The process for assembling the complete mitochondrial genome sequence using the eight mitochondrial contigs/fragments.

<p>The process for assembling the complete mitochondrial genome sequence using the eight mitochondrial contigs/fragments.</p

A multiple alignment of plastid genome (KR233156) and four mtDNAs (KU831325, KJ820683, JF920286, and AP012988) of <i>Brassica oleracea</i> consists of several rearranged pieces.

<p>Each genome is laid out horizontally with homologous blocks represented as coloured rectangles. Regions inverted relative to plastid are set below those that match in the forward orientation. Lines collate aligned segments between plastid and different mtDNAs. Sections of white within blocks and gaps between blocks indicate lineage specific sequence.</p

Whole-genome sequencing of <i>Brassica oleracea</i> var. <i>capitata</i> reveals new diversity of the mitogenome - Fig 4

<p>In the left panel (a) identification of variation in the KU831325 mtDNA in various inbred lines. A, B, and E are cabbage, C is broccoli, D is kale, F is cabbage of the Ogura CMS line, G is cauliflower, H is brussels sprouts, I is kohlrabi, and J is kailan and M is the reference ladder with 100 bp. The genotypes with the red asterisks were used for cloning, sequencing, and assemblage of mitochondrial DNA in <i>B</i>. <i>oleracea</i>. There are two bands for contig 2–3, with the lower band being the target amplified according to the primer product size presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194356#pone.0194356.t001" target="_blank">Table 1</a>. In right panel (b) showing relative expression with significant variation of different contigs/fragments present in different genotypes.</p

Map of the mitochondrial genome of <i>B</i>. <i>oleracea</i> var. <i>capitata</i>.

<p>Boxes on the inside and outside of the outer circle represent ORF genes. Gene colors correspond to the functional categories listed in the legend. The inner circle displays the GC content represented by dark gray bars. The Fig was created using OGDraw v1.2 (adopted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194356#pone.0194356.ref045" target="_blank">45</a>]).</p

Molecular phylogenetic analysis by Maximum Likelihood method of the four <i>B</i>. <i>oleracea</i> mtDNAs according to the genomic sequences and the distribution of their contigs, the black coloured value is node length and blue coloured one is boost-strap value.

<p>Molecular phylogenetic analysis by Maximum Likelihood method of the four <i>B</i>. <i>oleracea</i> mtDNAs according to the genomic sequences and the distribution of their contigs, the black coloured value is node length and blue coloured one is boost-strap value.</p

Whole-genome sequencing of <i>Brassica oleracea</i> var. <i>capitata</i> reveals new diversity of the mitogenome

<div><p>Plant mitochondrial genomes (mtDNAs) vary in sequence structure. We assembled the <i>Brassica oleracea</i> var. <i>capitata</i> mtDNA using a mean coverage depth of 25X whole genome sequencing (WGS) and confirmed the presence of eight contigs/fragments by BLASTZ using the previously reported KJ820683 and AP012988 mtDNA as reference. Assembly of the mtDNA sequence reads resulted in a circular structure of 219,975 bp. Our assembled mtDNA, NCBI acc. no. KU831325, contained 34 protein-coding genes, 3 rRNA genes, and 19 tRNA genes with similarity to the KJ820683 and AP012988 reference mtDNA. No large repeats were found in the KU831325 assembly. However, KU831325 showed differences in the arrangement of bases at different regions compared to the previously reported mtDNAs. In the reference mtDNAs KJ820683 and AP012988, contig/fragment number 4 is partitioned into two contigs/fragments, 4a and 4b. However, contig/fragment number 4 was a single contig/fragment with 29,661 bp in KU831325. PCR and qRT-PCR using flanking markers from separate parts of contig/fragment number 4 confirmed it to be a single contig/fragment. In addition, genome re-alignment of the plastid genome and mtDNAs supported the presence of heteroplasmy and reverse arrangement of the heteroplasmic blocks within the other mtDNAs compared to KU831325 that might be one of the causal factors for its diversity. Our results thus confirm the existence of different mtDNAs in diverse <i>B</i>. <i>oleracea</i> subspecies.</p></div

Syntenic block comparative analysis in <i>Brassica oleracea</i> mtDNAs.

<p>The map was generated using Circos. (a) Syntenic block of <i>B</i>. <i>oleracea</i> mtDNA ‘AP012988’ with three other <i>B</i>. <i>oleracea</i> mtDNAs. (b) Syntenic block of <i>B</i>. <i>oleracea</i> mtDNA ‘KJ820683’ with three other <i>B</i>. <i>oleracea</i> mtDNAs. (c) Syntenic block of <i>B</i>. <i>oleracea</i> mtDNA ‘JF920286’ with three other <i>B</i>. <i>oleracea</i> mtDNAs. (d) Syntenic block of <i>B</i>. <i>oleracea</i> mtDNA ‘KU831325’ with three other <i>B</i>. <i>oleracea</i> mtDNAs.</p

Block-wise amino acid similarities with the plastid (Plst) nucleotide sequences (bp) into mitochondrial (Mt) genome in <i>B</i>. <i>oleracea</i> and the nucleotide similarities of the KU831325 with other reference mitogenomes (Mts).

<p>Block-wise amino acid similarities with the plastid (Plst) nucleotide sequences (bp) into mitochondrial (Mt) genome in <i>B</i>. <i>oleracea</i> and the nucleotide similarities of the KU831325 with other reference mitogenomes (Mts).</p

Primers used for identification of the <i>B</i>. <i>oleracea</i> var. <i>capitata</i> mtDNAs.

<p>Primers used for identification of the <i>B</i>. <i>oleracea</i> var. <i>capitata</i> mtDNAs.</p