Characterization of a new high copy Stowaway family MITE, BRAMI-1 in Brassica genome

  • Perumal Sampath1,

    Affiliated with

    • Sang-Choon Lee1,

      Affiliated with

      • Jonghoon Lee1,

        Affiliated with

        • Nur Kholilatul Izzah1,

          Affiliated with

          • Beom-Soon Choi2,

            Affiliated with

            • Mina Jin3,

              Affiliated with

              • Beom-Seok Park3 and

                Affiliated with

                • Tae-Jin Yang1Email author

                  Affiliated with

                  BMC Plant Biology201313:56

                  DOI: 10.1186/1471-2229-13-56

                  Received: 12 September 2012

                  Accepted: 18 March 2013

                  Published: 2 April 2013

                  Abstract

                  Background

                  Miniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome.

                  Results

                  We identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs.

                  Conclusion

                  A Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species.

                  Keywords

                  Miniature Inverted-repeat Transposable Element (MITE) MITE insertion polymorphism (MIP) Brassica species Evolution BRAMI-1

                  Background

                  The large-scale sequencing of eukaryotic genomes has revealed that transposable elements (TEs) are present ubiquitously and occupy large fractions of genomes: 5% in yeast, 35% in rice, 45% in human, and up to 85% in maize [19]. TEs are classified into two classes based on their transposition mechanism. Class I mobile genetic elements, or retrotransposons, are replicated through RNA intermediates by a copy-and-paste mechanism, whereas Class II mobile genetic elements, or DNA transposons, move directly from DNA via a cut-and-paste mechanism [1, 2, 10].

                  Miniature inverted-repeat transposable elements (MITEs) are Class II DNA transposons that are non-autonomous, with defective or absent of coding genes. MITEs were identified in the maize genome [11] and later found in Arabidopsis, rice, grape, mosquito, fish, bacteria and human as well as in several other genomes [1, 1215]. Due to their extremely high copy numbers, MITEs can account for a significant fraction of a eukaryotic genome (i.e. >8% of the rice genome) even though the size of element itself is small [16]. Individual MITEs are usually less than 600 bp and A/T rich, with terminal inverted repeats (TIRs) and 2–11 bp target site duplication (TSD) sequences [1, 10]. MITEs, which are relatively stable in the genome, are often closely associated with genic regions and thus can affect gene expression patterns [16, 17]. Some MITEs are involved in up-regulation of host genes by providing additional recognition sequences or polyA signals to host genes [14, 18, 19]. MITE insertion into regulatory regions may cause disruption or promotion of gene expression [18]. Recent studies have found that MITEs are also a source of small interfering RNA (siRNA) evolution and may play an important role in gene regulation and epigenetic mechanisms [16, 2022]. MITE transposition into a new region of the genome causes insertion polymorphisms among accessions of same species that can be useful tools for development of various markers [23, 24].

                  The Brassicaceae family includes 338 genera and 3700 species, which serve as sources of vegetable, fodder, condiments and oil, with wide range of morphologies, such as Chinese cabbage, mustard, cabbage, broccoli, oilseed rape, and other leafy vegetables. The model plant, Arabidopsis thaliana is a close relative of the Brassica species and belongs to the same family. As a model Brassica crop, the B. rapa genome sequence spanning 256 Mb euchromatin chromosome spaces was completed recently and released to the public [25].

                  Comparative analysis of Brassica species with A. thaliana has revealed up to two additional rounds of recent genome duplication: one triplication and one allopolyploidization that is the major factor responsible for the increased genome size of Brassica[2527]. In addition, TEs also contribute to increase the genome size of the Brassica species and to genome evolution [28]. The completed genome sequence of B. rapa revealed that at least 39.5% of the genome contains TEs [25].

                  In this study, we identified a new MITE named Bra ssica rapa MITE (BRAMI)-1, which is present in more than 1400 copies in the genome of each of three Brassica species. We inspected its characteristics and distribution and inferred its potential involvement in the evolution of duplicated genes in the highly replicated Brassica genome. We also discovered high amounts of insertion polymorphism inter- and intra-species, which can serve a good source of genetic markers in the Brassica species.

                  Results

                  Characterization of BRAMI-1 in Brassica

                  We identified a 260 bp MITE in the Brassica rapa BAC clone, KBrB059A03 using MUST, a de novo program for MITE identification, and additional manual inspection. MITE characterization on B. rapa contig (KBrB059A03) using MUST yielded 291 candidate MITEs and further careful manual inspection of each candidate MITE for TIR and TSD using self-BLAST (http://​blast.​ncbi.​nlm.​nih.​gov/​) led to the identification of BRAMI-1. Comparison of BRAMI−1 against the repeat database (http://​www.​girinst.​org/​) showed 77% similarity to a reported Stowaway MITE, ATTIRTA-1 in A. thaliana[29]. Perfect MITE insertion was confirmed by comparing one of the representative B. rapa genes (Bra013859) harboring a BRAMI-1 insertion with the related empty sites in its non-insertion paralogs (NIPs) (Bra010475 and Bra019193) from B. rapa syntenic blocks and its ortholog (At4g25050) in A. thaliana (Figure 1a, b). The MITE included 33 bp of highly conserved A/T rich (>69%) TIRs and was flanked with a unique di-nucleotide TA target site duplication (TSD), which are distinct characteristics of the Stowaway super-family MITEs (Figure 1b, c). The secondary structure of the MITE was predicted using mfold (Figure 1d), which showed a potential DNA hairpin-like secondary structure.
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig1_HTML.jpg
                  Figure 1

                  Identification and characterization of the BRAMI- 1 elements. (a) Dotplot analysis of Bra013859 and the related empty sites in its two non-insertion paralog (NIP) genes, Bra019193 and Bra010475 from B. rapa and its orthologue At4g25050 from A. thaliana (b) The structure of BRAMI-1 showing its characteristic properties, TA Target site duplication (c) Conserved 33 bp TIR sequences shown by Weblogo analysis (d) Hypothetical secondary structure and expected loop formation predicted by mfold.

                  BLASTn searches revealed a total of 697 BRAMI-1 elements in the 256 Mb B. rapa genome sequence. In silico mapping of these elements on the B. rapa pseudo-chromosomes showed that they were evenly distributed in the euchromatin regions of the B. rapa genome (Figure 2). The physical positions of the 697 BRAMI-1 elements in the B. rapa genome are listed in Additional file 1. On average, 70 BRAMI-1 elements were found on each pseudo-chromosome. MITE density analysis (chromosome size/no. of MITEs per chromosome) shows chromosome 3 (31.72 Mb), which is the second largest in size, has the high MITE density (MITE/0.28 Mb), while the largest chromosome 9 (37.12 Mb) had the less MITE density (MITE/0.44 Mb).
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig2_HTML.jpg
                  Figure 2

                  In silico mapping of BRAMI- 1 elements in 256 Mb of B. rapa pseudo-chromosomes. Arrows indicate the positions of the 25 members used for MIP analysis. The exact physical positions of the 697 BRAMI-1 members are listed in Additional file 1.

                  We found 516 and 15 copies in 470 Mb of B. oleracea and 15 Mb of B. napus shotgun sequences, respectively. Based on this, the total numbers of the BRAMI-1 MITE members were estimated as 1440, 1464 and 2490 in the whole genomes of B. rapa, B. oleracea and B. napus, respectively (Table 1). By contrast, in A. thaliana we found only 70 copies of ATTIRTA-1, the closest Arabidopsis relative of BRAMI-1. Simple comparison revealed that the copy numbers of these MITEs in Brassica genomes are 20–35 times more than that of Arabidopsis.
                  Table 1

                  Summary of observed and predicted copy numbers of the BRAMI- 1 elements in Brassica relatives

                  MITE

                  BRAMI-1

                  ATTIRTA-1

                   

                  B. oleracea

                  B. rapa

                  B. napus

                  A. thaliana

                  Database type

                  GSS

                  Pseudo-chromosomes

                  GSS

                  Whole genome

                  Database size

                  470 Mb

                  256 Mb

                  15 Mb

                  119 Mb

                  Total copies

                  399

                  697

                  11

                  70

                  (>80% similarity)

                  123

                  401

                  4

                  34

                  Average length of the GSS sequence

                  700 bp

                  N/A

                  700 bp

                  N/A

                  Estimated Genome Size [30]

                  696 Mb

                  529 Mb

                  1132 Mb

                  157 Mb

                  Estimated copies in the whole genome

                  1464

                  1440

                  2490

                  44

                  BLASTn was performed at the local database (http://​imcrop.​snu.​ac.​kr).

                  N/A: not applicable.

                  Phylogenetic analysis of the BRAMI-1 elements

                  Phylogenetic analysis was conducted for 528 nearly intact MITE members that have >80% similarity to BRAMI-1: 401 members from B. rapa, 123 from B. oleracea, and four from B. napus. In addition, 34 ATTIRTA-1 members from A. thaliana were included. The ATTIRTA-1 members formed a separate clade from the Brassica members, and they were very diverse among themselves. By contrast, BRAMI-1 members from the three Brassica species were highly conserved and were interspersed with each other (Figure 3) indicating they were rapidly amplified in the Brassica genome after divergence from Arabidopsis. Due to their high sequence similarity, we could not distinguish any separate clades for the BRAMI-1 family members in the Brassica species.
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig3_HTML.jpg
                  Figure 3

                  Phylogenetic tree of BRAMI -1 elements from Brassica species and ATTIRTA -1 from A. thaliana. Relatively intact MITE members showing 80% similarity to the characteristic MITE structure were used for the analysis. A total of 528 BRAMI-1 members including 401, 123, and 4 from B. rapa (red), B. oleracea (blue), and B. napus (black), respectively, and 34 ATTIRTA-1 members (green) were compared. Sequence alignment was conducted using ClustalW and then the phylogenetic tree was generated using the neighbor joining method with 500 bootstrap replicates.

                  BRAMI-1 insertion in genic regions of the B. rapa genome

                  We inspected the insertion sites of the 697 BRAMI-1 elements in the B. rapa genome using the annotated B. rapa genome database [31]. The analysis showed that 548 members (78.6%) were located in gene-rich regions, less than 3 kb from genes. Among them, 71 (10.2%) were inside the gene structure, specifically in introns, and 281 (40.3%) were within less than 1 kb of a gene (Table 2).
                  Table 2

                  Summary of the insertion positions of 697 BRAMI- 1 elements in the B. rapa genome

                  Insertion position

                  Number of elements

                  Percentage of elements

                  Gene

                  71

                  10.2

                  Near Genic Regions (<1 kb)a

                  281

                  40.3

                  Near Genic Regions (1 kb to <2 kb)a

                  134

                  19.2

                  Near Genic Regions (2 kb to <3 kb)a

                  62

                  8.9

                  Intergenic Region (>3 kb)a

                  149

                  21.4

                  Total

                  697

                  100.0

                  a Distance from nearest gene.

                  We closely inspected the 71 genic insertions by comparing with their NIPs from triplicated chromosomal blocks. Similar numbers of insertions were identified in tri-, di-, and mono-copy genes (20, 26, and 24 insertions, respectively; Table 3) indicating that multi-copy genes did not preferentially contain BRAMI-1 insertions. Comparison of genes containing the BRAMI-1 insertion and their NIPs genes in the triplicated blocks revealed that all of the elements resided in intronic regions.
                  Table 3

                  Insertion positions and names of the 71 genes harboring BRAMI -1 elements in intronic regions and list of their orthologous genes in Arabidopsis and NIPs in the triplicated blocks of the B. rapa genome

                   

                  MITE No

                  Chr No

                  MITE start

                  MITE end

                  Ortholog from A. thaliana

                  Triplicated blocks inB. rapa z

                        

                  LF

                  MF1

                  MF2

                  THREE COPY GENES

                  23

                  A01

                  8110084

                  8109821

                  At4g25050

                  Bra013859

                  Bra019193

                  Bra010475

                   

                  83

                  A02

                  4177719

                  4177456

                  At5g17300

                  Bra008563

                  Bra006394

                  Bra023610

                   

                  176

                  A03

                  5778627

                  5778365

                  At5g55050

                  Bra002937

                  Bra035549

                  Bra028994

                   

                  188

                  A03

                  8910390

                  8910639

                  At2g37940

                  Bra005148

                  Bra017148

                  Bra000029

                   

                  219

                  A03

                  17370042

                  17370087

                  At3g15820

                  Bra027205

                  Bra021138

                  Bra001607

                   

                  220

                  A03

                  17370087

                  17370331

                  At3g15820

                  Bra027205

                  Bra021138

                  Bra001607

                   

                  299

                  A04

                  13405505

                  13405756

                  At2g30110

                  Bra018338

                  Bra021611

                  Bra022779

                   

                  303

                  A04

                  14136797

                  14136887

                  At2g31500

                  Bra018236

                  Bra021727

                  Bra022844

                   

                  346

                  A05

                  7674854

                  7674713

                  At2g29980

                  Bra018348

                  Bra021599

                  Bra022767

                   

                  347

                  A05

                  7674974

                  7675006

                  At2g29980

                  Bra018348

                  Bra021599

                  Bra022767

                   

                  349

                  A05

                  8733844

                  8733992

                  At4g04640

                  Bra029511

                  Bra000802

                  Bra018503

                   

                  368

                  A05

                  16917820

                  16918070

                  At3g20770

                  Bra035746

                  Bra023927

                  Bra001802

                   

                  425

                  A06

                  14973843

                  14973752

                  At5g64740

                  Bra024324

                  Bra037793

                  Bra031904

                   

                  443

                  A06

                  22195786

                  22196049

                  At3g28050

                  Bra025321

                  Bra033037

                  Bra039062

                   

                  450

                  A06

                  24123977

                  24123793

                  At2g01430

                  Bra024888

                  Bra026666

                  Bra017451

                   

                  452

                  A06

                  24666391

                  24666142

                  At5g46630

                  Bra025009

                  Bra022052

                  Bra017537

                   

                  473

                  A07

                  8537339

                  8537527

                  At1g22340

                  Bra031388

                  Bra012324

                  Bra016424

                   

                  474

                  A07

                  9114152

                  9114414

                  At1g20670

                  Bra025837

                  Bra012243

                  Bra016456

                   

                  566

                  A08

                  20712404

                  20712596

                  At1g07920

                  Bra018669

                  Bra031602

                  Bra030707

                   

                  654

                  A10

                  1570075

                  1569913

                  At1g06080

                  Bra015473

                  Bra032437

                  Bra030638

                  TWO COPY GENES

                  46

                  A01

                  18882651

                  18882770

                  At5g52140

                  Bra028293

                  Bra022579

                  -

                   

                  55

                  A01

                  23626196

                  23626459

                  At3g16180

                  Bra027185

                  Bra021168

                  -

                   

                  61

                  A01

                  25069244

                  25069502

                  At3g02180

                  -

                  Bra021476

                  Bra001035

                   

                  87

                  A02

                  5079784

                  5080047

                  At5g20540

                  -

                  Bra006563

                  Bra020109

                   

                  113

                  A02

                  9852697

                  9852644

                  At1g66370

                  Bra004162

                  Bra039763

                  -

                   

                  153

                  A02

                  25486260

                  25486523

                  At5g23940

                  Bra009716

                  -

                  Bra029388

                   

                  168

                  A03

                  2298612

                  2298349

                  At5g12420

                  -

                  Bra006160

                  Bra023377

                   

                  200

                  A03

                  11193078

                  11192830

                  At2g47460

                  Bra004456

                  -

                  Bra000453

                   

                  234

                  A03

                  20936196

                  20936267

                  At5g23260

                  Bra013028

                  Bra026507

                  Bra029365

                   

                  235

                  A03

                  20936271

                  20936494

                  At5g23260

                  Bra013028

                  Bra026507

                  Bra029365

                   

                  249

                  A03

                  24785451

                  24785715

                  At4g22950

                  -

                  Bra019343

                  Bra020826

                   

                  319

                  A04

                  18584148

                  18584406

                  At2g45550

                  Bra004921

                  Bra039330

                  -

                   

                  444

                  A06

                  22352521

                  22352784

                  At3g27640

                  Bra025293

                  -

                  Bra039073

                   

                  460

                  A07

                  1577014

                  1576769

                  At2g18230

                  Bra039627

                  -

                  Bra037229

                   

                  467

                  A07

                  6402416

                  6402153

                  At1g29120

                  -

                  Bra030121

                  Bra010851

                   

                  490

                  A07

                  12392864

                  12392917

                  At3g57530

                  Bra007334

                  -

                  Bra003287

                   

                  536

                  A08

                  12108300

                  12108552

                  At4g35150

                  -

                  Bra017699

                  Bra034678

                   

                  545

                  A08

                  15271728

                  15271631

                  At4g36760

                  Bra011704

                  -

                  Bra010574

                   

                  596

                  A09

                  8214071

                  8213980

                  At1g61890

                  Bra027073

                  Bra028379

                  -

                   

                  597

                  A09

                  8214185

                  8214078

                  At1g61890

                  Bra027073

                  Bra028379

                  -

                   

                  604

                  A09

                  16501688

                  16501868

                  At5g46350

                  Bra025021

                  Bra022033

                  Bra017561

                   

                  605

                  A09

                  16667316

                  16667053

                  At5g46040

                  -

                  Bra022016

                  Bra017582

                   

                  606

                  A09

                  16682960

                  16683223

                  At5g46040

                  -

                  Bra022016

                  Bra017582

                   

                  608

                  A09

                  19871591

                  19871427

                  At1g32780

                  Bra023290

                  Bra010185

                  -

                   

                  615

                  A09

                  24192808

                  24192545

                  At1g23380

                  Bra024593

                  -

                  Bra016348

                   

                  666

                  A10

                  8067999

                  8067852

                  At5g57655

                  Bra002710

                  Bra020426

                  -

                   

                  670

                  A10

                  8789727

                  8789464

                  At5g59340

                  Bra002576

                  Bra020321

                  -

                  ONE COPY GENES

                  40

                  A01

                  14766344

                  14766081

                  -

                  -

                  -

                  Bra029909

                   

                  41

                  A01

                  14767003

                  14766741

                  -

                  -

                  -

                  Bra029909

                   

                  129

                  A02

                  16530545

                  16530808

                  At4g01590

                  -

                  -

                  Bra008554

                   

                  178

                  A03

                  5992774

                  5992961

                  -

                  -

                  -

                  Bra029035

                   

                  223

                  A03

                  18448338

                  18448491

                  At3g20360

                  -

                  -

                  Bra001785

                   

                  578

                  A09

                  3996947

                  3996684

                  At2g11810

                  -

                  -

                  Bra037199

                   

                  266

                  A03

                  29733949

                  29734212

                  -

                  -

                  Bra017680

                  -

                   

                  268

                  A03

                  30599723

                  30599787

                  At4g36940

                  -

                  Bra017808

                  -

                   

                  472

                  A07

                  7616363

                  7616100

                  -

                  -

                  Bra012436

                  -

                   

                  49

                  A01

                  21397784

                  21397864

                  At3g19870

                  -

                  Bra038237

                  -

                   

                  120

                  A02

                  12485621

                  12485884

                  At1g72110

                  -

                  Bra008008

                  -

                   

                  128

                  A02

                  15935110

                  15935361

                  At1g80200

                  -

                  Bra008487

                  -

                   

                  148

                  A02

                  24005490

                  24005690

                  -

                  -

                  Bra020642

                  -

                   

                  285

                  A04

                  7140471

                  7140542

                  -

                  Bra028251

                  -

                  -

                   

                  378

                  A05

                  20048141

                  20048392

                  -

                  Bra027271

                  -

                  -

                   

                  445

                  A06

                  22716443

                  22716194

                  At3g26610

                  Bra025216

                  -

                  -

                   

                  501

                  A07

                  16958538

                  16958801

                  At1g65590

                  Bra004121

                  -

                  -

                   

                  513

                  A07

                  20340243

                  20340188

                  At1g74790

                  Bra015893

                  -

                  -

                   

                  655

                  A10

                  4270200

                  4270396

                  At1g02390

                  Bra033323

                  -

                  -

                   

                  656

                  A10

                  4270415

                  4270484

                  At1g02390

                  Bra033323

                  -

                  -

                   

                  657

                  A10

                  4410198

                  4410053

                  -

                  Bra033297

                  -

                  -

                   

                  672

                  A10

                  9364412

                  9364675

                  -

                  Bra002467

                  -

                  -

                   

                  673

                  A10

                  9364744

                  9365007

                  -

                  Bra002467

                  -

                  -

                   

                  677

                  A10

                  10858935

                  10858966

                  -

                  Bra002214

                  -

                  -

                  z The triplicated chromosome blocks are denoted according to the classification of the B. rapa genome annotation databases (BRAD) [25, 31]. The triplicated chromosome blocks are classified as one least fractionized block (LF) and two moderately fractionized blocks (MF1, MF2). Genes with BRAMI-1 insertion in introns are denoted in bold and their NIPs are denoted in plain letters.

                  For example, Bra024324 gene was annotated as having 13 exons and included the BRAMI-1 insertion in the 7th intron. Its two NIPs (Bra031904, Bra037793) and its Arabidopsis ortholog (At5g64740, CELLULOSE SYNTHASE 6) have similar structures in which the exonic regions share conserved sequences with Bra024324 (Figure 4a). Another gene, Bra010574, which has the BRAMI-1 insertion in 5th intron, showed conserved CDS sequences without any change of gene structure compared to its NIPs (Bra011704) and its Arabidopsis ortholog (At4g36760, 15 ORF, N-1-NAPHTHYLPHTHALAMIC ACID BINDING PROTEIN) (Figure 4b).
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig4_HTML.jpg
                  Figure 4

                  Microsynteny between the genomic regions with BRAMI -1 insertions and homologous blocks in B. rapa and A. thaliana . (a) Genomic region including 3 kb upstream from the start codon and 3 kb downstream from the stop codon of Bra024324 compared with those of its two paralogs and Arabidopsis ortholog. (b) Genomic region including 2 kb upstream from the start codon and 0.3 kb downstream from the stop codon of Bra010574 compared with those of its paralog and Arabidopsis ortholog. Genomic organization, such as exon and intron location, is based on annotation information in BRAD for B. rapa and TAIR for A. thaliana. Red lines indicate exons of each gene annotation. The gray bars connecting boxes on genome sequences indicate synteny blocks present in both sequences. The position of the MITE insertion is indicated by both an asterisk and a green block. The map was generated based on nucleotide sequence similarity determined by BLASTn search.

                  Transcriptional changes of B. rapa genes containing BRAMI-1 insertions

                  Even though most of the BRAMI-1 insertions were found in introns or UTRs, some modification of gene expression might still be mediated by BRAMI-1. Therefore, we analyzed expression level changes by comparison to NIPs using a B. rapa microarray database. Among the 46 multicopy genes with BRAMI-1 insertions (20 tri-copy genes and 26 di-copy genes), only six were present along with their NIPs in the microarray database. Of the six genes with BRAMI-1 insertions, only Bra039627 showed similar expression to that of its NIPs, regardless of stress treatments. One gene, Bra024324, showed decreased expression and four genes, Bra027185, Bra039330, Bra034678, and Bra010574, showed increased expression compared to that of their NIPs (Figure 5).
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig5_HTML.jpg
                  Figure 5

                  Comparison of expression profiles between genes with BRAMI- 1 insertions and their NIPs. (a) Expressions of Bra024324 and its two NIPs, Bra031904 and Bra037793, were analyzed by searching a microarray database of B. rapa treated with cold (4°C), salt (250 mM NaCl), drought (air-drying), or ABA (100 μM). (b) Expression of Bra010574 and its NIP, Bra011704, were compared. MITE+ and MITE- indicate genes with the BRAMI-1 insertion and their NIPs, respectively.

                  The expression of Bra024324, which contains a BRAMI-1 insertion, was severely decreased compared to that of its NIPs, Bra031904 and Bra037793, under normal conditions and also under the four stress treatment conditions, indicating that Bra024324 gene expression was maintained at a very low level even though the BRAMI-1 insertion did not affect exons (Figure 5a). By contrast, expression of Bra010574, with a BRAMI-1 insertion, was more than 3-fold higher than expression of its NIP Bra011704 under control and all four treatment conditions (Figure 5b).

                  Survey of MITE insertion polymorphisms (MIPs) and estimation of activation dates

                  To analyze BRAMI-1’s transposition activity and insertion time, we designed 50 MIP primers, 25 for B. rapa and 25 for B. oleracea, from the flanking regions of the BRAMI-1 insertions, especially insertions in genic regions (Additional file 2). The positions of the 25 B. rapa MIPs are denoted as arrows on the in silico map (Figure 2). Almost all of the primer pairs revealed polymorphisms (48 in 50 pairs; 96%) among seven accessions belonging to three Brassica species, indicating that the BRAMI-1 members have been continuously activated during diversification of the Brassica genome. Moreover, there was high polymorphism within species, with seven (14%), six (12%), and ten (20%) polymorphisms among two accessions of B. napus, two accessions of B. rapa, and three accessions of B. oleracea, respectively.

                  We grouped the 50 MIPs into three different groups: Bs (common to both species), Br (B. rapa-unique), and Bo (B. oleracea-unique), to deduce the tentative insertion times (Figure 6a). The Br and Bo MIPs were further classified into two subgroups, -I and –II, based on the presence or absence of the insertion in their allopolyploid species B. napus. Among the 25 B. rapa MIPs, 3, 17, and 5 were Bs, Br-I, and Br-II type insertions, respectively, and among the 25 B. oleracea MIPs, 6, 18, and 1 were Bs, Bo-I, and Bo-II types, respectively. Overall, 18% were shared in the Brassica genus, and 82% were species-unique insertions (Figure 6b).
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2229-13-56/MediaObjects/12870_2012_1263_Fig6_HTML.jpg
                  Figure 6

                  MITE insertion polymorphism (MIP) analysis and estimation of insertion time. MIP patterns were classified into 5 groups (Bs, Br- I, II and Bo- I, II), based on existence of MIPs between species. (a) Gel electrophoresis of five MIPs (Bo-23, Br-6, Br-3, Bo-10, Bo-21, ordered from the top, for more information on the MIP IDs refer to Additional file 2). The lane numbers (1 to 8) indicate plant materials used, as described in Table 1. A, C, and AC represent the genomes of B. rapa, B, oleracea, and B. napus, respectively. AT indicates A. thaliana. M, molecular size marker. The presence or absence of an insertion is denoted by a black or gray arrowhead, respectively. (b) Estimated insertion timing for the five MIP groups during the evolution of Brassica species [27, 36, 37].The number within the parentheses indicates the corresponding number of MITE members belonging to the particular group (based on the analysis in panel a).

                  Phylogenetic analysis based on the 50 MIP profiles revealed four distinct clusters at the 0.30 genetic similarity coefficient level (Additional file 3). Arabidopsis was separated from Brassica accessions with a genetic similarity coefficient of 0.16. Three Brassica species each formed a distinct cluster with two or three accessions belonging to each species, corresponding well with the phylogeny of Brassica species. Each MIP reflects the insertion time at that genomic position and thus MIP-based genotyping and phylogenetic analysis will be a good tool for study of genetic diversity in the Brassica genus. We also confirmed that the MIPs are clearly distinguishable on agarose gels, heritable and reproducible, characteristics beneficial as DNA markers. A MIP between two B. oleracea accessions, Bo-19, segregated according to a normal Mendelian 1: 2: 1 ratio in a survey of 94 F2 progeny of a cross between the two accessions (Additional file 4).

                  Discussion

                  Structure, distribution and evolution of BRAMI-1 in the B. rapa genome

                  BRAMI-1 exhibits the basic characteristics of conventional Stowaway-like MITEs, which include small size, TIRs, and TSDs, and also possesses a potential DNA hairpin-like secondary structure. BRAMI-1 elements have a highly conserved 33 bp TIR region that is rich in A + T nucleotides (>69%) and a 194 bp internal region. In plants, most MITEs are classified as either Tourist-like or Stowaway-like. Tourist-like MITEs are regarded as deletion derivatives of full-length autonomous TEs, such as mPing derived from Pong and PIF[13, 32, 33]. The origin of Stowaway-like MITEs is unclear due to the lack of sufficient sequence similarity to known autonomous TEs [1, 34]. However, numerous Stowaway-like MITEs can be cross-mobilized by distantly related Mariner-like elements (MLEs) to generate high copy numbers [13, 35]. However, we could not identify the trans-acting autonomous element for the BRAMI-1 elements in this study.

                  Rapid amplification of BRAMI-1 elements in the Brassica genus

                  The genus Brassica is an excellent model plant to study polyploidization-mediated genome evolution because allotetraploid species like B. juncea, B. napus, and B. carinata evolved very recently from the three diploid species B. rapa, B. oleracea, and B. nigra, and even the diploid Brassica species have triplicated genome features that arose approximately 13 million years ago (MYA) [26, 27, 36]. The estimated copy numbers of the BRAMI-1 elements were similar in two closely related Brassica species: 1440 and 1464 in B. rapa and B. oleracea, respectively supporting that BRAMI-1 elements were actively amplified in both Brassica species [27, 36, 37]. This is the first MITE found to exhibit very high copy numbers in Brassica, although one medium copy number Brassica Stowaway MITE, named Brasto, was recently characterized [38].

                  BRAMI-1 shares 77% similarity with the A. thaliana MITE ATTIRTA-1, suggesting that they evolved from a common ancestor of the Brassica and Arabidopsis lineage. Phylogenetic analysis revealed that ATTIRTA-1 and BRAMI-1 elements have clearly different evolutionary histories. The ATTIRTA-1 elements showed a high amount of variation even though their copy numbers were small compared to those of the BRAMI-1 members, indicating that the ATTIRTA-1 members were maintained in the Arabidopsis genome without further amplification after the split from the Brassica lineage 13–17 MYA [27, 36]. By contrast, members derived from B. rapa (red), B. oleracea (blue), and B. napus (black) are highly conserved and interrelated with each other, demonstrating that the members were actively amplified in the Brassica lineage after divergence from Arabidopsis (Figure 3). This is consistent with a report showing highly active TE amplification in B. oleracea[28]. We assume that several transpositional bursts may have been responsible for the amplification of the BRAMI-1 members in the Brassica lineage [16, 39, 40].

                  The putative role of BRAMI-1 in B. rapa genome evolution

                  There have been many reports of MITEs involved in the evolution of genes and genomes. MITEs are often inserted in genic regions such as promoter regions, UTRs, introns, or exons and can influence the expression of genes [1, 2, 16, 19, 34]. MITE insertion into the various functional regions of a gene can modify its transcriptional activity, cause silencing, and up- or down-regulation of gene expression [34, 41]. We found 697 BRAMI-1 elements were dispersed across the whole genome (Figure 2). A total of 626 members (90%) were identified in 177 Mb of intergenic spaces and 71 members (10%) were identified in 79 Mb of gene spaces in the 256 Mb B. rapa pseudo chromosome sequences. Among the 697 elements, 548 members (78.6%) were located within 3 kb of genic regions and all the 71 copies found in genic regions were resided in introns. The 33 Mb intronic regions exhibited 65% A + T composition, which was much higher than that of 46 Mb exonic regions (54% A + T composition). This insertion target site preference for non-coding sequences of genic regions is similar to the insertion preference of mPing in rice, which is more often found in A + T rich non-coding sequence than in G + C rich exonic regions [19].

                  We showed that BRAMI-1 insertion might be one of the causal forces for modification of gene expression. When we compared the expressions of several genes harboring BRAMI-1 within their genic regions with those of NIPs, most of the genes with BRAMI-1 insertions showed different expression patterns than their NIP counterparts (Figure 5). Comparison of microsynteny between regions with BRAMI-1 insertions and their non-insertion homologous genes in B. rapa and A. thaliana showed relatively conserved coding sequences but more sequence variation in introns and UTRs, including from the BRAMI-1 insertions (Figure 4). The observed changes in transcription levels might arise from BRAMI-1 insertions into intronic or UTR regions, similar to a recent report showing an enhancing effect of mPing near rice genes [19]. Further intensive study of whole transcriptome profiles will be necessary to address MITE effects on gene expression.

                  BRAMI-1 elements are active up to the present in Brassica genera

                  MIP patterns showing insertions specific to certain species or accessions elucidate the timing of insertion events. Among 50 MIPs, nine (18%) BRAMI-1 elements were found in both B. rapa and B. oleracea, indicating that they were inserted into the regions before B. rapa and B. oleracea diverged from each other 4 MYA [27, 36]. The other 41 (82%) were unique to one species or the other, indicating they were inserted after the divergence of the two lineages. Among the 41 species-specific members, six (8%) showed no insertion in B. napus (the allopolyploid product of B. rapa and B. oleracea) indicating that they inserted into each genome after allopolyplidization 0.01 MYA [36] (Figure 6). Some MIPs were found between accessions of same species, and the MIPs segregated normally in an F2 population, opening a new window for MIP-based marker development for marker-assisted selection and other breeding applications in Brassica crops. Overall, the MIPs revealed that BRAMI-1 elements were gradually inserted into the Brassica genome during various events and remained active up to the present.

                  Conclusions

                  We characterized a high copy Stowaway family MITE, named as BRAMI-1, in three Brassica crops and showed its putative role in the evolution of the highly duplicated Brassica genome based on comparative genomics analysis. MIP analysis revealed that the BRAMI-1 elements were dispersed into whole Brassica genome by gradual amplification. We also propose effective utilization of the elements as DNA markers for breeding and evolution of duplicated genes.

                  Methods

                  Identification and characterization of BRAMI-1

                  We analyzed a repeat-rich B. rapa BAC clone sequence, KBrB059A03 (AC189406), to find high copy repeat elements using MUST, a de novo program for MITE analysis, with the default parameters [42]. The BAC clone contained 139 kb of highly repetitive sequence. The structure of the TIRs was analyzed using weblogo [43]. The hypothetical DNA hairpin-like structure was predicted using the mfold application [44].

                  The new MITE was used as a query to retrieve its family members from a local database (http://​im-crop.​snu.​ac.​kr/​) that includes 256 Mb of 10 pseudo-chromosome sequences from B. rapa, 425 Mb of B. oleracea shotgun sequences, 15 Mb of B. napus shotgun sequences, and the whole genome sequence of A. thaliana, using the approach suggested by Wicker et al. (2007) [10]. BLASTn with default parameters [45] and a threshold E-value of 1E-10 was employed to search for MITE family members. The insertion sites of 697 elements and their flanking regions were annotated using the B. rapa genome database [31].

                  Estimation of copy number

                  The copy number of BRAMI-1 in the B. rapa genome (529 Mb) was estimated from the number of copies identified in 256 Mb of 10 pseudo-chromosome sequences from B. rapa[25]. The copy numbers in the B. oleracea and B. napus genomes were estimated by considering the hit numbers in the available genome shotgun sequences. A total of 425 Mb of B. oleracea sequences derived from 680,894 genome shotgun sequences with an average length of 700 bp [46] and 15 Mb of B. napus shotgun sequences derived from 52,099 genome shotgun sequences (GSS) with an average length of 700 bp were downloaded from GenBank (NCBI) and used as local databases. The copy numbers of BRAMI-1 in B. oleracea and B. napus were estimated using the previously reported formula [28]: [(1/genome coverage)/2] x number of hits {[1 + [(average GSS) -TIR length x2)/(average GSS length + TIR length x2)]}. Relatively intact copies with more than 80% coverage of the BRAMI-1 structure were collected from the three Brassica species for phylogenetic analysis. Multiple sequence alignment was conducted using ClustalW and phylogenetic analysis was performed based on the neighbor joining method in MEGA5 [47]. In A. thaliana, ATTIRTA-1 was the most closely related element to BRAMI-1, so it was included in the phylogenetic analysis. Tree topologies were evaluated using bootstrap analysis with 500 replicates for the neighbor-joining method [47].

                  Expression analysis of B. rapa genes with BRAM1-1 insertions

                  We investigated expression modification of genes that had a MITE insertion inside of the gene structure by comparison with their syntenic paralogs using a 24 K microarray database (http://​nabic.​rda.​go.​kr) [48]. The microarray database represented ca. 24,000 unigenes generated from cDNA libraries of B. rapa ssp. pekinensis (inbred line ‘Chiifu’) and provided transcriptome profiling of changes induced by abiotic stress treatment. A given probe sequence and its ID in the microarray were searched using the coding sequence of the gene as a query. The perfect match (PM) values of probes were retrieved and processed to identify expression patterns, as described previously [48].

                  MITE Insertion polymorphism (MIP)

                  To inspect insertion polymorphisms and thus infer activation times, we used seven Brassica accessions belonging to three species and A. thaliana ecotype Columbia (Table 4). DNA was extracted from fresh leaf samples using the CTAB method [49]. In addition, a total of 94 F2 progeny from a cross between B. oleracea accessions C1234 and C1184 were used for segregation pattern analysis of MIPs.
                  Table 4

                  Plant materials used for MIP analysis

                   

                  Genome

                  Species

                  Accessions (cultivars)

                  1

                  AACC

                  B. napus

                  Tapidor

                  2

                  AACC

                  B. napus

                  Ningyou7

                  3

                  AA

                  B. rapa

                  Chiifu

                  4

                  AA

                  B. rapa

                  Kenshin

                  5

                  CC

                  B. oleracea

                  C1234

                  6

                  CC

                  B. oleracea

                  C1184

                  7

                  CC

                  B. oleracea

                  C1235

                  8

                  AT

                  A. thaliana

                  Columbia

                  We designed 50 primer pairs, 25 using shotgun sequences of B. oleracea (Bo 1–25) and 25 using the B. rapa pseudo-chromosome sequences (Br 1–25), from the flanking sequences of BRAM1-1 insertion sites using the Primer3 software program [50] (Additional file 2). PCR was conducted in 20 μL total volume containing 10 ng DNA, 10 pmol each primer, 250 μM dNTPs, and 1 unit Taq DNA polymerase (VIVAGEN, Republic of Korea). PCR conditions were as follows: 5 min at 94°C, 38 cycles of 95°C for 30 sec, 56°C-62°C for 30 sec, and 72°C for 60 sec, with a final extension at 72°C for 5 min, using a MG96G thermo cycler (LongGene Scientific Instruments, China). PCR products were analyzed using 1% agarose gel electrophoresis and visualized on a UV trans-illuminator after ethidium bromide staining.

                  For MIP marker analysis, each band was scored as ‘1’ or ‘0’ for presence or absence, respectively. Jaccard’s similarity coefficient and a dendrogram of the genetic relationship according to Unweighted Pair Group Method with Arithmetic Average (UPGMA) analysis were determined by the NTSYS-pc program (Numerical Taxonomy & Multivariate Analysis System) [51].

                  Declarations

                  Acknowledgments

                  This work was supported by the Technology Development Program (No. 309008–05) for Agriculture and Forestry, Ministry of Food, Agriculture, Forestry and Fisheries, Republic of Korea and and a grant from the Next-Generation BioGreen 21 Program (No. PJ0090762012), Rural Development Administration, Republic of Korea. Perumal Sampath is supported by a Korean Government Scholarship (KGSP) 2008 from the National Institute for International Education (NIIED), Ministry of Education, Science, and Technology, Republic of Korea.

                  Authors’ Affiliations

                  (1)
                  Dept. of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University
                  (2)
                  National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University
                  (3)
                  National Academy of Agricultural Science, Rural Development Administration

                  References

                  1. Feschotte C, Jiang N, Wessler SR: Plant transposable elements: where genetics meets genomics. Nat Rev Genet 2002,3(5):329–341.PubMedView Article
                  2. Feschotte C: Transposable elements and the evolution of regulatory networks. Nat Rev Genet 2008,9(5):397–405.PubMedView Article
                  3. Haberer G, Young S, Bharti AK, Gundlach H, Raymond C, Fuks G, Butler E, Wing RA, Rounsley S, Birren B: Structure and architecture of the maize genome. Plant Physiol 2005,139(4):1612–1624.PubMedView Article
                  4. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W: Initial sequencing and analysis of the human genome. Nature 2001,409(6822):860–921.PubMedView Article
                  5. Martiel JL, Blot M: Transposable elements and fitness of bacteria. Theor Popul Biol 2002,61(4):509–518.PubMedView Article
                  6. Morrell PL, Buckler ES, Ross-Ibarra J: Crop genomics: advances and applications. Nat Rev Genet 2011,13(2):85–96.PubMed
                  7. SanMiguel P, Gaut BS, Tikhonov A, Nakajima Y, Bennetzen JL: The paleontology of intergene retrotransposons of maize. Nat Genet 1998,20(1):43–45.PubMedView Article
                  8. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA: The B73 maize genome: complexity, diversity, and dynamics. Science 2009,326(5956):1112–1115.PubMedView Article
                  9. Matsumoto TWJ, Kanamori H: The map-based sequence of the rice genome. Nature 2005,436(7052):793–800.View Article
                  10. Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O: A unified classification system for eukaryotic transposable elements. Nat Rev Genet 2007,8(12):973–982.PubMedView Article
                  11. Bureau TE, Wessler SR: Tourist: a large family of small inverted repeat elements frequently associated with maize genes. Plant Cell 1992,4(10):1283–1294.PubMed
                  12. Tu Z: Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae. Proc Natl Acad Sci USA 2001,98(4):1699–1704.PubMedView Article
                  13. Jiang N, Feschotte C, Zhang X, Wessler SR: Using rice to understand the origin and amplification of miniature inverted repeat transposable elements (MITEs). Curr Opin Plant Biol 2004,7(2):115–119.PubMedView Article
                  14. Oki N, Yano K, Okumoto Y, Tsukiyama T, Teraishi M, Tanisaka T: A genome-wide view of miniature inverted-repeat transposable elements (MITEs) in rice rice, Oryza sativa ssp japonica. Genes Genet Syst 2008,83(4):321–329.PubMedView Article
                  15. Benjak A, Boue S, Forneck A, Casacuberta JM: Recent amplification and impact of MITEs on the genome of grapevine (Vitis vinifera L.). Genome Biol Evol 2009, 1:75–84.PubMedView Article
                  16. Lu C, Chen J, Zhang Y, Hu Q, Su W, Kuang H: Miniature Inverted–Repeat Transposable Elements (MITEs) Have Been Accumulated through Amplification Bursts and Play Important Roles in Gene Expression and Species Diversity in Oryza sativa. Mol Biol Evol 2012,29(3):1005–1017.PubMedView Article
                  17. Mo YJ, Kim KY, Shin WC, Lee GM, Ko JC, Nam JK, Kim BK, Ko JK, Yu Y, Yang TJ: Characterization of Imcrop, a Mutator-like MITE family in the rice genome. Genes Genomics 2012,34(2):189–198.View Article
                  18. Yang G, Lee YH, Jiang Y, Shi X, Kertbundit S, Hall TC: A two-edged role for the transposable element Kiddo in the rice ubiquitin2 promoter. Plant Cell 2005,17(5):1559–1568.PubMedView Article
                  19. Naito K, Zhang F, Tsukiyama T, Saito H, Hancock CN, Richardson AO, Okumoto Y, Tanisaka T, Wessler SR: Unexpected consequences of a sudden and massive transposon amplification on rice gene expression. Nature 2009,461(7267):1130–1134.PubMedView Article
                  20. Piriyapongsa J, Jordan IK: Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 2008,14(5):814–821.PubMedView Article
                  21. Kuang H, Padmanabhan C, Li F, Kamei A, Bhaskar PB, Ouyang S, Jiang J, Buell CR, Baker B: Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: new functional implications for MITEs. Genome Res 2009,19(1):42–56.PubMedView Article
                  22. Lisch D, Bennetzen JL: Transposable element origins of epigenetic gene regulation. Curr Opin Plant Biol 2011,14(2):156–161.PubMedView Article
                  23. Casa AM, Brouwer C, Nagel A, Wang L, Zhang Q, Kresovich S, Wessler SR: The MITE family heartbreaker (Hbr): molecular markers in maize. Proc Natl Acad Sci USA 2000,97(18):10083–10089.PubMedView Article
                  24. Lyons M, Cardle L, Rostoks N, Waugh R, Flavell AJ: Isolation, analysis and marker utility of novel miniature inverted repeat transposable elements from the barley genome. Molecular genetics genomics 2008,280(4):275–285.View Article
                  25. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F: The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 2011,43(10):1035–1039.PubMedView Article
                  26. Yang TJ, Kim JS, Kwon SJ, Lim KB, Choi BS, Kim JA, Jin M, Park JY, Lim MH, Kim HI: Sequence-level analysis of the diploidization process in the triplicated FLOWERING LOCUS C region of Brassica rapa. Plant Cell 2006,18(6):1339–1347.PubMedView Article
                  27. Mun JH, Kwon SJ, Yang TJ, Seol YJ, Jin M, Kim JA, Lim MH, Kim JS, Baek S, Choi BS: Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication. Genome Biol 2009,10(10):R111.PubMedView Article
                  28. Zhang X, Wessler SR: Genome-wide comparative analysis of the transposable elements in the related species Arabidopsis thaliana and Brassica oleracea. Proc Natl Acad Sci USA 2004,101(15):5589–5594.PubMedView Article
                  29. Kapitonov VV, Jurka J: Molecular paleontology of transposable elements from Arabidopsis thaliana. Genetica 1999,107(1–3):27–37.PubMedView Article
                  30. Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J, Lopez R, Price HJ: Evolution of genome size in Brassicaceae. Ann Bot 2005,95(1):229–235.PubMedView Article
                  31. Feng C, Shengyi L, Jian W, Lu F, Silong S, Bo L, Pingxia L, Wei H, Xiaowu W: BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol 2011,11(136):1–6.
                  32. Jiang N, Bao Z, Zhang X, Hirochika H, Eddy SR, McCouch SR, Wessler SR: An active DNA transposon family in rice. Nature 2003,421(6919):163–167.PubMedView Article
                  33. Yang G, Zhang F, Hancock CN, Wessler SR: Transposition of the rice miniature inverted repeat transposable element mPing in Arabidopsis thaliana. Proc Natl Acad Sci USA 2007,104(26):10962–10967.PubMedView Article
                  34. Casacuberta JM, Santiago N: Plant LTR-retrotransposons and MITEs: control of transposition and impact on the evolution of plant genes and genomes. Gene 2003, 311:1–11.PubMedView Article
                  35. Yang G, Nagel DH, Feschotte C, Hancock CN, Wessler SR: Tuned for transposition: molecular determinants underlying the hyperactivity of a Stowaway MITE. Science 2009,325(5946):1391–1394.PubMedView Article
                  36. Rana D, Boogaart T, O'Neill CM, Hynes L, Bent E, Macpherson L, Park JY, Lim YP, Bancroft I: Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives. Plant J 2004,40(5):725–733.PubMedView Article
                  37. Truco MJ, Hu J, Sadowski J, Quiros CF: Inter- and intra-genomic homology of the Brassica genomes: Implications for their origin and evolution. Theor Appl Genet 1996,93(8):1225–1233.View Article
                  38. Sarilar V, Marmagne A, Brabant P, Joets J, Alix K: BraSto, a Stowaway MITE from Brassica: recently active copies preferentially accumulate in the gene space. Plant Mol Biol 2011,77(1–2):59–75.PubMedView Article
                  39. Fontdevila A: Hybrid genome evolution by transposition. Cytogenet Genome Res 2005,110(1–4):49–55.PubMedView Article
                  40. Hu G, Hawkins JS, Grover CE, Wendel JF: The history and disposition of transposable elements in polyploid Gossypium. National Research Council Canada = Genome Conseil national de recherches Canada 2010,53(8):599–607.View Article
                  41. Santiago N, Herraiz C, Goni JR, Messeguer X, Casacuberta JM: Genome-wide analysis of the Emigrant family of MITEs of Arabidopsis thaliana. Mol Biol Evol 2002,19(12):2285–2293.PubMedView Article
                  42. Chen Y, Zhou F, Li G, Xu Y: MUST: a system for identification of miniature inverted-repeat transposable elements and applications to Anabaena variabilis and Haloquadratum walsbyi. Gene 2009,436(1–2):1–7.PubMedView Article
                  43. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res 2004,14(6):1188–1190.PubMedView Article
                  44. Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003,31(13):3406–3415.PubMedView Article
                  45. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990,215(3):403–410.PubMed
                  46. Ayele M, Haas BJ, Kumar N, Wu H, Xiao Y, Van Aken S, Utterback TR, Wortman JR, White OR, Town CD: Whole genome shotgun sequencing of Brassica oleracea and its application to gene discovery and annotation in Arabidopsis. Genome Res 2005,15(4):487–495.PubMedView Article
                  47. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011.
                  48. Lee SC, Lim MH, Kim JA, Lee SI, Kim JS, Jin M, Kwon SJ, Mun JH, Kim YK, Kim HU: Transcriptome analysis in Brassica rapa under the abiotic stresses using Brassica 24 K oligo microarray. Mol Cells 2008,26(6):595–605.PubMed
                  49. Doyle J, Doyle J: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 1987, 19:11–15.
                  50. Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000, 132:365–386.PubMed
                  51. Rohlf F: NTSYS-pc, version 2.10 z. Setauket, New York: Exeter Software; 2002.

                  Copyright

                  © Sampath et al.; licensee BioMed Central Ltd. 2013

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                  Advertisement