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

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.

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.

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.

Figure 3

Phylogenetic tree of BRAMI-1 elements from Brassica species and ATTIRTA-1 fromA. 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.

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.

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.

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).