetFinder software (v1.6) was employed to predict the targets of miRNAs in Chinese

cabbage SBP-box genes, with the maximum mis-pairing set at 3.

2.6. SBP-box gene expression analysis in tissues

We used the Chiifu-401-42 RNA-seq transcriptions with the related fragments per kilobase per million measure (FPKM)

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values to study SBP-box genes expression patterns in six Chinese cabbage tissues from eight samples. According to

previous report, callus tissue was gathered via tissue culture; root, stem and leaf tissues were collected from seven-week old

plants, while root and stem were obtained from different batches; flower tissue was collected from blooming plants; silique

tissue was collected from 15-day plant after pollination (Tong et al., 2013). The SBP-box gene expression profile from each

sample was analyzed via the HemI program (http://hemi.biocuckoo.org/) with the average hierarchical clustering method.

2.7. Plant material and hormone treatment

The Chinese cabbage cultivar Chiifu-401-42 was used. Seeds were germinated and grown in pots containing a soil :

vermiculite mixture (2:1) in a controlled-environment growth chamber programmed for 14/10 h at 23/18°C for day/night,

and a relative humidity of 55–60% (Song et al., 2014b). At the five-leaf stage, hormone treatments were conducted by

spraying leaves with 100 µM salicylic acid (SA) or 50 µM methyl jasmonic acid (MeJA). The young leaf samples were

collected at 0 h, 6 h and 12 h after treatment, and then snap-frozen in liquid nitrogen before storage at -75°C for further

analysis.

2.8. SBP-box gene expression analysis by qPCR

An RNA kit (Tiangen, Beijing, China) was used to extract total RNA from isolated plant leaves, according to the

manufacturer’s instructions. The Prime Script RT reagent kit (Takara, Kyoto, Japan) reverse transcribed the RNAs into

cDNA. The Chinese cabbage actin gene (Bra028615) was used as an internal control to normalize the expression level of

the target genes among different samples. Primer 5.0 software was used to design specific primers according to the SBP-box

gene sequences (Table S3). Three biological and three technical replicates were performed. Each reaction was performed in

a 20-µL reaction mixture containing diluted cDNA sample as the template, SYBR Pre-mix Ex Taq (2×) (Takara) and

gene-specific primers. Quantitative real-time PCR (qPCR) was performed using a CFX96 Single color Real-Time PCR

Detection System (BioRad, Hercules, CA, USA) with the following cycling profile: 94°C for 30 s; followed by 40 cycles at

94°C for 10 s, 58°C for 30 s; and then a melting curve (61 cycles at 65°C for 10 s) was generated to check the specificity of

the amplification. The comparative Ct value method was employed to analyze the relative gene expression level. The RNA

level was expressed relative to the actin gene expression level as 2−∆∆CT

, according to previous analyses (Pfaffl, 2001). The

SBP-box gene expression cluster from each stress treatment was analyzed via the HemI software.

3. Results

3.1. Identification of SBP-box genes in Chinese cabbage

Bioinformatics analysis identified 29 SBP-box transcription factors in the Chinese cabbage genome among 41019 coding

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genes. All 29 candidates were confirmed and retained after checking against the SMART database. All of the Chinese

cabbage SBP-box genes were well conserved, since the E-value of the full sequence aligned with the SBP-box domain

varied from 4e-40

to 5.1e-19

. We named these genes “BrSBP” with a serial number, sorted by E-value (Tables 1 and S4). The

average length of the coding sequence (CDS) of the SBP-box genes (~1224 bp) is longer than that of all Chinese cabbage

genes (~1173 bp), while the average whole gene length (~1702 bp) is shorter than all Chinese cabbage genes (~2019 bp). In

other worlds, the SBP-box genes have longer exon and smaller introns. To determine the closest SBP-box genes in

Arabidopsis, we searched against the TAIR database (version 10) and found the most similar gene pair for each Chinese

cabbage SBP-box gene. Twenty-six Chinese cabbage SBP-box genes could be classified to LF, MF1 and MF2 subgenomes

while three genes were unclear. The present B. rapa genome (version 1.5) is annotated only with CDSs of genes; however,

RNA-seq transcriptome data provided the 5′-UTR sequence of 12,879 genes and the 3′-UTR sequence of 12485 genes. The

5′- and 3′-UTRs of eight SBP-box genes were extracted and are shown in Table S5. The average length of the 5′-UTRs was

326 bp, much longer than in all detected 5′-UTRs in the transcriptome sample. This suggested that transcription factor genes

with binding domains seem to have longer 5′-UTRs. However, the average length of the 3′-UTRs was about 129 bp, shorter

than the average of all estimated 3′-UTRs. To be more specific, the 5’-UTR of BrSBP17 was 599 bp, and this gene also had

the longest coding region among the SBP-box genes. BrSBP17 may have similar functions to AtSPL14, playing a role in

sensitivity to FB1 and the development of normal plant architecture.

According to the most recently assembled Chinese cabbage genome resources, the 29 Chinese cabbage SBP-box genes were

distributed unevenly on the 10 chromosomes. Five Chinese cabbage SBP-box genes were located on chromosome 7; four on

chromosomes 4, 5 and 9; three on chromosomes 2 and 6; two on chromosomes 3 and 10; one each on chromosomes 1 and 8;

and one on Scaffold000403. The positions of the 28 genes located on the chromosomes are shown in Fig. 1, providing a

visual insight into SBP-box gene distribution.

Physical and chemical characteristics of SBP proteins were analyzed and the result was summarized in Table 2. The

molecular formula, molecular weight, pI (theoretical isoelectric point), instability index, aliphatic index and GRAVY (grand

average of hydropathicity) were characterized for each SBP protein. Most proteins were alkaline, which is opposite to the

GRAS or HSF families. The instability index of all the proteins ranged from 46.57 to 111.23, revealing that all of them are

unstable molecules. The GRAVY scores were all less than 0, indicating that the SBP proteins are hydrophilic (Table 2).

3.2. Phylogenetic relationship and evolutionary divergence of SBP-domain family genes

For the phylogenetic analysis, we selected seven well-studied and representative plant species, including green alga (C.

reinhardtii), moss (Physcomitrella patens), four eudicots (Arabidopsis, Chinese cabbage, grape and poplar), as well as a

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monocotyledonous angiosperm, rice. To obtain a more precise phylogenetic tree of plant SBP proteins, we trimmed three

amino acids and used the more conserved SBP domains (76 amino acids). Certain SBP proteins were not included in the

analysis because their conserved domains contained too few amino acids to perform the phylogenetic analysis. All of the

Chinese cabbage SBP proteins were retained for further phylogenetic analysis (Table S2).

According to the phylogenetic analysis, the 128 SBP-box genes could be classified into seven groups (Fig. 2). Similar to

other higher plants, none of the Chinese cabbage SBP-box genes were present in Group 7, which only contains seven alga

genes. Six and seven SBP-box proteins from moss were classified into Group 6 and Group 1, respectively, suggesting that

Group 6 and Group 1 are secondary ancient groups along with Group 7.

The selection types and divergence timings of duplicated genes were estimated by calculating the Ks, Ka and Ka/Ks of gene

pairs. Among 193 gene pairs, only 2 were underwent neutral selection, 52 and 139 gene pairs underwent positive and

purifying selection, respectively. The divergent time is varied from 174.82 MYA (Million Years Ago) to 4.83 MYA.

Interesting, the Ka/Ks values of all 24 duplicated gene pairs were less than 1, indicates that they underwent purifying

selection. Besides, the divergent time is much closer than other gene pairs, with a range of 39.95 MYA to 4.83 MYA (Table

3).

3.3. Gene structure and motif analysis

To compare the 29 Chinese cabbage SBP-box genes, their exon-intron structures were predicted (Fig. 3). In general, genes

in the same group have similar exon and intron numbers, and even intron phases. Group 1 and Group 5 genes contain nine

or ten exons. The similarity of splicing patterns in Group 1 and Group 5 showed the close relationship between these two

groups. Group 3 was the biggest branch, containing genes with three or four exons. Group 4 and Group 6 have more

conserved splicing patterns of their two and three exons, respectively. Interestingly, according to the phylogenic analysis,

both BrSBP3 and BrSBP8 are related to AtSPL6 (three exons), while BrSBP3 has only one exon and BrSBP8 has three

exons. To validate the sequence information of BrSBP3, we performed a genomic DNA PCR and transcriptional RNA PCR.

The same sequence obtained from the DNA and RNA confirmed that BrSBP3 is a single-exon gene. We hypothesized that

an intron loss event occurred after the divergence of Arabidopsis and Chinese cabbage.

We searched for motifs to analyze the conserved features of Chinese cabbage SBP proteins using MEME (Fig. 4). The

majority of SBP proteins contains Motif 1, indicating that they have both Cys-Cys-Cys-His and Cys-Cys-His-Cys type zinc

finger, and the former seems less conserved than the latter. BrSBP24, BrSBP27, BrSBP28 and BrSBP29 contain Motif 2,

showing that they only have Cys-Cys-His-Cys type zinc finger. Motif 8 was only found in Group 3, indicating that the

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proteins in Group 3 might share a unique function. Moreover, some motifs only exists one unique group but were shared by

all the members within the group. This is the case of Motif 6, Motif 9, Motif 14 in Group 1 and Motif 10 in Group 3.

3.4. Identification of orthologous and paralogous SBP-box genes in plants

Comparative analysis was performed to identify the orthologous and paralogous gene pairs. We identified 23 orthologous

and four coorthologous SBP-box gene pairs among 21 Chinese cabbage genes and 16 Arabidopsis genes using OrthoMCL

with default settings. Moreover, nine orthologous and four coorthologous gene pairs among Chinese cabbage and rice genes

were identified, providing comparative genomics information of SBP-box genes between eudicots and monocots. Nine

paralogous pairs were found in Chinese cabbage, more than four pairs in Arabidopsis and six in rice. The relationships of

orthologous, coorthologous and paralogous SBP-box genes among the three species were visualized using the Circos

program (Table S7, Fig. 5).

An interaction network of Chinese cabbage SBP-box genes was constructed to improve our understanding of the

genome-wide regulation network. The Arabidopsis Interactions Viewer

(http://bar.utoronto.ca/interactions/cgi-bin/arabidopsis_interactions_viewer.cgi) is a database of 70944 predicted and 36306

confirmed Arabidopsis interacting proteins, providing deep insight and high confidence interactions among Brassicaceae

plants. We replaced Arabidopsis genes with corresponding orthologous and coorthologous Chinese cabbage genes after

constructing an Arabidopsis gene interaction network (Fig. 6). The PCC (Pearson correlation coefficients) of 22 gene pairs

were less than zero, whereas that of two genes were more than zero, which revealed SBP proteins mainly have a negative

interaction with other proteins in Chinese cabbage. Twenty-eight gene pairs were not calculated, indicating that many of

their regulation patterns remain unknown.

The interaction network showed that the number of proteins regulated by each SBP-box gene was significantly different.

For instance, BrSBP5, BrSBP10 and BrSBP12 potentially interacted with at least 10 proteins, suggesting their importance in

transcriptional-level regulation. BrSBP11 has a positive interaction with Bra008613 and Bra023570, both of which encode a

KANADI protein (KAN) that regulates organ polarity. It has been suggested that BrSBP11 is required for the development

of carpel and the outer integument of the ovule (Ha et al., 2007). Both BrSBP16 and BrSBP23 had opposite interactions

with Bra024536, which participates in a circadian clock-controlled flowering pathway under long days. Moreover, these

two SBP-box genes also have unclear interactions with three other genes. The gene pair of BrSBP12 and BrSBP10 also has

opposite interactions with 10 other Chinese cabbage genes that are orthologous genes with AT4G11330. These 12 genes

may play an import role in the protein phosphorylation process, and have protein kinase activity, protein serine/threonine

kinase activity, ATP binding, MAP kinase activity, transferase activity and transferring phosphorus-containing groups.

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3.5. Identification of SBP-box genes regulated by miRNAs

The UTR sequences of Chinese cabbage SBP-box genes were obtained through previously reported transcriptome (Table

S5). To study transcriptional regulation of SBP-box genes, we searched 29 Chinese cabbage SBP-box genes with all 157

available relevant miRNAs. We identified only the miR156/157 family and all positive 5p mature members could

potentially regulate the coding regions or 3′-UTRs of Chinese cabbage SBP-box genes (Table S8). In grape, miR156/157

sequences were complementary with nine SBP-box genes in their coding regions. Three members of Group 4, except for

VvSBP4, contain an miR156/157 target site in their 3′-UTR. In Arabidopsis, AtSPL3, AtSPL4 and AtSPL5 in Group 4 also

showed similar complementarity with miR156/157 members (Hou et al., 2013). All SBP-box genes from Group 2 and

Group 3, except for BrSBP18, were potential targets of miR156/157 members, and the target site is located in the last exon.

The loss of a potential exon of BrSBP18 leads to loss of regulation interaction with miRNAs. Interestingly, BrSBP7 from

Group 4 contains a target site in the 3′-UTR like AtSPL3, while other members have no target site in their largely different

3′-UTRs.

3.6. Expression patterns of Chinese cabbage SBP-box genes in different tissues

We searched expression values for each SBP-box gene using RNA-seq data. The FPKM method was performed to eliminate

errors of sequencing data size. According to the FPKM values, all the BrSBP genes were expressed in at least one tissue,

and 28 (96.6%) of these genes were expressed at relatively high levels (FPKM>1) in at least one tissue, including root, stem,

leaf, callus, flower and silique (Table S8, Fig. 7). Sixteen BrSBP genes expressed more abundantly in flowers than in any

other tissues, especially BrSBP9, BrSBP10 and BrSBP19, which suggested their necessity in the flowering process.

SBP-box genes in Group 1 had similar expression quantities in six tissues, suggesting that these four genes have similar

transcription patterns. Similar results were obtained for Group 3 and Group 5. The expression profile in callus is similar to

that in silique tissue, and is of more difference with stem and root, and seems big difference with flower.

3.7. Expression patterns of Chinese cabbage SBP-box genes treated with exogenous plant hormones

As SBP-box genes were found to potentially participate in some plant development pathways, quantitative real-time PCR

analysis was performed and showed that Chinese cabbage SBP-box genes are also sensitive to exogenous hormone methyl

jasmonic acid and salicylic acid (Hou et al., 2013). To increase our understanding of the expression profiles of the SBP-box

genes in Chinese cabbage under exogenous plant hormones, we performed qPCR with primer pairs within non-conserved

regions for each Chinese cabbage SBP-box gene, to ensure the specificity of PCR amplification..

Under SA treatment, the expressions of 16 genes increased and those of 13 decreased after 3 h, and the expressions of 20

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genes increased and nine decreased after 12 h compared with control. The BrSBP8 gene increased over 20 times compared

with the control, suggesting that it is a response acceptor. BrSBP1 decreased over 10 times after 3 h treatment compared

with the control, and then increased to almost 1.5 times higher than the control.

Under MeJA treatment, the expressions of 20 genes increased and those of 9 decreased after 3 h, while the expression of 11

genes increased and 18 decreased after 12 h, compared with the control. The expression of the SBP-box genes under the

exogenous hormone varied considerably among each group (Table S10, Fig. 8).

4. Discussion

In the present study, we analyzed the SBP-box transcription factor family in Chinese cabbage and seven other well-studied

plants, and investigated their expression profiles during Chinese cabbage developmental stages.

Our results confirmed that during the vegetative period, the expressions of two members from Group 3 that are similar to

AtSPL9 and AtSPL15 increased progressively and reached their highest levels at the heading stage, revealing that SBP-box

genes play an important role in heading time (Wang et al., 2014). Besides, a number of previous reports also found that

SBP-box genes play an important role in sporogenesis, gibberelic acid signaling and toxin response. A recent study on grape

SBP-box genes supported that some grape SBP-box genes responded to the pathogen as well as hormone (Hou et al., 2013).

In this study, Chinese cabbage SBP-box genes supported its widely different responses to exogenous hormone MeJA and

SA; however, the mechanism and pathway require further investigation.

A. thaliana has been thoroughly studied as a model plant, and deep insights have been gained into its metabolism and

development. As another plant in the Brassicaceae family, Chinese cabbage is similar to Arabidopsis phenotypically and

genetically (Wang et al., 2011). As expected, we found there is a high similarity between the Chinese cabbage SBP-box

proteins with those from Arabidopsis, suggesting that SBP-box genes from Arabidopsis and Chinese cabbage are closely

related thus share more similar expression profiles compared with other plants. Every SBP-box in Chinese cabbage has a

highly similar counterpart in Arabidopsis; thus, during evolution, no Chinese cabbage species-specific SBP-box genes have

evolved or emerged. Moreover, previous report showed that a lot of Chinese cabbage has three subgenomes originating

from the Arabidopsis genome, called least fractionated blocks (LF), the medium fractionated blocks (MF1) and the most

fractionated blocks (MF2). On the genome-wide scale, the LF subgenome retains 70% of the genes found in A. thaliana,

while the MF1 and MF2 subgenomes retain substantially lower proportions of retained genes (46% and 36%) (Wang et al.,

2011; Cheng et al., 2014). We search each Chinese cabbage SBP-box gene in the BRAD database, and 26 of them can be

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classified as subgenomes while three are unclear. Compared with 16 Arabidopsis SBP-box genes, the Chinese cabbage

retains 69% (11 genes), 50% (eight genes) and 44% (seven genes) of the genes in LF, MF1 and MF2 subgenomes; the actual

ratio would be higher when considering the other three unclassified SBP-box genes. SBP-box genes in the LF genome

showed consistency in their statistical data compared with all genes, while the ratio of SBP-box genes from MF1 and MF2

was higher. We observed that in three and eight homologous groups, one Arabidopsis protein has two or three homologous

genes in Chinese cabbage, respectively. Interestingly, in Group 1, only BrSBP5 was identified to be homologous with

AtSPL1 and AtSPL12. Similarly, only BrSBP11 was deduced to be homologous with AtSPL13 and AtSPL17. We infer that

some of the Chinese cabbage SBP-box genes underwent gene loss after genome triple duplication. The ratio of gene loss is

slightly lower than for other genes, indicating their importance in Chinese cabbage plant development.

The genome locations of the 29 Chinese cabbage SBP-box genes are far from each other, except BrSBP18 and BrSBP23.

The SBP-box domains of these two genes are very similar, but their gene structures are different. In the subgroup of

BrSBP18, BrSBP21, BrSBP22 and BrSBP23, BrSBP23 has four exons, BrSBP21 and BrSBP22 have three introns, and

BrSBP18 has three exons, two introns and a downstream sequence, according to the B. rapa genome. BrSBP18, BrSBP21

and BrSBP22 belong to LF, MF2, MF1 subgenomes, respectively, while BrSBP23 is still unknown. We hypothesized that

BrSBP18 and BrSBP23 may have undergone a tandem duplication, and that BrSBP23 belongs to the LF subgenome like

BrSBP18. Given the gene structure, the BrSBP23 retains an ancient gene structure, and BrSBP18 has evolved after tandem

duplication.

Changes in gene structures also cause changes in expression profiles. In the SBP-box gene group including BrSBP18,

BrSBP21 and BrSBP22, the tissue expression value of BrSBP18 was similar to BrSBP21, both of which were significantly

higher in flower and callus than BrSBP22. After treatment with exogenous hormone, the expression profiles became more

complicated, and BrSBP18 and BrSBP22 seemed more similar than BrSBP21. The diversification and complexity of

SBP-box genes expression under multiple circumstances suggested that the genes that have been retained after gene

duplication and loss have evolved to provide a more refined regulation of diverse stimuli in plant tissues.

In conclusion, we have performed a comprehensive analysis of SBP-box transcription factors in Chinese cabbage. The

physical and chemical characteristics of SBP proteins provide useful information for protein extraction. The results revealed

the SBP proteins participate in plant tissue development and are sensitive to exogenous hormones, suggesting that the SBP

proteins play an important role in plant development, signaling pathway regulation and defense responses under stress

conditions via interactions with other proteins. Our bioinformatics analysis provides new insights in the evolution and

diversity of SBP-box transcription factors, thus can serve as a basic resource for developing new cultivars via manipulation

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of SBP-box genes.

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program,

No. 2012AA100101), China Agriculture Research System CARS-25-A-12, Jiangsu Science and Technology Support

Program (BE2013429) and a Project Funded by the Priority Academic Program Development of Jiansu Higher Education

Institutions. We thank Dr. Dapeng Zhang from Sustainable Perennial Crops Laboratory, USDA-ARS, Beltsville Agricultural

Research Center, Beltsville, MD, USA for his advice and revision.

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Tables

Table 1. Genomic characteristics of SBP-box genes in Chinese cabbage

SBP ID Gene Name Group CDS length Whole gene length Protein length Domain E-value Chr Strand Gene start Gene end A. thaliana protein Identity Blastp E-value

BrSBP1 Bra004674 3 1110 1983 369 4.00E-40 A05 + 1325605 1327587 AT2G42200.1 78.91 8.00E-166

BrSBP2 Bra038101 4 552 622 183 6.70E-40 A05 - 10523110 10523731 AT3G15270.1 66.67 1.00E-67

BrSBP3 Bra004363 2 906 906 301 8.10E-40 A07 + 18244893 18245798 AT1G69170.1 51.23 4.00E-94

BrSBP4 Bra016891 3 1092 2052 363 1.10E-39 A04 + 17735745 17737796 AT2G42200.1 78.91 1.00E-179

BrSBP5 Bra041037 1 2670 3390 889 1.20E-39 Scaffold000403 - 9231 12620 AT2G47070.1 73.98 0

BrSBP6 Bra039656 4 543 994 180 1.20E-39 A06 - 712241 713234 AT1G53160.1 74.73 2.00E-62

BrSBP7 Bra005470 4 429 515 142 1.50E-39 A05 - 5668800 5669314 AT2G33810.1 75.94 3.00E-52

BrSBP8 Bra038324 2 981 1127 326 2.70E-39 A02 + 11266162 11267288 AT1G69170.1 55.5 2.00E-119

BrSBP9 Bra021880 4 426 513 141 6.20E-39 A04 + 15024917 15025429 AT2G33810.1 72.86 8.00E-45

BrSBP10 Bra027265 4 540 615 179 9.90E-39 A05 + 20012855 20013469 AT3G15270.1 82.32 5.00E-99

BrSBP11 Bra022766 2 1029 1329 342 1.80E-38 A03 + 6912451 6913779 AT5G50570.2 70.16 6.00E-140

BrSBP12 Bra021094 4 531 623 176 1.90E-38 A01 + 24002469 24003091 AT3G15270.1 81.71 3.00E-82

BrSBP13 Bra015732 1 2982 3670 993 4.40E-38 A07 + 21116717 21120386 AT1G76580.1 71.06 0

BrSBP14 Bra027478 3 1173 1465 390 5.60E-38 A09 + 14014860 14016324 AT5G43270.1 76.67 0

BrSBP15 Bra033671 3 1089 1576 362 6.90E-38 A06 + 25710537 25712112 AT5G43270.1 83.97 3.00E-135

BrSBP16 Bra030040 3 1173 1502 390 1.20E-37 A07 - 7000703 7002204 AT1G27360.4 73.25 0

BrSBP17 Bra025863 1 3096 3988 1031 1.80E-37 A06 + 8551929 8555916 AT1G20980.1 83.33 0

BrSBP18 Bra032823 3 987 1267 328 3.40E-37 A09 + 12595945 12597211 AT1G27370.4 70.57 9.00E-148

BrSBP19 Bra033221 6 987 1618 328 4.00E-37 A10 - 4830621 4832238 AT1G02065.1 69.75 2.00E-128

BrSBP20 Bra032662 6 477 1150 158 4.90E-37 A09 + 37068749 37069898 AT1G02065.1 84.06 1.00E-67

BrSBP21 Bra030041 3 1125 1376 374 1.60E-36 A07 + 6998542 6999917 AT1G27370.4 77.67 2.00E-149

BrSBP22 Bra010949 3 1101 1437 366 5.30E-36 A08 + 17238139 17239575 AT1G27370.4 67.92 1.00E-166

BrSBP23 Bra032822 3 1098 1361 365 6.30E-36 A09 - 12598090 12599450 AT1G27360.4 65.59 4.00E-164

BrSBP24 Bra022954 4 210 303 69 2.50E-34 A03 + 7931410 7931712 AT2G33810.1 86.96 5.00E-39

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BrSBP25 Bra023695 5 2445 4282 814 4.70E-34 A02 + 4574853 4579134 AT5G18830.1 78.49 0

BrSBP26 Bra002188 5 2322 4013 773 6.50E-34 A10 - 10997330 11001342 AT5G18830.1 77.95 0

BrSBP27 Bra003305 3 951 1247 316 4.60E-27 A07 - 12505202 12506448 AT3G57920.1 64.92 2.00E-134

BrSBP28 Bra014599 3 909 1243 302 8.30E-27 A04 + 1674231 1675473 AT3G57920.1 57.26 6.00E-95

BrSBP29 Bra008266 1 2574 3194 857 5.10E-19 A02 + 14212530 14215723 AT1G76580.1 80.16 0

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Table 2. Physical and chemical characteristics of SB-box proteins in Chinese cabbage

SBP ID Gene Name Group Formula Weight pIa Instability Aliphatic GRAVY

b

BrSBP1 Bra004674 3 C1698H2640N532O555S21 40060.1 8.92 54.65 46.75 -0.817

BrSBP2 Bra038101 4 C881H1439N295O282S12

21060.6 9.61 71.79 42.62 -1.258

BrSBP3 Bra004363 2 C1480H2287N447O463S15

34231.0 8.94 64.13 54.09 -0.820

BrSBP4 Bra016891 3 C1684H2606N528O552S22

39785.7 8.13 61.12 46.20 -0.835

BrSBP5 Bra041037 1 C4328H6815N1241O1351S38

99068.5 5.89 52.47 78.55 -0.456

BrSBP6 Bra039656 4 C859H1414N286O280S11

20581.0 9.55 56.79 49.28 -1.176

BrSBP7 Bra005470 4 C682H1095N229O231S9

16487.1 8.22 107.66 34.44 -1.417

BrSBP8 Bra038324 2 C1609H2497N475O506S15

37072.3 8.99 66.88 57.70 -0.775

BrSBP9 Bra021880 4 C673H1087N229O238S9

16482.9 6.25 111.23 31.21 -1.523

BrSBP10 Bra027265 4 C865H1419N293O278S11

20724.2 9.55 69.22 50.17 -1.209

BrSBP11 Bra022766 2 C1627H2510N472O523S12

37435.4 8.34 71.00 54.42 -0.654

BrSBP12 Bra021094 4 C863H1389N287O269S11

20441.9 9.63 59.20 44.38 -1.178

BrSBP13 Bra015732 1 C4722H7543N1391O1501S47

109324.0 8.84 58.04 71.95 -0.509

BrSBP14 Bra027478 3 C1904H2951N553O596S15

43605.6 8.43 51.77 57.77 -0.703

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BrSBP15 Bra033671 3 C1724H2659N497O562S13

39756.8 6.83 51.11 57.13 -0.659

BrSBP16 Bra030040 3 C1877H2950N560O606S19

43666.6 8.71 56.67 57.46 -0.780

BrSBP17 Bra025863 1 C4915H7855N1469O1550S52

113993.4 8.48 63.84 74.72 -0.496

BrSBP18 Bra032823 3 C1586H2458N474O500S16

36678.8 9.04 55.65 48.69 -0.853

BrSBP19 Bra033221 6 C1577H2413N481O506S11

36559.0 8.85 56.08 49.94 -0.814

BrSBP20 Bra032662 6 C741H1190N246O230S9

17513.6 10.13 66.60 53.23 -0.763

BrSBP21 Bra030041 3 C1804H2819N517O580S17

41575.3 7.99 46.57 60.45 -0.666

BrSBP22 Bra010949 3 C1753H2712N518O579S22

41013.2 6.02 61.57 49.26 -0.863

BrSBP23 Bra032822 3 C1760H2778N524O560S17

40783.6 9.02 50.48 60.27 -0.691

BrSBP24 Bra022954 4 C355H572N130O98S5

8389.5 10.65 100.69 36.81 -1.401

BrSBP25 Bra023695 5 C4011H6355N1125O1210S50

91301.3 7.40 50.62 78.16 -0.409

BrSBP26 Bra002188 5 C3771H5973N1075O1159S49

86485.3 6.38 58.90 77.01 -0.421

BrSBP27 Bra003305 3 C1512H2379N455O487S15

35204.1 9.36 53.94 59.56 -0.686

BrSBP28 Bra014599 3 C1436H2289N435O468S17

33680.6 9.37 60.23 59.70 -0.689

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BrSBP29 Bra008266 1 C4102H6538N1194O1292S41

94568.7 8.38 54.21 76.23 -0.408

apI, theoretical isoelectric point

bGRAVY, grand average of hydropathicity

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Table 3 Ka/Ks calculation and divergent time of the duplicated gene pairs

Seq1 Seq2 Ks Ka Ka/Ks Selection Time(MYA)

BrSBP13 BrSBP17 1.20 0.27 0.23 Purifying 39.95
























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Figure Legends

Fig. 1 Distribution of SBP-box genes on Chinese cabbage chromosomes. Genes colored in black, red, blue and green indicate they belong to LF, MF1, MF2 and unclear subgenomes,

respectively

Fig. 2 Phylogenetic tree of eight plants constructed based on amino acid sequences of SBP-box domains using the maximum-parsimony method. Different shapes and colors represent

SBP-box genes from different species

Fig. 3 Gene structure of Chinese cabbage SBP-box genes. Green boxes indicate the exon regions and black and blue lines indicate introns and downstream regions, respectively. The

lengths of the boxes and lines are scaled based on the length of the genes

Fig. 4 Distribution of conserved motifs of SBP-box genes in Chinese cabbage. (A) The phylogenetic tree and motifs located on each gene with relative combined P-values. (B) Amino

acid sequences of each motif

Fig. 5 Comparative analysis of synteny and expansion of SBP-box genes. Red, yellow and blue lines indicate paralogous, orthologous and coorthologous gene pair relationships,

respectively. (A) Ten Chinese cabbage (A01 to A10) and five Arabidopsis chromosomes (Chr1 to Chr5) maps showing highly conserved synteny. (B) Ten Chinese cabbage (A1 to A10)

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and rice chromosome (Chr1 to Chr12) maps showing lower conserved synteny

Fig. 6 The interaction network of SBP-box genes in Chinese cabbage according to the orthologs in Arabidopsis. Red and pink indicate that the Pearson correlation coefficient (PCC)

index is above or below 0, respectively; orange indicates that the PCC index of interaction has not been calculated

Fig. 7 Heat map representation and hierarchical clustering of SBP-box genes in eight samples from root, stem, leaf, callus, flower and silique. These expression profile data were

obtained from an Illumina RNA-seq transcriptome. The expression values were calculated by the FPKM measure before generating heat maps

Fig. 8 Heat map representation and hierarchical clustering of SBP-box genes after being treated with SA and MeJA. A qPCR experiment generated these expression profile data, and

the RNA level is expressed relative to the actin gene expression level as 2−∆∆CT

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Distribution of SBP-box genes on Chinese cabbage chromosomes. Genes colored in black, red, blue and green indicate they belong to LF, MF1, MF2 and unclear subgenomes, respectively.

1196x1219mm (72 x 72 DPI)

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Phylogenetic tree of eight plants constructed based on amino acid sequences of SBP-box domains using the maximum-parsimony method. Different shapes and colors represent SBP-box genes from different species.

304x303mm (300 x 300 DPI)

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Gene structure of Chinese cabbage SBP-box genes. Green boxes indicate the exon regions and black and blue lines indicate introns and downstream regions, respectively. The lengths of the boxes and lines are

scaled based on the length of the genes.

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Distribution of conserved motifs of SBP-box genes in Chinese cabbage. (A) The phylogenetic tree and motifs located on each gene with relative combined P-values. (B) Amino acid sequences of each motif.

271x509mm (300 x 300 DPI)

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Comparative analysis of synteny and expansion of SBP-box genes. Red, yellow and blue lines indicate paralogous, orthologous and coorthologous gene pair relationships, respectively. (A) Ten Chinese cabbage (A01 to A10) and five Arabidopsis chromosomes (Chr1 to Chr5) maps showing highly conserved synteny.

(B) Ten Chinese cabbage (A1 to A10) and rice chromosome (Chr1 to Chr12) maps showing lower conserved synteny.

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The interaction network of SBP-box genes in Chinese cabbage according to the orthologs in Arabidopsis. Red and pink indicate that the Pearson correlation coefficient (PCC) index is above or below 0, respectively;

orange indicates that the PCC index of interaction has not been calculated.

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Heat map representation and hierarchical clustering of SBP-box genes in eight samples from root, stem, leaf, callus, flower and silique. These expression profile data were obtained from an Illumina RNA-seq

transcriptome. The expression values were calculated by the FPKM measure before generating heat maps.

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Heat map representation and hierarchical clustering of SBP-box genes after being treated with SA and MeJA. A qPCR experiment generated these expression profile data, and the RNA level is expressed relative to the

actin gene expression level as 2−∆∆CT.

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Type Gene 1 Gene 2 Index

paralog Arabidopsis|AT1G20980 Arabidopsis|AT1G76580 1




paralog rapa|Bra002188 rapa|Bra023695 108





paralog rapa|Bra021094 rapa|Bra027265 0.646



paralog rapa|Bra038101 rapa|Bra039656 0.599

paralog rice|LOC_Os01g69830 rice|LOC_Os08g41940 0.466





paralog rice|LOC_Os08g41940 rice|LOC_Os09g32944 175

ortholog Arabidopsis|AT1G02065 rapa|Bra033221 0.835

ortholog Arabidopsis|AT1G20980 rapa|Bra008266 118






















ortholog Arabidopsis|AT1G20980 rice|LOC_Os08g40260 1.422



Table S7. Orthologs, coorthologs and paralogs information

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ortholog rapa|Bra005470 rice|LOC_Os07g32170 0.3









coortholog Arabidopsis|AT1G27360 rapa|Bra032822 1.289

coortholog Arabidopsis|AT1G27370 rapa|Bra032822 187



coortholog Arabidopsis|AT5G43270 rapa|Bra033671 133

coortholog Arabidopsis|AT3G60030 rice|LOC_Os01g18850 1.477

coortholog Arabidopsis|AT5G43270 rice|LOC_Os06g49010 0.523

coortholog rapa|Bra002188 rice|LOC_Os05g33810 1.865




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miRNA name Mature miRNA Sequence Pre-miRNAbra-miR156a-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156a

bra-miR156a-3p GCUUACUCUCUCUCUGUCACC bra-MIR156a

bra-miR156b-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156b

bra-miR156b-3p GCUCACUCUCUAUCUGUCACC bra-MIR156b

bra-miR156c-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156c

bra-miR156c-3p GCUCACUGCUCUAUCUGUCAGA bra-MIR156c

bra-miR156d-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156d

bra-miR156d-3p GCUCACUCUCUAUCUGUCACC bra-MIR156d

bra-miR156e-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156e

bra-miR156e-3p UGCUCACCUCUCUUUCUGUCAGU bra-MIR156e

bra-miR156f-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156f

bra-miR156f-3p UGCUCACUGCUCUUUCUGUCAGA bra-MIR156f

bra-miR156g-5p UGACAGAAGAGAGUGAGCAC BrSBP1,BrSBP3,BrSBP4,BrSBP8,BrSBP11,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28,BrSBP7-3UTRbra-MIR156g

bra-miR156g-3p GCUCACUGCUCUAUCUGUCAGA bra-MIR156g

bra-miR157a UUGACAGAAGAUAGAGAGCAC BrSBP1,BrSBP4,BrSBP8,BrSBP14,BrSBP15,BrSBP16,BrSBP21,BrSBP22,BrSBP23,BrSBP27,BrSBP28bra-MIR157a

Table S8. Characterisitics of miRNAs that target Chinese SBP-box genes

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Chromosome Strand Pre-miRNA Start Pre-miRNA Stop Pre-miRNA LengthA02 + 2981964 2982092 128

A02 + 2981964 2982092 128

A06 + 17870437 17870559 122

A06 + 17870437 17870559 122

A03 - 28483094 28483198 104

A03 - 28483094 28483198 104

A06 + 17870686 17870783 97

A06 + 17870686 17870783 97

A08 - 13494080 13494199 119

A08 - 13494080 13494199 119

A09 + 31052647 31052824 177

A09 + 31052647 31052824 177

A01 + 2836909 2837019 110

A01 + 2836909 2837019 110

Table S8. Characterisitics of miRNAs that target Chinese SBP-box genes

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Pre-miRNA SequenceAAGGUAAGGGGAGGUGACAGAAGAGAGUGAGCACACAUGGUGGCUUUCUUGCAUGCUUUUUUCAAUUAGGGUUUCAUGCUUGAAGCUAUGUGUGCUUACUCUCUCUCUGUCACCCCUUCUCUCUCUCU

AAGGUAAGGGGAGGUGACAGAAGAGAGUGAGCACACAUGGUGGCUUUCUUGCAUGCUUUUUUCAAUUAGGGUUUCAUGCUUGAAGCUAUGUGUGCUUACUCUCUCUCUGUCACCCCUUCUCUCUCUCU

GAGUGCUGAGGAAUUGAUGGUGACAGAAGAGAGUGAGCACACAUGGUGGCUUUCUUGCAUAUUUGAAGAUGUCAUGCUUGAAGCUAUGUGUGCUCACUCUCUAUCUGUCACCGCCUUCUCUC


CGCAUAGAAACUGACAGAAGAGAGUGAGCACACAAAGGCACUUUGCAUGUUCGAUGCAUUUGCUUCUCUUGCGUGCUCACUGCUCUAUCUGUCAGAUUCCGGCU


UGGUGACAGAAGAGAGUGAGCACACAUGGUGGCUUUCUUGCAUAUUGGAAGAUCUCAUGCUUGAAGCUAUGUGUGCUCACUCUCUAUCUGUCACCCC


GAAAUGCAACAGAGAAAACUGACAGAAGAGAGUGAGCACUUGCAAGGCACUGUUAUGUGUCAAUAACUUUGCGUGUGCGUGCUCACCUCUCUUUCUGUCAGUUGCCUGACUUCUCGCUU


UUGUGAGUGAAUGAGCUGGGGCAAAAGAAACACACAGAAACUGACAGAAGAGAGUGAGCACACAAAAGUAAAUUGCAUAUGUUGCAUUUGCUUCUCUUGCGUGCUCACUGCUCUUUCUGUCAGAUUCCCGUGCUGAUCUCUUUGGCCUGUCCUUGUUCCUCUUUGUCUCAGUCUCUC


CGCAAAGAAACUGACAGAAGAGAGUGAGCACACAAAGGCACUUUGCAUGUUCGAUGCAUUUGCUUCUCUUGCGUGCUCACUGCUCUAUCUGUCAGAUUCCGGCUCCGAUU


GUGAUGCUGGUUGUUGACAGAAGAUAGAGAGCACUAAGGAUGACAUGCAAGUACAUAUGUAUGUAUCAUCACACCGCCUGUGGAUGAUUACAAAAAUAGCCUGCAUGUCUACUCUUUCGUGCUCUCUAUACUUCUGUCACCACCAUUAU

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Gene Root_1 Root_2 Stem Leaf_1 Leaf_2 Callus Flower Silique

BrSBP1 0.756616 0.764038 15.0976 4.25738 7.05537 0 7.33216 0

BrSBP2 0 0 0.13992 0.559191 0.599698 0 1.36194 0

BrSBP3 2.35488 2.46055 3.12274 1.72164 0.703342 1.20368 4.2021 1.38701

BrSBP4 2.86584 3.27663 8.74825 7.54017 9.78902 0.71844 8.70391 0

BrSBP5 40.4507 38.7602 40.2669 34.422 28.4204 31.9049 45.2483 24.8002

BrSBP6 0.555228 0 0.431904 0 0 2.71212 0.279091 0

BrSBP7 5.67669 4.19335 9.7713 7.92492 7.41201 18.475 24.3286 3.98694

BrSBP8 4.64453 2.86966 2.1524 1.56066 0.75868 0.954481 2.92099 1.07831

BrSBP9 2.46272 3.78704 2.76902 1.68866 2.51034 0 75.9181 1.72907

BrSBP10 0.279937 0.147213 0.29074 0 0 0 14.07 0

BrSBP11 3.07888 3.98698 14.9462 6.87185 8.01645 0.257272 1.43811 0

BrSBP12 0.287145 0 0.149744 0.296271 0.482139 0 0 0

BrSBP13 17.8825 19.6114 22.0673 17.5282 20.8956 18.1867 46.329 10.1702

BrSBP14 0.708541 0.712533 6.56756 0.944426 2.86279 0.109963 2.05023 0

BrSBP15 3.87062 1.67019 6.5319 6.30419 3.58726 0.360407 2.7989 0.158493

BrSBP16 8.60371 8.21788 11.3742 4.61719 3.31481 5.38817 23.6801 1.45159

BrSBP17 30.716 39.6156 50.4626 32.8997 39.04 49.6573 57.9488 21.3327

BrSBP18 4.23615 2.31291 3.02501 1.03271 0.752593 0.270679 13.6143 2.14069

BrSBP19 0.747556 0.711665 1.7201 0 0 0.135339 23.1948 0.713562

BrSBP20 0.679233 0.558269 3.83746 0 1.18322 0 10.5569 0

BrSBP21 1.17007 1.90281 2.90863 0.771821 1.2707 0.577771 11.3088 0.609959

BrSBP22 1.63719 1.80136 2.42208 1.24443 0.816303 1.77853 3.42554 0.93864

BrSBP23 7.33671 9.50304 13.6003 10.6689 8.13694 0.832696 26.1633 0.784741

BrSBP24 0 73.711 59.0046 16.7625 89.1882 7.51926 28.0411 0

BrSBP25 7.93662 7.16234 3.46062 2.53186 2.74284 3.80629 5.53642 6.44741

BrSBP26 8.95531 9.84367 6.12856 5.35434 4.23337 4.99299 8.66192 6.7492

BrSBP27 1.43464 1.49524 1.43252 1.75641 1.18598 0 1.45116 0

BrSBP28 26.0727 35.8825 5.29174 2.85761 3.92107 0.299681 2.78992 0

BrSBP29 6.50194 6.84278 5.98547 6.88159 6.58872 5.19653 14.2673 4.7719

Table S9. Expression of SBPa-box genes in six tissues

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Gene SAa 3 h SA 12 h MeJA

b 3 h MeJA 12 h

BrSBP1 -0.082048307 1.586593272 0.332001533 -0.90575646

BrSBP2 0.525150304 0.719921539 0.95001895 0.971563887

BrSBP3 0.312020043 4.423922723 0.06135397 -0.566176672

BrSBP4 0.208623456 1.71874819 1.651550653 -0.521829746

BrSBP6 0.062747502 -0.499256332 1.590944301 0.077065533

BrSBP5 2.668475078 2.544081718 2.092812845 1.126650148

BrSBP7 2.470618073 7.208109924 0.783347664 0.000275066

BrSBP8 28.70696521 24.5346532 1.482512546 1.119120912

BrSBP9 0.104856098 3.159062877 0.310480183 -0.722355355

BrSBP10 -0.619223541 -0.610073783 -0.790971084 -0.798871297

BrSBP11 -0.143460329 4.678674599 -0.258900661 2.172925509

BrSBP12 1.263635695 1.74716407 0.067990426 -0.601012806

BrSBP13 1.174687987 7.375553646 1.238127636 -0.932895878

BrSBP14 -0.312662225 4.400034028 0.962358765 -0.616150842

BrSBP15 4.414024618 7.581301757 -0.077750295 -0.94278136

BrSBP16 -0.836699415 -0.731858467 -0.621660137 -0.32936298

BrSBP17 -0.728251999 -0.621780637 -0.870466133 -0.436144243

BrSBP18 -0.698474835 -0.470774363 -0.89126174 -0.96548447

BrSBP19 -0.745750025 -0.798319215 -0.926347165 -0.960513984

BrSBP20 3.772063028 3.827317022 1.536205581 2.631789293

BrSBP21 0.423286204 1.554348403 0.308550563 0.404381329

BrSBP22 -0.885036902 0.430875183 -0.066454462 -0.781981998

BrSBP23 -0.003496521 -0.586740279 0.169355929 -0.987418482

BrSBP24 1.137888567 -0.136454808 0.169476325 -0.643889687

BrSBP25 1.077347451 1.676471886 0.570154493 0.717097986

BrSBP26 1.897665857 3.257541604 0.545055564 0.677839091

BrSBP27 -0.819749777 -0.880469972 1.049237284 -0.951634171

BrSBP28 -0.049813968 0.340789281 0.260477554 1.25567975

BrSBP29 -0.292123776 0.649723862 -0.377716823 -0.106192825

asalicylic acid

bmethyl jasmonic acid

Table S10. Expression of SBP-box genes under the treatment of plant hormone

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Draft - University of Toronto T-Space · Draft Genome-wide analysis of the SBP ... Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology ... 2006), DPTF database (Zhu