Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica

Abstract

Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2–7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes.

Introduction

In oilseed brassicas, a yellow-seeded form is preferred over a black- or brown-seeded counterpart mainly because of a thinner seed coat and higher oil content (Friedt and Snowdon, 2009; Velasco and Ferna'ndez-Martı'nez, 2009). Importantly, proanthocyanidins (PAs) play a critical role in this differential pigmentation process (Auger et al., 2010; Fang et al., 2012; Lu et al., 2012).

Proanthocyanidins (PAs) are end-products of a well-studied branch of the flavonoid biosynthetic pathway in higher plants (Winkel-Shirley, 2001; Lepiniec et al., 2006; Saito et al., 2013). In Arabidopsis, a close relative of the Brassica species, 19 single-copy genes have been associated with PA (Appelhagen et al., 2014, 2015; Ichino et al., 2014). These genes can be divided into three classes based on their functions: structural, transcriptionally regulatory, or genes responsible for PA modification, transport, and oxidation. PA genes have also been cloned from a dozen other plant species (Hichri et al., 2011; Falcone Ferreyra et al., 2012) such as maize, and soybean (Yang et al., 2010; Senda et al., 2012). In contrast to single-copy genes in Arabidopsis, several plant species have multiple homologs for a given PA gene. For example, there are nine CHS homologs in soybean (Yi et al., 2010).

In Brassica species homologous cloning is used to isolate PA genes by such as DFR/TT3 (Yan et al., 2008; Akhov et al., 2009), ANS/TT18 (Yan et al., 2011), ANR/BAN (Nesi et al., 2009), TT10 (Zhang et al., 2013), TT2 (Wei et al., 2007), TT8 (Padmaja et al., 2014), TT12 (Chai et al., 2009), TT16 (Deng et al., 2012; Chen et al., 2013), TTG1 (Zhang et al., 2009; Yan et al., 2014) and TTG2 (Li et al., 2015). However, homologous cloning has drawbacks. It needs prior knowledge of sequences of homologous gene, and is slow and difficult to amplify all members of a gene family, particularly in polyploid species, e.g., Brassica juncea, an allotetraploid species. To address these limitations, next-generation sequencing has been widely adopted. Up to date the genomes of over 100 plant species, including B. rapa (Wang et al., 2011), B. olearcea (Liu et al., 2014), and B. napus (Chalhoub et al., 2014) have been sequenced. Very recently, the genome sequence of B. nigra has also been released (http://www.ncbi.nlm.nih.gov/genome/10988). Whole-genome sequence annotation facilitates in genome-wide identification of PA genes (Velasco et al., 2007; Guo et al., 2014). However, the PA genes of Brassica species have not been analyzed in great detail. Furthermore, the complete genome sequencing of Brassica juncea has not been achieved to date. Yang et al. (2014) has conducted a survey of genome sequences in B. juncea. Genome survey sequencing (GSS) can provide information about gene content, functional elements and molecular markers (Jiao et al., 2012; Hirakawa et al., 2015), as well as compare genes of related species for the phylogenetic reconstruction of other non-model species.

Reverse transcription-polymerase chain reaction (RT-PCR), real-time fluorescent quantitative PCR, and transcriptome sequencing (RNA-seq) can analyze the spatial and temporal expression pattern, functions and interactions among various genes (Agarwal et al., 2014). RNA-seq is widely used to estimate transcript amounts and to obtain a quantitative account of transcript amounts in organisms, organs, tissues, or specific cell types, frequently comparing transcript amounts among different samples (Martin et al., 2013; Weber, 2015).

In the present study, GSS was conducted on the inbred line of B. juncea var. Purple-leaf Mustard (PM), and a total of 69,193 coding genes, including 72 PA genes, were predicted by annotation of GSS. Approximately 19 primer pairs specific for PA genes were then designed to screen a bacterial artificial chromosome (BAC) library of B. juncea, which was constructed from the same inbred line. In total, 284 BAC clones were identified and 55 B. juncea PA genes were confirmed by sequencing of fragments amplified from representative BAC clones. Its genomic or chromosomal positions were predicted by mapping to the sequenced B. rapa, B. nigra, or B. napus genomes, which was used as reference genomes to perform phylogenetic analysis on the full-length gene sequences and the end sequences of gene-carrying BACs. The expression level of PA genes were estimated in the seed coat and compared between the yellow- and brown-seed coat by fragments per kilobase of exon model per million mapped reads (FPKM) analysis of RNA-seq data in B. juncea and B. napus. Identification, mapping, and expression analysis of the PA genes in the present study may facilitate in better understanding the genetic mechanism underlying proanthocyanidin biosynthesis, profile, and accumulation in various Brassica species.

Materials and methods

Plant accessions

The inbred line of B. juncea var. PM was used for GSS and construction of the BAC library. RNA was extracted from the seed coat of the inbred line of B. juncea var. Sichuan Yellow (SY, yellow-seeded) and its brown-seeded near-isogenic lines (NILA and NILB), the black-seeded B. napus cv. Xiangyou 15 and two of its F₇ recombinant inbred linesRIL52 and RIL55 15 days after pollination (DAP, torpedo to late torpedo stage) (Liu et al., 2009; Nesi et al., 2009). The plants were grown in a greenhouse under a photoperiod of 16 h/8 h (day/night cycle) at 22°C.

Genome sequencing, sequence assembly, gene prediction, and annotation

Paired-end (PE) libraries were prepared using total DNA from PM, which were then constructed according to the instructions provided by Illumina (San Diego, CA, USA) with a 500-bp insert size and 125-bp read length. Sequence analyses were conducted using the Illumina HiSeq 2000 platform.

The obtained reads were subjected to quality control as follows: bases with quality scores <10 were filtered out by FastQC-0.11.3 (Schmieder and Edwards, 2011). Adaptor sequences in the reads were trimmed using fastx clipper of the FASTX-Toolkit 0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit). After trimming, reads including N nucleotide lengths of <100 bases were excluded, and the remaining high-quality data was used for de novo sequence assembly by SOAP (Schmieder and Edwards, 2011). Protein-encoding sequences in the assembled genomic sequences of PM were predicted by Augustus 2.7 (Stanke and Waack, 2003) using the A. thaliana training set under the default parameters. Reciprocal best-hit analysis (Moreno-Hagelsieb and Latimer, 2008) was performed to compare the results of the prediction by using B. rapa training sets.

Construction, pooling, and screening of the BAC library

The B. juncea BAC library named ZBjuH was constructed from the inbred line of the PM that were treated with the restriction endonuclease HindIII (Luo and Wing, 2003). This library consists of 71,808 clones with an average insert size of 126 kb genomic DNA, and an estimated 10.8-fold coverage of the B. juncea genome. The clones were arranged in 187 384-well plates. The clones were organized into three-dimensional BAC pools of plates, rows, and columns. The superplate consisted of 19 DNA samples, each representing 10 BAC plates, except for superplate 19, which only consists of 7 384-well plates. The first dimension consisted of the BAC clone plate of 187 DNA samples. The second and third dimensions consisted of 8 and 12 DNA samples, respectively, for the pooled 16 rows and 24 columns of the BAC clones. Screening of single BAC clones was performed in a five-step PCR process (Figure S1). The PCR primers were designed according to the conserved sequences of the PA genes that were annotated from the B. juncea GSS (Table S1). PCR reactions were performed in a total volume of 10 μL with a reaction mixture as follows: 10 × PCR buffer (1.0 μL), dNTP mix (10 mM each, 0.15 μL), 1 U Taq DNA polymerase (Takara, Japan), 1 μL template, 10 mM forward primer (0.5 μL), 10 mM reverse primer (0.5 μL) and ddH₂O up to 10 μL. A “touchdown” PCR amplification program is used as follows: 94°C for 5 min; 6 cycles of 30 s at 94°C, 40 s at 62°C with a 1°C decrease in the annealing temperature per cycle, and 1 min at 72°C; 30 cycles of 30 s at 94°C, 45 s at 56°C, and 1 min at 72°C; and a final extension at 72°C for 10 min. The PCR products were observed by electrophoresis on 1.5% agarose gels using ethidium bromide and UV visualization. The BAC clones from which the fragment of expected size was amplified were considered positive BAC clones.

Grouping and sequencing for full-length gene of positive BAC clones

Gene fragments amplified from the positive BAC clones were sequenced and aligned with annotated PA genes using DNAMAN4.0 (LynnonBiosoft, USA) to confirm whether the cloned and the annotated gene were the same copy. When a cloned gene harbored a single nucleotide difference (SNP) and/or insertion or deletion (Indels) in its sequence from the corresponding annotated gene, the cloned and the annotated genes are considered different. For each PA gene, one or two BAC clones were selected for sequencing of the full-length genes by the high-quality, longer read Sanger method (Life Technologies, Shanghai).

Identification and phylogenetic analysis of PA genes in B. napus, B. nigra, and B. rapa

The sequences of cloned B. juncea PA genes were mapped to the released B. napus (http://www.genoscope.cns.fr/blat-server/cgi-bin/colza/webBlat), B. nigra (http://www.ncbi.nlm.nih.gov/genome/10988), or B. rapa (http://brassicadb.org/brad/blastPage.php) reference genome to search for homologous B. napus, B. nigra or B. rapa PA genes with an identity ≥90%. Phylogenetic analysis of homologous PA genes in B. juncea, B. rapa, B. napus, and Arabidopsis was performed by using neighbor-joining (NJ) method as provided in MEGA 5.2 (Tamura et al., 2011), and the reliability of the phylogenetic trees was evaluated by the bootstrap method, with 1000 replications. The B. juncea PA genes on the same branch (clade) of the phylogenetic tree were classified into a homologous group.

Sequencing and mapping of BAC ends

The BACs used for full-length sequencing of the gene were also sequenced for end-sequencing on an ABI 3730X DNA analyzer (Life Technologies, Shanghai). The sequencing primers were modified pIndigoBAC536 cloning vector-derived sequencing primers M13R (5′-CAGGAAACAGCTAT-GACC-3′) and S2 (5′-CGAATTCGAGCTCGGTACCC-3′). The sequence obtained by using the primer M13R was designated as left end (L) of the BAC clone, whereas the sequence by S2 was considered the right end (R). BAC end-sequences (BESs) were also mapped to the recently sequenced B. napus (http://www.genoscope.cns.fr/blat-server/cgi-bin/colza), B. nigra (http://www.ncbi.nlm.nih.gov/genome/10988) or B. rapa (http://brassicadb.org) reference genome to assign a genomic location when at least 100 bp aligned to the reference genome, with at least 75% identity. If hits were obtained at multiple locations in any one of the reference genomes, then a BES was assigned to the position of the hit with the highest identity. The position of a BES was indicated by the first and the last assigned nucleotide (nt) on each reference genome.

Expression analysis of PA genes in seed coat

Isolation, reverse transcription and RNA-seq analysis of RNA from fresh seed coats were performed as described by Liu et al. (2013). The expression level of every PA gene in the seed coat was calculated using the FPKM method (Mortazavi et al., 2008). To compare transcript abundance of cloned PA genes in seed coat between the yellow-seeded inbred SY and its brown-seeded near-isogenic lines (NILA and NILB), the respective mapped reads from the SY/NILA and the SY/NILB pairs for each gene were counted using TopHat v2.0.9 (Kim et al., 2013). Fold changes for each gene between NILs and SY were computed as the ratio of the FPKM values. When the FPKM value of NILs or SY was 0, the substitute 0.001 was used for estimation of fold change. To display changes of PA gene expression in seed coat, the heatmap was constructed by using Heml software (“Normalization:” Logarithmic Base 2, “DEMO:” Canvas) (Deng et al., 2014).

The primers used in RT-PCR expression analysis are listed in Table S2. The following cycling parameters were used for amplification of the PA genes: 1 cycle of 4 min at 94°C; 38 cycles of 50 s at 94°C, 50 s at 58°C, 1 min at 72°C; one cycle of 6 min at 72°C. The PCR products were verified by gel electrophoresis as earlier described.

Results

Identification and cloning of PA genes in B. juncea

A total of 56.2 Gb high-quality sequencing data were assembled into 835 Mb of genomic sequence, with contig and scaffold N50 sizes of 2584 bp and 16,777 bp in B. juncea (Table S3). A total of 233,309 coding genes were predicted by Augustus 2.7 (Table S3) and annotated by alignment of the deduced amino acid sequence to B. rapa genes (http://brassicadb.org/brad/). Approximately 69,193 records were screened out, with sequence identity greater than 70% and alignment length greater than 100 amino acids, which correspond to 32,798 B. rapa genes (Table S4). For a B. rapa gene, an averaged 2.1 homologs, at most 11 homologs, were detected in the B. juncea genome. Among the 69,193 predicted B. juncea genes, 72 were identified as PA genes (Table S5). The number of B. juncea genes that were homologous to a given Arabidopsis PA gene varied from two (DFR, TT1, TT2, TT8, TTG1, and TT12) to six (TT4, TT6, and ANR) (Table S5). Furthermore, two annotated B. juncea genes of TT6 and TT7 were located within the same scaffold (Table S5).

A total of 284 positive BAC clones were identified using 19 PA gene-specific primer pairs from ZBjuH BAC library (Table 1). The amplified fragments were sequenced, and 284 clean sequences with sizes between 192 and 1487 bp were obtained. Alignment showed that these fragments represented 55 B. juncea PA genes, corresponding to 16 Arabidopsis PA genes, with each Arabidopsis PA gene having 2–7 B. juncea homologs (Table 1). All cloned B. juncea PA genes, except for BjuTT4-2, BjuTT4-7, and BjuTT16-6, showed genomic sequences that were similar to the corresponding predicted PA genes. These amplified sequences were not evenly distributed among genes. For 6 genes, only one sequence was each identified, whereas at least 10 sequences were detected for 7 other genes. The remaining 42 genes were each carried by 2–9 BAC clones (Table 1), which is consistent with coverage of the genome by the BAC library used. No BAC clones were identified for six the annotated genes (TT4_g135394, TT5_g158015, ANR_g228640, ANR_g226654, TT19_g144296, and TT19_g167454) (Figure S3).

Table 1.

Grouping of the PA gene carrier BAC clones screened by PCR from Brassica juncea.

Gene Type	Arabidopsis homolog	Primer pair used	Predicted gene	Cloned gene	No. BACs	BAC clone(s) carrying the gene
Structural	TT4/CHS	STT4	g125911	BjuTT4-1	5	ZBjuH038D07, ZBjuH052L16, ZBjuH187G14, ZBjuH187G15, ZBjuH187H15
			–	BjuTT4-2	14	ZBjuH036L22, ZBjuH062E10, ZBjuH068P04, ZBjuH090C04, ZBjuH103N17, ZBjuH115L06, ZBjuH117A13, ZBjuH125A20, ZBjuH130C13, ZBjuH167H05, ZBjuH167H11, ZBjuH167H12, ZBjuH175I06, ZBjuH187A11
			g94262	BjuTT4-3	16	ZBjuH037O10, ZBjuH040M24, ZBjuH042E09, ZBjuH054M24, ZBjuH058B18, ZBjuH102A19, ZBjuH103N21, ZBjuH110J17, ZBjuH111B11, ZBjuH119C13, ZBjuH124I11, ZBjuH129K08, ZBjuH139O13, ZBjuH143K02, ZBjuH162N03, ZBjuH165A05
			g160192	BjuTT4-4	5	ZBjuH031A21, ZBjuH031B12, ZBjuH036O12, ZBjuH048I18, ZBjuH095E01
			g112186	BjuTT4-5	2	ZBjuH044O21, ZBjuH053C09
			g134422	BjuTT4-6	2	ZBjuH053C08, ZBjuH115L05
			–	BjuTT4-7	4	ZBjuH049I15, ZBjuH049J15, ZBjuH090K23, ZBjuH121I20
	TT5/CHI	STT5	g10826	BjuTT5-1	1	ZBjuH186N11
			g147891	BjuTT5-2	1	ZBjuH181K10
			g94675	BjuTT5-3	10	ZBjuH027P19, ZBjuH036L22, ZBjuH041J15, ZBjuH058J20, ZBjuH066I18, ZBjuH066O10, ZBjuH080G05, ZBjuH156B18, ZBjuH158J20, ZBjuH177D05
			g153768	BjuTT5-4	5	ZBjuH096N21, ZBjuH106O08, ZBjuH108L09, ZBjuH119I24, ZBjuH122O18
	TT6/F3H	STT6	g93144	BjuTT6-1	7	ZBjuH020C14, ZBjuH048G02, ZBjuH048M11, ZBjuH058K21, ZBjuH120F22, ZBjuH165E24, ZBjuH181K08
			g230814	BjuTT6-2	5	ZBjuH058P02, ZBjuH059D03, ZBjuH076G06, ZBjuH087J23, ZBjuH144L06
			g34078	BjuTT6-3	1	ZBjuH031F14
			g58779	BjuTT6-4	8	ZBjuH022O18, ZBjuH025L04, ZBjuH047N11, ZBjuH088F14, ZBjuH095M08, ZBjuH106B12, ZBjuH132K03, ZBjuH132P11
			g51817	BjuTT6-5	6	ZBjuH106N13, ZBjH131P01, ZBjuH143I07, ZBjuH146J13, ZBjuH149K20, ZBjuH171M24
	TT7/F3'H	STT7	g118579	BjuTT7-1	9	ZBjuH012H01, ZBjuH025M21, ZBjuH045C24, ZBjuH063G22, ZBjuH095P11, ZBjuH105C09, ZBjuH156A19, ZBjuH159L04, ZBjuH175L17
			g105339/ g105340	BjuTT7-2	4	ZBjuH080O14, ZBjuH081G21, ZBjuH092C04, ZBjuH153O14
	TT3/DFR	SDFR	g119544	BjuTT3-1	7	ZBjuH029J10, ZBjuH043G11, ZBjuH118M13, ZBjuH119K03, ZBjuH157O03, ZBjuH157P04, ZBjuH184C12
			g127201	BjuTT3-2	3	ZBjuH134O05, ZBjuH175D09, ZBjuH183H13
	TT18/ANS	STT18	g16568	BjuTT18-1	4	ZBjuH054O02, ZBjuH091D16, ZBjuH181K13, ZBjuH187D05
			g178347	BjuTT18-2	3	ZBjuH020C14, ZBjuH181I12, ZBjuH181K08
			g86816	BjuTT18-3	3	ZBjuH091K10, ZBjuH097N14, ZBjuH178L19
			g114026	BjuTT18-4	5	ZBjuH054H16, ZBjuH093H16, ZBjuH177N08, ZBjuH182I21, ZBjuH187H15
	ANR	SANR	g97466	BjuANR-1	3	ZBjuH022P08, ZBjuH082J01, ZBjuH123C06
			g177273	BjuANR-2	2	ZBjuH148I16, ZBjuH165M04
			g228640	BjuANR-3	4	ZBjuH071P08, ZBjuH116E04, ZBjuH116I23, ZBjuH185I01
			g19699	BjuANR-4	1	ZBjuH034P21
	TT10	STT10-1	g60604	BjuTT10-1	6	ZBjuH003E23, BjuH033G01, ZBjuH048L18, ZBjuH057G09, ZBjuH083G18, ZBjuH152B03
		STT10-2	g161120	BjuTT10-2	9	ZBjuH006C17, ZBjuH019G11, ZBjuH055H16, ZBjuH107M03 ZBjuH121G03, ZBjuH126F02, ZBjuH140A18, ZBjuH140E23, ZBjuH144O05,
		STT10-1	g169945	BjuTT10-3	11	ZBjuH021A16, ZBjuH024M11, ZBjuH025E16, ZBjuH037G20 ZBjuH066O13, ZBjuH080H07, ZBjuH084L21, ZBjuH092L15, ZBjuH101B03, ZBjuH144O03, ZBju155P16
		STT10-2	g6758	BjuTT10-4	1	ZBjuH176D10
Regulatory	TT1	STT1	g65737	BjuTT1-1	4	ZBjuH021J20, ZBjuH036J21, ZBjuH157B22, ZBjuH180A05
			g10440	BjuTT1-2	3	ZBjuH097N03, ZBjuH147E23, ZBjuH176G24
	TT2	STT2	g27300	BjuTT2-1	2	ZBjuH085H24, ZBjuH137N11
			g136881	BjuTT2-2	7	ZBjuH028M22, ZBjuH034J15, ZBjuH061O14, ZBjuH068M24, ZBjuH135H01, ZBjuH149C17, ZBjuH172K23
	TT8	STT8-1	g113056	BjuTT8-1	5	ZBjuH004L18, ZBjuH038M05, ZBjuH068D18, ZBjuH122I23, ZBjuH173H05
		STT8-2	g109603	BjuTT8-2	3	ZBjuH005J18, ZBjuH033E04, ZBjuH036F18
	TT16	STT16-1	g141603	BjuTT16-1	2	ZBjuH051G23, ZBjuH130K12
		STT16-1	g157583	BjuTT16-2	6	ZBjuH046H18, ZBjuH070H21, ZBjuH082B14, ZBjuH099A21, ZBjuH153H13, ZBjuH171M11
		STT16-2	g150784	BjuTT16-3	2	ZBjuH091L03, ZBjuH163M05
		STT16-2	–	BjuTT16-4	4	ZBjuH057K05, ZBjuH057K06, ZBjuH098G12, ZBjuH160B19
		STT16-1	g231621	BjuTT16-5	7	ZBjuH061F23, ZBjuH064O06, ZBjuH070C13, ZBjuH094F17, ZBjuH094N07, ZBjuH131N02, ZBjuH135M11
		STT16-1	g170816	BjuTT16-6	11	ZBjuH013K01, ZBjuH030F17, ZBjuH049C21, ZBjuH077C18, ZBjuH077C23, ZBjuH081M09, ZBjuH093J03, ZBjuH142O22, ZBjuH144E18, ZBjuH152O04, ZBjuH171A06
	TTG1	STTG1	g228836	BjuTTG1-1	2	ZBjuH030O08, ZBjuH130K10
			g55489	BjuTTG1-2	6	ZBjuH129A18, ZBjuH135B10, ZBjuH140O11, ZBjuH182K06, ZBjuH185M13, ZBjuH185M14
	TTG2	STTG2	g112447	BjuTTG2-1	1	ZBjuH088A24
			g173809	BjuTTG2-2	3	ZBjuH101A24, ZBjuH131A11, ZBjuH170G21
			g118314	BjuTTG2-3	13	ZBjuH025O05, ZBjuH032N08, ZBjuH039D04,ZBjuH063L13, BjuH065B18, ZBjuH065I18, ZBjuH066D01, ZBjuH067B22, ZBjuH076O21, BjuH135G23, ZBjuH174G04, ZBjuH174O02, ZBjuH184O06
			g156630	BjuTTG2-4	5	ZBjuH028C13, ZBjuH043G17, ZBjuH77P08, ZBjuH147A11, ZBjuH162F23
Transporter	TT12	STT12	g29228	BjuTT12-1	4	ZBjuH046J03, ZBjuH047J16, ZBjuH148K24, ZBjuH148O16
			g146440	BjuTT12-2	3	ZBjuH124J12, ZBjuH125I12, ZBjuH150E09
	TT19	STT19	g72809	BjuTT19-1	13	ZBjuH006M03, ZBjuH037H06, ZBjuH061A09, ZBjuH064M21, ZBjuH066G20, ZBjuH092A06, ZBjuH093G08, ZBjuH095N01, ZBjuH140M06, ZBjuH161G15, ZBjuH165B07, ZBjuH172L17, ZBjuH185D12
			g159509	BjuTT19-2	6	ZBjuH062L17, ZBjuH120J17, ZBjuH143N08, ZBjuH170C22, ZBjuH179C03, ZBjuH181C15
			g118434	BjuTT19-3	5	ZBjuH021A20, ZBjuH070G12, ZBjuH122M08, ZBjuH164K02, ZBjuH168M17

The BAC clone in bold was used to sequence full-length sequence of the gene.

One or two BAC clones were chosen for each of the above mentioned PA gene groups of BAC clones and sequenced by walking to obtain full-length gene sequence. Alignment of the resultant full-length gene with its respective GSS sequence indicated that two predicted genes was in fact from the same gene because each of them was only a portion of the same gene (Table S6). Finally, 55 PA genes were confirmed in B. juncea by BAC sequencing (Table 2).

Table 2.

Proanthocyanidins-associated genes identified in B. rapa, B. juncea, and B. napus.

A. thaliana	B. rapaa	B. junceab	B. napusc
ENZYMES
AtTT4/CHS (AT5G13930)	Bra020688(A02)	BjuTT4-1/ TT4_g135394	BnaA02g30320D /BnaC02g05070D
	Bra023441(A02)	BjuTT4-2/ BjuTT4-5	BnaC02g38730D/ BnaCnng01290D
	Bra006224(A03)	BjuTT4-3/ BjuTT4-6	BnaA03g04590D/ BnaC03g06120D
	Bra008792(A10)	BjuTT4-4/ BjuTT4-7	BnaA10g19670D/ BnaC09g43250D
	Bra036307(A09)		BnaA09g29340D
AtTTT5/CHI (AT3G55120)	Bra017728(A03)	BjuTT5-1	BnaAnng08210D /BnaC07g45760D
	Bra003209(A07)	BjuTT5-2/ TT5_g158015	BnaA07g37900D/BnaCnng45660D
	Bra007142(A09)	BjuTT5-3/BjuTT5-4	BnaA09g34840D/BnaC08g26010D
AtTT6/F3H (AT3G51240)	Bra012862(A03)	BjuTT6-1/ BjuTT6-4	BnaA03g41250D/BnaC07g32140D
	Bra036828(A09)	BjuTT6-2/ BjuTT6-5	BnaA09g31780D/BnaC08g22640D
	Bra007813(A09)	BjuTT6-3	BnaA09g55810D
AtTT7/F3′H (AT5G07990)	Bra009312(A10)	BjuTT7-1/BjuTT7-2	BnaA10g23330D/BnaC09g47980D
AtTT3/DFR (AT5G42800)	Bra027457(A09)	BjuTT3-1/ BjuTT3-2	BnaA09g15710D/BnaC09g17150D
AtTT18/ANS (AT4G22880)	Bra013652(A01)	BjuTT18-1/BjuTT18-3	BnaA01g12530D/BnaC01g14310D
	Bra019350(A03)	BjuTT18-2/BjuTT18-4	BnaA03g45610D/BnaC07g37670D
AtANR (AT1G61720)	Bra021318(A01)	BjuANR-1/BjuANR-2	BnaA03g60670D/BnaC04g18950D
	Bra031403(A01)	BjuANR-3/BjuANR-4	BnaA01g36200D/BnaC01g29820D
AtTT10 (AT5G48100)	Bra020720(A02)	BjuTT10-1/BjuTT10-3	BnaAnng08030D /BnaC02g38340D
	Bra037510(A06)	BjuTT10-2/BjuTT10-4	BnaA06g30430D
TRANSCRIPTIONAL FACTORS
AtTT1 (AT1G34790)	Bra028067(A09)	BjuTT1-1/ BjuTT1-2	BnaAnng02100D/ BnaC06g08390D
AtTT2 (AT5G35550)	Bra035532(A08)	BjuTT2-1/BjuTT2-2	BnaA08g29930D/BnaC08g07960D
AtTT8 (AT4G09820)	Bra037887(A09)	BjuTT8-1/BjuTT8-2	BnaA09g22810D/BnaC09g24870D
AtTT16 (AT5G23260)	Bra029365(A02)	BjuTT16-1/ BjuTT16-5	BnaAnng30140D/ BnaC02g41690D
	Bra013028(A03)	BjuTT16-2/ BjuTT16-6	BnaA03g39500D/BnaC02g42240D
	Bra026507(A09)	BjuTT16-3/ BjuTT16-4	BnaA09g05410D/BnaC09g04950D
AtTTG1 (AT5G24520)	Bra009770(A06)	BjuTTG1-1/ BjuTTG1-2	BnaC07g29950D
AtTTG2 (AT2G37260)	Bra023112(A03)	BjuTTG2-1/ BjuTTG2-3	BnaA03g17120D/BnaC03g20650D
	Bra005210(A05)	BjuTTG2-2/ BjuTTG2-4	BnaA05g07220D/BnaC04g08020D
TRANSPORTERS
AtTT12 (AT3G59030)	Bra003361(A07)	BjuTT12-1/ BjuTT12-2	BnaA07g18120D/BnaC06g17050D
AtTT19 (AT5G17220)	Bra023602(A02)	BjuTT19-1/BjuTT19-3	BnaA02g03440D/BnaC02g07090D
	Bra008570(A10)	BjuTT19-2/ TT19_g144296	BnaA10g17440D/BnaC09g40740D

^afrom http://brassicadb.org/brad/;

^bthis study;

^cfrom http://www.genoscope.cns.fr/brassicanapus/.

Genomic locations of PA genes in Brassica species

BLAST of these cloned 55 B. juncea PA genes against the B. rapa or B. napus reference genome identified 31 and 58 homologous genes in B. rapa and B. napus, respectively (Table 2). The neighbor-joining tree of the PA genes from B. juncea, B. rapa, B. napus, and Arabidopsis showed that TT4 genes were clustered into five homologous groups, TT5, TT6, and TT16 each into three groups; TT10, TT18, TTG2, and TT19 each into two groups; and the remaining TT3, TT7, ANR, TT1, TT2, TT8, TTG1, and TT12 genes were clustered into only one homologous group, indicating that these genes were highly conserved in terms of genomic sequence (Figure S2).

Mapping of these cloned 55 B. juncea PA genes to the B. rapa, B. nigra, or B. napus reference genome indicated that 30 and 29 PA genes were homologous to the genes located in A-genome chromosomes except A04 of B. rapa and B. napus, respectively, whereas 23 of the other 25 genes were located in the B-genome chromosomes except B05 and B07 of B. nigra, the remaining two gene (BjuTT5-4 and BjuTT2-2) were anchored on scaffold_30.1 and scaffold_500.1 of B. nigra, respectively, which have not yet been mapped onto a chromosome (Table 3, Figure 1). These PA genes have >95 identity (Table 3). Moreover, 23 of these A-genome PA genes were, respectively, located on the same chromosomes in B. rapa and B. napus, but additional genes may be located in either the same or different A-genome chromosomes or C-genome chromosomes because their positions have not been mapped to the B. napus reference genome (Table 3). The B-genome and the C-genome contributed 25 and 29 PA genes to B. juncea and B. napus genome, respectively, which is approximately equal to the number of PA genes from the A-genome.

Table 3.

Mapping to the Brassica rapa, B. nigra, or B. napus reference genome of full-length sequences of the B. juncea PA genes cloned in this study.

B. juncea gene	BAC sequenced	Sequence length (bp)	Position in B. rapa/B. nigra reference genome	Coverage (%)	Identity (%)	Putative genome or chromosome	Corresponding B. rapa homolog	Position in B. napus reference genome	Coverage (%)	Identity (%)	Putative genome or chromosome	Corresponding B. napus homolog
BjuTT4-1	ZBjuH187G14	1269	A02(23204660-23205926)	98.7	98.9	A02	Bra020688	A02(21961707-21962975)	100	99.2	A02	BnaA02g30320D
BjuTT4-2	ZBjuH175I06	1454	A02(2357734-2359185)	98.0	97.9	A02	Bra023441	C02(2648298-2649755)	99.7	96.6	–	BnaC02g38730D
BjuTT4-3	ZBjuH037O10	1458	A03(2596137-2597594)	92.4	99.3	A03	Bra006224	A03(2138849-2140306)	100	99.8	A03	BnaA03g04590D
BjuTT4-4	ZBjuH036O12	1263	A10(12657235-12655973)	99.0	99.0	A10	Bra008792	A10(13887677-13888939)	100	99.3	A10	BnaA10g19670D
BjuTT4-5	ZBjuH053C09	1516	B02(31676065-31677577)	100	97.8	B02	–	C02(2648298-2649755)	96.2	93.7	–	BnaCnng01290D
BjuTT4-6	ZBjuH053C08	1352	B03(41854375-41855800)	94.8	96.2	B03	–	C03(2967996-2969460)	92.3	94.3	–	BnaC03g06120D
BjuTT4-7	ZBjuH090K23	1267	B08(26615339-26614079)	100	96.9	B08	–	A10(13887677-13888939)	99.7	93.5	–	BnaC09g43250D
TT4-135394		1387	B06(30337366-30338627)	97.3	94.4	B03	–	A03(2138849-2140306)	96.7	94.8	–	BnaC02g05070D
BjuTT5-1	ZBjuH186N11	1358	A03(30108206-30109397)	98.9	99.0	A03	Bra017728	C07(43667810-43668920)	93.2	95.5	–	BnaC07g45760D
BjuTT5-2	ZBjuH181K10	1526	A07(15175119-15173091)	71.1	97.1	A07	Bra003209	A07_random(1352839-1353912)	70.4	100	A07	BnaA07g37900D
BjuTT5-3	ZBjuH080G05	1621	A09(29057157-29055564)	98.6	97.8	A09	Bra007142	A09(25461126-25462763)	99.0	98.7	A09	BnaA09g34840D
BjuTT5-4	ZBjuH106O08	1625	scaffold_30.1(142586-144208)	99.7	98.8	B	–	C08(27510384-27511983)	98.5	91.9	–	BnaC08g26010D
TT5-g158015		1667	B04(26277974-26279709)	96.1	95.2	B04	–	Un_random(67254442-67255968)	91.6	92.4	–	BnaCnng45660D
BjuTT6-1	ZBjuH058K21	1343	A03(21908585-21910045)	82.4	97.0	A03	Bra012862	A03(20668741-20670084)	99.9	99.4	A03	BnaA03g41250D
BjuTT6-2	ZBjuH087J23	1514	A09(27095567-27097080)	100.0	100.0	A09	Bra036828	C08(25256551-25258093)	98.1	97.6	–	BnaA09g31780D
BjuTT6-3	ZBjuH031F14	2998	A09(32529280-32526116)	94.0	98.0	A09	Bra007813	A09_random(3426703-3429867)	94.7	99.8	A09	BnaA09g55810D
BjuTT6-4	ZBjuH022O18	1820	B03(6099152-6097595)	93.2	98.5	B03	–	A09(23688234-23689696)	80.4	92.7	–	BnaC07g32140D
BjuTT6-5	ZBjuH143I07	1454	B08(40828252-40826791)	99.1	98.1	B08	–	C07(35957629-35959030)	96.4	91.9	–	BnaC08g22640D
BjuTT7-1	ZBjuH159L04	2742	A10(14358845-14356094)	89.4	99.2	A10	Bra009312	A10(15436550-15439304)	99.5	98.8	A10	BnaA10g23330D
BjuTT7-2	ZBjuH080O14	2989	B08(28566520-28563560)	98.6	98.4	B08	–	C09(47019883-47026924)	44.4	94.1	–	BnaC09g47980D
BjuTT3-1	ZBjuH029J10	1556	A09(10927890-10926334)	98.3	98.3	A09	Bra027457	A09(9168455-9170011)	99.9	100	A09	BnaA09g15710D
BjuTT3-2	ZBjuH183H13	1689	B06(24256936-24255214)	96.1	99.5	B06	–	C09(10927890-10926334)	92.5	94.2	–	BnaC09g17150D
BjuTT18-1	ZBjuH054O02	1422	A01(6887113-6885692)	98.5	98.5	A01	Bra013652	A01(6294305-6295726)	100	100	A01	BnaA01g12530D
BjuTT18-2	ZBjuH181K08	1161	A03(24797395-24796232)	96.3	96.2	A03	Bra019350	A03(23215394-23216555)	99.9	97.3	A03	BnaA03g45610D
BjuTT18-3	ZBjuH091K10	1143	B02(37385323-37384165)	99.2	95.3	B02	–	C01(9585700-9587061)	83.9	93.8	–	BnaC01g14310D
BjuTT18-4	ZBjuH177N08	1152	B08(43361751-43360600)	98.1	95.8	B08	–	C07(39327212-39328382)	98.4	94.2	–	BnaC07g37670D
BjuANR-1	ZBjuH082J01	1433	A01(21514882-21513450)	99.2	99.2	A01	Bra021318	A03_random(5666520-5667956)	99.7	99.2	A03	BnaA03g60670D
BjuANR-2	ZBjuH148I16	1466	A01(17603658-17602193)	99.3	99.3	A01	Bra031403	A01(1630437-1631902)	100	100	A01	BnaA01g36200D
BjuANR-3	ZBjuH116E04	1499	B01(22657990-22659506)	98.6	96.8	B01	–	C01(28124388-28125894)	99.5	90.5	–	BnaC01g29820D
BjuANR-4	ZBjuH034P21	1400	B08(31434047-31435447)	99.9	99.1	B08	–	C04(18894005-18895401)	99.8	94.6	–	BnaC04g18950D
BjuTT10-1	ZBjuH083G18	3491	A02(22971851-22968361)	99.9	98.8	A02	Bra020720	Un_random(41316930-41322490)	62.8	94.9	–	BnaAnng08030D
BjuTT10-2	ZBjuH055H16	2297	A06(20410060-20412553)	91.3	99.7	A06	Bra037510	A06(20553612-20555918)	99.6	99.2	A06	BnaA06g30430D
BjuTT10-3	ZBjuH021A16	2838	B06(30198717-30195455)	87.0	97.4	B06	–	C02(41316930-41322490)	51.0	91.5	–	BnaC02g38340D
BjuTT10-4	ZBjuH176D10	2293	B08(38281497-20412553)	98.9	99.0	B08	–	A06(20553612-20555918)	99.4	93.1	–	–
BjuTT1-1	ZBjuH180A05	1761	A09(18767007-18765243)	99.8	97.5	A09	Bra028067	Un_random(4808305-4809953)	93.6	97.9	–	BnaAnng02100D
BjuTT1-2	ZBjuH147E23	1707	B06(8841257-8839558)	100	97.4	B06	–	C06(9366519-9368246)	98.8	93.2	–	BnaC06g08390D
BjuTT2-1	ZBjuH085H24	945	A08(8306171-8305232)	96.5	98.9	A08	Bra035532	A08_random(1033684-1034627)	100	99.5	A08	BnaA08g29930D
BjuTT2-2	ZBjuH034J15	944	scaffold_500.1(70364-69421)	100	100	B	–	C08(11760224-11761157)	98.9	93.3	–	BnaC08g07960D
BjuTT8-1	ZBjuH004L18	3551	A09(15769736-15773288)	80.2	97.7	A09	Bra037887	A09(15413735-15417282)	99.9	99.3	A09	BnaA09g22810D
BjuTT8-2	ZBjuH005J18	2768	B03(8122342-8125109)	100	99.7	B03	–	C09(23189158-23191902)	99.1	94.1	–	BnaC09g24870D
BjuTT16-1	ZBjuH130K12	1954	A02(25055704-25053743)	98.3	99.5	A02	Bra029365	Un_random(101450485-101452437)	99.9	99.6	–	BnaAnng30140D
BjuTT16-2	ZBjuH099A21	2258	A03(20961426-20959132)	92.9	99.4	A03	Bra013028	A03(19707165-19709160)	88.4	98.5	A03	BnaA03g39500D
BjuTT16-3	ZBjuH091L03	2004	A09(3307401-3305402)	45.1	97.6	A09	Bra026507	A09(2642192-2644190)	99.7	98.9	A09	BnaA09g05410D
BjuTT16-4	ZBjuH098G12	1981	B01(19554897-19552670)	89.0	96.9	B01		C09(2859965-2861956)	99.4	91.3	–	BnaC09g04950D
BjuTT16-5	ZBjuH094N07	2129	B06(30711288-30713462)	97.5	97.6	B06	–	Un_random(101450485-101452437)	91.7	92.1	–	BnaC02g41690D
BjuTT16-6	ZBjuH077C18	1980	B08(41662625-41664607)	99.8	98.1	B08	–	C02(44915780-44917790)	98.5	92.6	–	BnaC02g42240D
BjuTTG1-1	ZBjuH130K10	1582	A06(17740552-17739539)	99.7	98.8	A06	Bra009770	A06(18525005-18526105)	69.6	93.9	A06	–
BjuTTG1-2	ZBjuH129A18	1014	B08(42051009-42049996)	100	98.6	B08	–	C07(34623713-34624389)	66.8	92.8	–	BnaC07g29950D
BjuTTG2-1	ZBjuH088A24	1516	A03(8752727-8754251)	96.4	96.0	A03	Bra023112	A03(8032043-8033567)	99.4	97.5	A03	BnaA03g17120D
BjuTTG2-2	ZBjuH101A24	1466	A05(4037093-4035604)	96.5	97.2	A05	Bra005210	A05(3894221-3895658)	99.4	96.3	A05	BnaA05g07220D
BjuTTG2-3	ZBjuH063L13	1528	B03(32083362-32081848)	100	97.2	B03	–	C03(10964876-10966395)	99.2	93.1	–	BnaC03g20650D
BjuTTG2-4	ZBjuH043G17	1501	B04(5191336-5189901)	100	95.1	B04	–	C04(6027042-6028503)	97.4	90.9	-	BnaC04g08020D
BjuTT12-1	ZBjuH047J16	2487	A07(16102336-16104823)	95.1	95.5	A07	Bra003361	A07(14915288-14917797)	99.1	96.9	A07	BnaA07g18120D
BjuTT12-2	ZBjuH124J12	2505	B04(25039840-25037376)	98.4	92.8	B04	–	C06(19784039-19786887)	87.9	94.1	–	BnaC06g17050D
BjuTT19-1	ZBjuH095N01	808	A02(3117740-3118547)	98.6	98.6	A02	Bra023602	A02(1531517-1532324)	100	99.9	A02	BnaA02g03440D
BjuTT19-2	ZBjuH170C22	800	A10(11678470-11677671)	99.9	99.9	A10	Bra008570	A10(12914260-12915058)	99.9	99.7	A10	BnaA10g17440D
BjuTT19-3	ZBjuH122M08	1030	B02(33205499-33206525)	100	96.7	B02	–	C02(3777795-3778584)	85.6	95.2	–	BnaC02g07090D
TT19-g144296		825	B08(25314277-25313454)	100	99.4	B08	–	A10(12914260-12915058)	96.4	91.1	–	–

Figure 1.

Putative chromosomal positions of cloned proanthocyanidins-associated genes in Brassica juncea. BjuTT5-4 and BjuTT2-2 were not located on the chromosome in B. juncea.

To confirm the above genomic locations, the BAC clones used for sequencing full-length genes were also sequenced for BESs. The resulting BESs between 587 and 1233 bp in length were also mapped in a similar way. Mapping of the BESs to the B. rapa reference genome showed that both BESs of 23 A-genome B. juncea PA genes were mapped around the genomic position as mapped by the full-length sequence of the corresponding genes. However, one BES of the BACs carrying two A-genome genes, i.e., BjuTT2-1 and BjuTTG1-1 was mapped to an unfixed scaffold, whereas one BES of the BACs carrying the remaining five A-genome genes, i.e., BjuTT5-2, BjuTT6-1, BjuANR-2, BjuTT10-1, and BjuTTG2-1 was mapped to an unexpected genomic position (Table 4). Mapping of the BESs to the B. napus reference genome generated a more complicated picture. For only 15 A-genome B. juncea PA genes, both BESs were mapped around the genomic position as mapped by the full-length sequence of the corresponding genes. One or both BESs of the BACs carrying 7 A-genome genes, i.e., BjuTT5-1, BjuTT6-2, BjuTT7-1, BjuTT16-2, BjuTT1-1, BjuTT2-1, and BjuTTG1-1 were mapped to an unfixed scaffold, whereas one or both BESs of the BACs carrying the remaining 8 A-genome genes were mapped to an unexpected A-genome chromosome, or a C-genome chromosome in B. napus reference genome (Table 4). Mapping of the BESs to the B. nigra reference genome showed that both BESs of 19 B-genome B. juncea PA genes were mapped around the genomic position as mapped by the full-length sequence of the corresponding genes, one BES of the BACs carrying three B-genome genes, i.e., BjuTT4-6, BjuTT18-4, and BjuTT7-2 was mapped to an unexpected genomic position in the B. nigra reference genome, and then one BES of the BACs carrying the remaining three B-genome genes, i.e., BjuTT5-4, BjuTT1-2, and BjuTT2-2 was mapped to an unfixed scaffold (Table 4).

Table 4.

Mapping to the B. rapa, B. nigra, or B. napus reference genome of end sequences of the PA gene carrier BACs from B. juncea.

B. juncea gene	BAC sequenced	Left end			Right end			Putative Genome or chromosome
		Length (bp)	Position in B. rapaa, B. napusb or B. nigra reference genome	Identity (%)	Length (bp)	Position in B. rapaa, B. napubs or B. nigra reference genome	Identity (%)
BjuTT4-1	ZBjuH187G14	1048	A02(23296428-23295380)a	98.9	1113	A02(23152779-23153899)a	97.7	A02
			A02(22075270-22077756)b	99.5		A02 (21892025-21893145)b	99.6
BjuTT4-2	ZBjuH175I06	1071	A02(2363318-2361982)	97.5	964	A02(2240655-2241616)	96.1	A02
			A02 (887331-888410)	99.6		A02 (755926-756886)	99.2
BjuTT4-3	ZBjuH037O10	1080	A03(2659211-2658129)	99.0	587	A03(2525990-2526576)	99.1	A03
			A03(2210720-2211802)	100		A03(2079518-2080104)	97.5
BjuTT4-4	ZBjuH036O12	1031	A10(12537033-12538063)	97.4	1059	A10(12660016-12659255)	97.8	A10
			A10(13765877-13766904)	98.1		A10(13890039-13890915)	98.2
BjuTT4-5	ZBjuH053C09	1062	B02(31653057-31654113)	94.2	994	B02(31799935-31798937)	95.2	B02
BjuTT4-6	ZBjuH053C08	1110	B03(41773983-41775087)	97.5	1097	Repeat sequence	–	B03
BjuTT4-7	ZBjuH090K23	1065	B08(26731199-26730093)	93.2	1111	B08(26612733-26613845)	92.3	B08
BjuTT5-1	ZBjuH186N11	1023	A03(30133579-30132560)	98.0	1112	A03(30031062-30032157)	98.3	A03
			Un_random(22402145-22403163)	98.6		A03(28012499-28013608)	97.6
BjuTT5-2	ZBjuH181K10	1071	A07(15073257-15074276)	97.0	926	A09(31899835-31899128)	97.4	A07
			A07(13943509-13944466)	95.7		A06(7618337-7619247)	97.5
BjuTT5-3	ZBjuH080G05	997	A09(29107181-29106192)	99.0	948	A09(28952074-28953021)	97.3	A09
			A09(25510915-25511912)	98.2		A09_random(28952074-28953021)	99.1
BjuTT5-4	ZBjuH106O08	1233	scaffold_30.1(166852-165708)	92.5	930	scaffold_30.1(64946-65862)	94.8	B
BjuTT6-1	ZBjuH058K21	1177	A07(2473977-2472803)	96.2	1179	A03(21939848-21938684)	97.7	A03
			A07(2765573-2766747)	98.0		A03(20708198-20709378)	98.9
BjuTT6-2	ZBjuH087J23	863	A09(27193799-27193372)	96.4	1002	A09(27076762-27077609)	98.9	A09
			Un_random(93706755-93707622)	96.9		A09(23672668-23672668)	92.7
BjuTT6-3	ZBjuH031F14	1036	A09(32538591-32537560)	97.4	963	A09(32402616-32403171)	98.6	A09
			A09_random(3439921-3440956)	98.7		A09(28629617-28630172)	99.1
BjuTT6-4	ZBjuH022O18	1051	B03(6156413-6155347)	97.2	1068	B03(6011976-6012882)	99.1	B03
BjuTT6-5	ZBjuH143I07	1034	B08(40752199-40753230)	96.6	1115	B08(40891718-40890578)	95.6	B08
BjuTT7-1	ZBjuH159L04	950	A10(14281026-14287855)	94.3	875	A10(14428932-14428063)	99.2	A10
			A10(15377459-15378411)	95.4		Un_random(110160592-110161461)	99.1
BjuTT7-2	ZBjuH080O14	1072	B08(28651670-28650600)	94.8	1084	B05(13990978-13992051)	97.5	B08
BjuTT3-1	ZBjuH029J10	952	A09(10801814-10802675)	99.1	902	A09(10945043-10944178)	96.0	A09
			A09_random(1204329-1205280)	99.7		A09(9183884-9184783)	97.5
BjuTT3-2	ZBjuH183H13	973	B06(24254316-24255289)	99.9	984	B06(24372970-24371986)	99.9	B06
BjuTT18-1	ZBjuH054O02	1063	A01(6900126-6899636)	97.6	896	A01(6775183-6775797)	94.7	A01
			A04 (19187764-19188396)	97.2		A01_random(376002-376873)	96.3
BjuTT18-2	ZBjuH181K08	1071	A03(24832511-24831559)	93.6	928	A03(24707499-24708449)	98.2	A03
			A03(23248562-23249571)	95.6		A03_random(1774070-1775012)	96.3
BjuTT18-3	ZBjuH091K10	1151	B02(37271971-37273119)	99.5	1033	B02(36953007-36952312)	92.9	B02
BjuTT18-4	ZBjuH177N08	1179	B08(43363553-43362571)	94.8	1036	Scaffold_215.1 (85454-84890)	99.1	B08
BjuANR-1	ZBjuH082J01	981	A01(21610018-21609039)	100	890	A01(21496749-21497598)	93.7	A01
			A03_random(5751842-5752821)	98.8		A03_random(5650455-5651236)	98.1
BjuANR-2	ZBjuH148I16	1088	A01(17599566-17600642)	98.5	996	A05(6382585-6383570)	96.7	A01
			A01_random(1627810-1628889)	97.7		C06(11884727-11885709)	94.0
BjuANR-3	ZBjuH116E04	1136	B01(23295810-23295103)	96.8	1143	B01(22662292-22661178)	93.5	B01
BjuANR-4	ZBjuH034P21	1015	B08(31415998-31416996)	95.1	996	B08(31573791-31572839)	99.6	B08
BjuTT10-1	ZBjuH083G18	895	A02(22936601-22937747)	96.8	926	A07(287213-288132)	82.6	A02
			Un_random(20422597-20428316)	99.7		A10(7084906-7085831)	99.4
BjuTT10-2	ZBjuH055H16	998	A06(20316953-20317946)	95.7	974	A06(20441440-20440468)	99.6	A06
			A06(20471573-20472569)	96.0		A06 (20582545-20583508)	96.9
BjuTT10-3	ZBjuH021A16	1006	B06(30166234-30167223)	92.7	988	B06(30284319-30283311)	93.7	B06
BjuTT10-4	ZBjuH176D10	1037	B07(12155684-12156717)	99.0	1008	B07(12306990-12305968)	98.1	B07
BjuTT1-1	ZBjuH180A05	1064	A09(18800232-18799233)	96.8	888	A09(18714197-18714808)	95.7	A09
			Un_random(4883830-4884831)	96.2		Un_random(4606100-4606768)	95.5
BjuTT1-2	ZBjuH147E23	1021	Scaffold_312.1 (121001-121983)	97.7	880	Repeat Sequence	–	B06
BjuTT2-1	ZBjuH085H24	1008	Scaffold000519(4310-5318)	99.2	933	A08(8207949-8208882)	99.3	A08
			Un_random(53133429-53134437)	99.8		A08(7146333-7147266)	99.7
BjuTT2-2	ZBjuH034J15	1041	Scaffold_500.1 (142630-141751)	98.1	974	Scaffold_1045.1 (32982-32009)	99.2	B
BjuTT8-1	ZBjuH004L18	926	Repeat sequence	–	962	A09(15796870-15796730)	91.4	A09
			A09_random(2192731-2193692)	98.7		A09(15375879-15376928)	99.4
BjuTT8-2	ZBjuH005J18	923	B03(8048419-8049250)	98.0	729	B03(8126536-8125803)	98.5	B03
BjuTT16-1	ZBjuH130K12	1069	A02(25075678-25075282)	84.8	1046	A02(24938143-24939195)	97.8	A02
			A02(23647099-23647956)	95.6		A02(23509181-23510233)	98.7
BjuTT16-2	ZBjuH099A21	973	A03(20916287-20917258)	100	971	A03(21065354-21064524)	98.1	A03
			Un_random(122117253-122118230)	99.1		A03(19805901-19806859)	96.9
BjuTT16-3	ZBjuH091L03	1089	A09(3425481-3424388)	96.9	1096	Repeat sequence	–	A09
			A09(2759676-2760769)	97.5		A04(12367561-12368716)	95.1
BjuTT16-4	ZBjuH098G12	1116	B01(19570278-19569159)	95.7	1094	B01(19452807-19453868)	93.9	B01
BjuTT16-5	ZBjuH094N07	1014	B06(30590171-30591180)	98.3	998	B06(30721361-30720370)	95.9	B06
BjuTT16-6	ZBjuH077C18	1022	B08(41697103-41696075)	97.8	1076	B08(41543305-41544388)	97.4	B08
BjuTTG1-1	ZBjuH130K10	1077	A06(17786017-17784921)	96.1	1073	Scaffold000172(118821-119882)	96.3	A06
			A06(18565145-18566064)	94.5		Un_random(101024139-101025200)	96.8
BjuTTG1-2	ZBjuH129A18	1083	B08(41995537-41996211)	93.2	1087	B08(42142183-42141110)	97.3	B08
BjuTTG2-1	ZBjuH088A24	1139	A01(12598037-12597696)	78.9	1145	A03(8757693-8756631)	96.4	A03
			C07(33256608-33257744)	92.1		A03(8043552-8044706)	99.1
BjuTTG2-2	ZBjuH101A24	1047	A05(4101759-4100924)	98.8	1074	A05(3964877-3966181)	90.9	A05
			A05(3967225-3968262)	97.6		A05(3832372-3833452)	98.6
BjuTTG2-3	ZBjuH063L13	1099	B03(32169026-32168705)	82.7	1080	B03(32059185-32060257)	96.2	B03
BjuTTG2-4	ZBjuH043G17	1094	B04(5231833-5230759)	96.8	1118	B04(5118011-5119109)	94.9	B04
BjuTT12-1	ZBjuH047J16	1005	A07(16179990-16178972)	96.9	1010	A07(16062416-16062903)	98.6	A07
			A07(14996804-14997785)	96.6		A07(14870425-14871409)	98.6
BjuTT12-2	ZBjuH124J12	1057	B04(24922182-24923204)	90.5	1030	B04(25075022-25073994)	97.8	B04
BjuTT19-1	ZBjuH095N01	1093	Repeat sequence	–	969	A02(3047112-3048020)	96.7	A02
			Repeat sequence	–		A02(1465948-1468567)	96.5
BjuTT19-2	ZBjuH170C22	1054	A10(11649528-11650476)	94.0	985	A10(11804011-11803026)	99.8	A10
			A10(12892359-12893412)	100		C09(43030716-43031727)	93.4
BjuTT19-3	ZBjuH122M08	1033	B02(33362984-33362101)	92.3	888	B02(33197233-33197697)	98.5	B02

^aPosition in B. rapa reference genome is listed in the first line.

^bPosition in B. napus reference genome is listed in the second line.

Expression of PA genes in seed coat of B. juncea and B. napus

Fragments Per Kilobase of a transcript per Million (FPKM) analysis indicated that 55 annotated B. napus PA genes (excluding BnaCnng01290D and BnaA09g29340D), and all cloned B. juncea PA genes except BjuTT5-1 and BjuTT5-4 were expressed in seed coat (Figure 2, Table S7). However, transcript abundance significantly varied among PA genes, as well as accessions. In general, the expression level of structural and transporter genes were higher than that of transcriptional factor genes in black- and brown-seeded accessions analyzed. No transcripts of BjuTT3, BjuANR, BjuTT18-1, BjuTT19-1, and BjuTT19-3 were detected in the seed coat of yellow-seeded SY. In addition, a 7-fold or greater difference in expression level of BjuTT3, BjuTT18, BjuANR, and BjuTT19 as well as BjuTT4-2, BjuTT4-3, BjuTT4-4, and BjuTT5-3 were found between SY and its brown-seeded near-isogenic lines (Figure 2, Table S7), implying that these differentially expressed genes are involved in seed pigmentation. Moreover, six additional genes, i.e., BjuTT4-5, BjuTT6-1, BjuTT6-4, BjuTT8-1, BjuTT16-3, and BjuTT16-6, were upregulated by at least 2-fold in seed coat of NILA, whereas four other genes (BjuTT4-5, TT4_g135394, BjuTT6-4, and BjuTT8-2) were upregulated by at least 2-fold in seed coat of NILB compared with SY (Figure 2, Table S7). RT-PCR analysis confirmed the differential expression profile of BjuTT3, BjuTT18, BjuANR, and BjuTT19 that was carried out using FPKM analysis (Figure 3).

Figure 2.

Expession heatmap of gene expression based on FPKM data. NILA, NILB, SY represent the seed coat of B.juncea, and XY15, RIL52, RIL55 represent the seed coat of B.nupus. The color key represents FPKM normalized log₂ transformed counts.

Figure 3.

RT-PCR analysis of genes for proanthocyanidin biosynthesis in the seed coats of Brassica juncea. SY, Sichuan Yellow; NILA and NILB, Near-Isogenic Lines A and B. Seed coats were separated from seeds at 15 days after pollination.

Discussion

In the present study, we identified 55, 58, and 31 PA genes in B. juncea, B. napus, and B. rapa through a combination of experimental and bioinformatics approaches, analyzed their phylogenetic relationship and genomic locations in Brassica, and detected and compared their expression in seed coats of different accessions by RNA-seq. Cloning of these genes not only lays a foundation for the elucidation of the molecular mechanism underlying PA accumulation/profile and seed pigmentation in Brassica species, but also facilitates in the functional characterization of each PA gene.

The PA genes in Arabidopsis (16) were almost doubled in B. rapa (31) and nearly quadrupled in B. juncea (55) and B. napus (58). The ancestral A, B, and C genomes of the Brassica species contributed a comparable number of PA genes. These findings are consistent with mesopolyploid nature of B. rapa and the allopolyploid nature of B. juncea and B. napus, implying that polyploidization plays an important role in expansion of PA genes. However, the number of PA genes in allopolyploid B. juncea and B. napus does not amount to the sum of PA genes from both ancestral species due to gene loss by genomic fractionation during allopolyploidization. Bra036307 and Bra009770 might have been lost in B. juncea and B. napus, respectively.

Phylogenetic analysis and genomic localization of B. juncea PA genes indicated that 30 and 29 B. juncea PA genes were homologous to genes located in the A-genome chromosomes of B. rapa and B. napus, respectively (Figure S2, Table 3). However, both BESs of 23 and 15 A-genome B. juncea PA genes were mapped around the B. rapa and B. napus genomic position, as mapped by the full-length sequence of the corresponding genes, respectively (Table 4). The other BESs were mapped to other chromosomes or not detected in the B. rapa and B. napus reference genome. These findings indicate that although B. rapa, B. juncea, and B. napus have the common A-genome, the chromosomes of each of these species do not harbor the same structure (Zou et al., 2016). On the other hand, assembly of the present reference genomes of Brassica species need improving.

For 6 of the annotated PA genes in B. juncea GSS, no BAC clones were identified. Sequence analysis revealed that the annotated genes ANR_g228640, ANR_g226654, and TT19_g167454 were false genes or artifacts that arose by misassembled sequences because these annotated genes only contain a part of the protein domains of the corresponding genes and its alignment ratios were significantly lower than other predicted genes (Table S5). No BACs carrying the annotated gene TT4_g135394, TT5_g158015, or TT19_g144296 were detected, most probably because the sequenced fragments amplified from positive BACs were too short to distinguish different members of a gene family (Table 1), or maybe because the primers used in screening the BAC library were not appropriate. In contrast, the cloned BjuTT4-1, BjuTT4-7, and BjuTT16-5 genes were not predicted from our GSS dataset, illustrating that these genes were missed in our genome sequence survey of B. juncea genome, most probably because of insufficient sequencing depth or assembly errors.

In Arabidopsis, three additional PA genes TT15 (DeBolt et al., 2009), TT9 (Ichino et al., 2014), and TT13/aha10 (Appelhagen et al., 2015) have recently been cloned. Their Brassica homologs were not investigated in the present study. In our next study, we will clone and analyze these genes to complete the set of PA genes in Brassica spp. Initial screening of our BAC library identified seven BAC clones for each of these three genes. Sequencing of the fragments amplified from these BACs is underway.

RNA-seq and FPKM analyses showed that BnaCnng01290D, BnaA09g29340D, BjuTT5-1 and BjuTT5-4 were not expressed in the seed coat, indicating that these genes might not be involved in seed pigmentation. Interestingly, the BjuTT3, BjuTT18, and BjuANR genes were not expressed in yellow-seeded testa, but expressed very high in brown-seeded testa of B. juncea (Figure 2, Table S7), which is consistent with previous results (Yan et al., 2008, 2011; Akhov et al., 2009; Liu et al., 2009, 2013; Jiang et al., 2013), suggesting that seed color is determined by expression of genes that encode enzymes that catalyze PA biosynthesis. Concomitant with the absence of expression of these enzyme-encoding genes in yellow-seeded testa, the early stage genes, BjuTT4-2 and BjuTT4-3, which encode chalcone synthase, and transporter genes, BjuTT19-1 and BjuTT19-3, which encode glutathione transferase, were remarkably downregulated or not expressed in yellow-seeded testa (Table S7). These findings illustrate that these genes are co-regulated with BjuTT3, BjuTT18, and BjuANR, and their expression is not essential to the production of biosynthetic substrates and epicatechin transport in yellow-seeded testa. Other BjuTT19 and BjuTT4 genes did not show differential expression between yellow- and brown-seeded testa (Figure 2, Table S7), implying that these genes are not involved in seed pigmentation and that their biological roles require further investigation.

To answer the questions why all the BjuTT3, BjuTT18, and BjuANR genes are not fully expressed in yellow-seeded testa and why these genes are mutated, transcriptionally regulated, or both, we also cloned full-length genomic sequences of these genes from SY and compared them with the corresponding sequences from PM. Comparative analysis showed no differences, except for a 33-bp and 2-bp difference in BjuTT18-2 and BjuTT3-1. In Arabidopsis, the genes TT3, TT18, and ANR are transcriptionally regulated by TT2-TT8-TTG1 complex (Xu et al., 2013). Comparison between SY and PM uncovered a 1275-bp insertion in exon 7 of BjuTT8-1 and a C-T transition in exon 7 of BjuTT8-2 of SY, which is almost in agreement with findings from Padmaja et al. (2014) who speculated that the TT8 gene controls seed pigmentation in B. juncea.

Conclusions

A total of 55 genes homologous to 16 Arabidopsis proanthocyandin-associated genes were identified and cloned from B. juncea. Approximately 58 and 31 PA genes were detected in B. napus and B. rapa genome databases. Around 30 of these cloned B. juncea genes were located in the A-genome chromosomes, except A04, whereas the remaining 25 were mapped to the B-genome chromosomes, except B05 and B07. A majority of these genes were expressed in the seed coat of B.juncea and B. napus. Tissue-specific expression of the TT4, TT5, and TT19 genes were observed in B. juncea and B. napus. BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 were transcriptionally regulated in the seed coat and not expressed or downregulated in yellow-seeded testa. In summary, the present study facilitates in better understanding the molecular mechanism underlying PA accumulation/profile and seed pigmentation, as well as in further characterization of the structure, variations, and functions of PA genes in Brassica spp.

Author contributions

ZL and CG designed the research. XL, YL, MY, and DS performed the research and analyzed the data. XH took part in screening of the BAC library. SL and SC provided the genes primers and assisted with sequencing of the BAC clones. ZL and XL wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No.31101176 and No.31271762).

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.01831/full#supplementary-material

Figure S1

The PCR screening systems and process of BAC library. Screening of a specific clone by five-step PCR is shown: Step 1, screening of 19 superplates: A positive signal was detected in the superplate3 (plates SP 021–030). Steps 2–4, screening against superplate3 by 3D-PCR: Positive signals were identified in 1D, 2D, and 3D; these consisted of Plate034, C15&16, and RI&J, respectively, indicating that Plate034, column 15/16, and row I/J contained the specific BAC DNA. Step 5, screening of four candidate BACs: A positive signal was detected in the one of the four candidate BACs (ZBjuH034I15, ZBjuH034I16, ZBjuH034J15, and ZBjuH034J16). Consequently, a BAC clone containing the specific sequence was identified as the clone of ZBjuH034J15.

Figure S2

Phylogenetic trees of proanthocyanidin-associated genes from Brassica juncea, B. rapa, B. napus, and Arabidopsis thaliana. Phylogenetic reconstruction of proanthocyanidin biosynthetic genes from Brassica juncea, Arabidopsis thaliana, B. rapa, and B. napus. Phylogenetic trees were constructed from genomic sequences of PA genes using Neighbor-Joining (NJ) algorithm and 1000 bootstrap replications provided in MEGA5.2. (a) TT4; (b) TT5; (c) TT6; (d) TT7; (e) TT3; (f) TT18; (g) ANR; (h) TT10; (i) TT1; (j) TT2; (k) TT8; (l) TT16; (m) TTG1; (n) TTG2; (o) TT12; (p) TT19.

Figure S3

Sequences of annotated but unidentified proanthocyanidins-associated genes in Brassica juncea.

Table S1

Sequences of the primer pairs used in screening for proanthocyanidin-associated genes of Brassica juncea BAC library.

Table S2

Sequences of the primer pairs used in expression analysis of proanthocyanidin-associated genes of Brassica juncea.

Table S3

Global statistics of the genomic assembly of Brassica juncea.

Table S4

Annotated genes of Brassica juncea genome survey sequences (xls).

Table S5

Annotated proanthocyanidin-associated genes from Brassica juncea genome survey sequences (xls).

Table S6

Mapping to the GSS sequence of full-length sequences of Brassica juncea PA genes cloned in this study (xls).

Table S7

Transcript abundance of proanthocyanidin-associated genes in the transcriptome of Brassica napus and B. juncea seed coats.