BACKGROUND
cultivated barley  was domesticated from its wild ancestral form  ca.
<dig>  years ago  <cit> .
cultivated and wild barley share closely related genomes that do not exhibit crossing or recombination barriers  <cit> .
barley is a self-pollinated diploid with seven pairs of chromosomes comprising a large haploid genome of  <dig>  gbp.
the barley genome has been analyzed by bac fingerprinting, bac-end sequencing, whole-genome shotgun sequencing and transcriptome analysis  <cit> .
the genome data also include haplotype sequence information for several cultivated and wild barley lines, which provides  <dig> million snps among haplotypes.
the possibility of multiple domestication events for barley has been discussed based on phylogenic analyses of cloned genes  <cit> .
the wild ancestral form of barley is widely distributed in the near east to central asia, with a center in the fertile crescent.
during the evolution of wild barley, its mitochondrial genomes might have diversified.
for example, ahokas  <cit>  detected cytoplasmic male sterility, one of the main traits derived from the mitochondrial genome, in a wild barley germplasm.
this finding indicates that the organellar genomes have differentiated in wild barley during its long evolutionary process.
although the gene contents of the mitochondrial genomes of flowering plants are nearly identical, gene order and genome configuration are highly variable even within a species due to genome rearrangement  <cit> .
tsunewaki et al.
<cit>  explored the evolutionary dynamics of the wheat mitochondrial genome with respect to its structural differentiation during cereal evolution by comparing the nucleotide sequences of wheat genes to those of rice and maize.
the nuclear genome of barley is closely related to those of wheat and its relatives, i.e., members of the triticeae tribe.
the mitochondrial genome structure of common wheat  cv.
chinese spring is almost identical to that of the chinese wheat cultivar yumai, with differences of seven snps and  <dig> indels in non-coding regions  <cit> .
noyszewski et al.
<cit>  sequenced the triticeae mitochondrial genomes of aegilops longissima, t. turgidum and an alloplasmic line with the ae.
longissima cytoplasm that carries the t. turgidum nucleus, finding evidence suggesting that the alloplasmic condition accelerates evolution towards forming new mitochondrial genomes.
however, to date, little information about the mitochondrial genome in barley has been published.
in a study investigating the chloroplast genome, which together with the mitochondrial genome composes the plasmon, middleton et al.
<cit>  found that the chloroplast sequences of cultivated and wild barley are closely related .
the divergence time of the two barley sequences was estimated to be  <dig>  ±  <dig>  years using semi-penalized likelihood.
a comparison of the chloroplast genome sequences of cultivated barley and common wheat identified four deletions and five insertions greater than 50 bp in the chloroplast genome sequence of common wheat.
this similarity in chloroplast sequence indicates that cultivated and wild barley are more closely related to each other than cultivated barley and cultivated wheat.
the haplotypes used in the current study were the japanese malting barley cultivar ‘haruna nijo’ and the wild barley accession ‘h602’.
haruna nijo has been extensively used as a crossing parent for developing high-quality malting barley cultivars in japan.
the origin of this cultivar can be traced to the haplotype of a malting cultivar introduced more than 100 years ago from europe.
haruna nijo has also been used to generate genomic resources, e.g., a bac library  <cit> , full-length cdnas  <cit> , and whole genome shotgun sequence analysis  <cit> .
wild barley accession h <dig> has been used as a haplotype that is distantly related to haruna nijo and thus, a high-resolution transcript map of haruna nijo/h <dig> has been developed  <cit> .
both haplotypes may represent cultivated and wild barley gene pools, suggesting that they are appropriate materials for revealing the diversity of mitochondrial genomes in barley.
in this study, we determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley and compared their structures.
we then analyzed the gene contents and unique regions of each genome in detail at the sequence level.
since no mitochondrial genome sequence has been published for barley, we compared the resulting sequences to the published mitochondrial sequences of related species, i.e., wheat and its relatives, to clarify the evolutionary process of barley mitochondria.
methods
plant materials and mitochondrial dna extraction
mitochondrial dna samples were extracted from etiolated seedlings barley cultivar ‘haruna nijo’ and wild barley accession ‘h602’ according to the method of bonen and gray  <cit> , with minor modifications.
barley seeds were obtained from the national bioresource project of barley, mext of japan.
using discontinuous  sucrose density gradient centrifugation, dnase-i treated mitochondria were collected from the interface between  <dig>  and  <dig>  m sucrose.
the mitochondrial dna samples were purified by etbr/cscl density-gradient ultracentrifugation.
for diversity analysis of snps, seed samples from  <dig> barley cultivars and four wild accessions preserved at the institute of plant science and resources, okayama university, japan were used.
sequencing, assembly and completion
both  <dig> sequencing with the gs-flx titanium platform , and illumina sequencing with the miseq platform  were performed to generate longer reads by gs-flx  <dig> system and correct read errors with high redundant shorter reads by miseq system.
the libraries for  <dig> sequencing were prepared with gs titanium rapid library preparation kit  and sequenced using gs flx titanium picotiterplate kit 70x <dig>  and gs flx titanium sequencing kit xl+  following the manufacturer’s instructions.
gs flx titanium rapid library mid adapter kit , gs flx titanium lv empcr kit  v <dig>  and gs flx titanium empcr breaking kit lv/mv 12pc  were used for adapter ligation and emulsion pcr during library and sequencing preparation.
the libraries for illumina sequencing were prepared with nextera xt dna sample preparation kit  and nextera xt index kit  and sequenced with miseq reagent kit v <dig> 500 cycles  following the manufacturer’s instructions.
the reads from  <dig> sequencing were assembled with newbler  <dig>  .
the generated contigs were filtered by average depth .
blastn analysis was performed to exclude contamination of chloroplast genome sequences.
filtered contigs were temporally ordered by hooking shared terminator reads in each contig.
modified touchdown pcr  <cit>  was performed to connect the contigs and to validate the snps with the following program: initial denaturation at 98 °c for 2 min, 10 cycles of a melting step at 98 °c for 10 s, an annealing step at 65 °c decreased 1 °c/cycle for 15 s and an extension step at 72 °c for 1 min, followed by 20 cycles of denaturation at 95 °c for 10 s, annealing at 55 °c for 20 s and extension at 72 °c for 1 min, with a final extension at 72 °c for 10 min.
the 10 μl reaction mixture for pcr included 1 ng mitochondrial dna as template, 1× primestar® max dna polymerase  and  <dig>  μm specific primers listed in additional file 1: tables s1a, b and additional file 2: tables s <dig>  the pcr amplicons were checked by electrophoresis in  <dig>  % agarose gels  in tbe running buffer.
direct sequencing of the pcr amplicons was performed on a 3130xl genetic analyzer .
to increase the accuracy of genome sequencing, illumina-reads from h <dig> and haruna nijo were mapped and aligned onto the reference mitochondrial draft genome of h <dig>
genome annotation and comparative analysis
the genes encoding mitochondrial proteins and rrnas were identified using blastn and mitofy  <cit>  based on the known annotation of the mitochondrial genes of wheat cv.
chinese spring  <cit> .
the trnascan-se  <dig>  server  <cit>  was used to identify trnas.
repeat sequences  were identified by align sequences nucleotide blast using mitochondrial sequences from haruna nijo.
partial sequences sharing more than 10 % of nucleotides with the original genes were identified as pseudogenes.
for transposable element analysis, censor  <cit>  was used with four sequence sources  including poaceae, oryza, panicoideae and triticum.
to identify novel orfs, ‘getorf’ analysis in emboss  was conducted with the following parameters: predicted protein more than  <dig> aa in length; methionine as the start codon; circular dna as the query sequence.
after getorf analysis, the orfs overlapping with known genes were eliminated.
alignments and phylogenetic analysis
phylogenetic analysis was carried out using concatenated nucleotide sequences of the  <dig> protein-coding genes  extracted from the complete mitochondrial genome sequences deposited in the public databases.
all positions containing gaps and missing data were eliminated, yielding a total of  <dig>  positions in the final dataset.
alignments and phylogenetic tree construction were performed by the maximum likelihood method with the mega <dig> program  <cit> .
snp analysis
a modified touchdown pcr procedure, as described previously, was performed for snp analysis with specific primer pairs  using 50 ng genomic dna from cultivated and wild accessions of barley.
pcr amplicons were sequenced with a 3130xl genetic analyzer.
RESULTS
sequencing and assembly of the mitochondrial genome in barley
the mitochondrial genomes of wild and cultivated barley were analyzed by  <dig> sequencing .
a total of  <dig>  and  <dig>  reads corresponding to  <dig> mbp  and  <dig> mbp  were generated from h <dig> and haruna nijo, respectively.
these reads were independently de novo assembled using newbler  <dig>   with average contig sizes of  <dig> and 1624 bp, n <dig> contig sizes of  <dig> and 1789 bp and largest contig sizes of  <dig>  and  <dig>  bp for h <dig> and haruna nijo respectively.
after trimming of duplicated sequences, these contigs were aligned in  <dig> contigs  for h <dig> and  <dig> contigs  for haruna nijo .
the contig sequences were aligned, followed by sequencing of the pcr products with a 3130xl genome analyzer .
draft master circular maps  of the mitochondrial genome were generated for both h <dig> and haruna nijo.
the illumina miseq reads were mapped onto the master circles and re-aligned to develop high quality mitochondrial sequences.
from the mitochondrial sequencing libraries of h <dig> and haruna nijo,  <dig> , <dig> and  <dig> , <dig> paired reads were generated, respectively.
reads of h <dig> were trimmed with clc genomic workbench  <dig>  , and the resulting pairs of  <dig>  reads were mapped onto the draft master circle of h <dig>  a  <dig>  bp circular molecule with a g/c content of  <dig>  %  was developed for h <dig>  using the same methods, another  <dig>  bp circular molecule  was developed for haruna nijo with pairs of  <dig> , <dig> trimmed reads.
the alignment of two mitochondrial circular molecules identified three snps between h <dig> and haruna nijo at the positions of nt  <dig> ,  <dig>   and  <dig>  .
snp <dig> was located in a genic region , and snp <dig> and snp <dig> were located in intergenic regions.
these snps were validated by sanger sequencing of pcr products, including the alleles amplified by specific primers .
annotation of the mitochondrial genome
the master circles were annotated by blast analysis against the mitochondrial genome annotation databases mitofy  <cit>  and trnascan-se  <dig>   <cit> , and  <dig> protein-coding genes, three ribosomal rnas and  <dig> transfer rnas were identified .
of the  <dig> protein-coding genes,  <dig> encoded members of the electron transport chain and atp synthase, including the following: nine subunits of complex i including nad <dig>  nad <dig>  nad <dig>  nad <dig>  nad4l, nad <dig>  nad <dig>  nad <dig> and nad9; an apocytochrome b subunit  of complex iii; three subunits of complex iv including cox <dig>  cox <dig> and cox3; and five subunits of complex v including atp <dig>  atp <dig>  atp <dig>  atp <dig> and atp <dig>  four additional protein-coding genes were involved in cytochrome c biogenesis, including ccmb, ccmc, ccmfc and ccmfn.
there were  <dig> genes for ribosomal proteins, namely rps <dig>  rps <dig>  rps <dig>  rps <dig>  rps <dig>  rps <dig>  rps <dig>  rpl <dig>  rpl <dig> and pseudogene rpl2p, as well as two other protein-coding genes, matr and mttb.
among the protein-coding genes, there were two copies of nad <dig>  nad <dig>  ccmc, rps <dig> and rpl <dig>  among rrna and trna genes, there were three copies of rrm <dig>  rrn <dig>  trnd, trnfm <dig>  trnk and trnq and two copies of rrn <dig> and trnr.
of the two copies of cox2b, the one nearest to cox2a was regarded as active cox2b, whereas the other, which is located far from cox2a, was regarded as a pseudogene due to the general rule of cis-splicing between cox2a and cox2b.fig.
<dig> positions of genes on the barley mitochondrial genome map .
genes encoding proteins , rrna  and trna  are shown outside  and inside  the circle.
arrowhead indicates gene or exon, p indicates pseudogene, a, b, c, d and e indicate cis- and trans-spliced genic exon and underscores followed by numbers indicate gene copies
prediction of new genes
we used the ‘getorf’ command in embos to identify unique genes in the barley mitochondrial genome, leading to the prediction of  <dig> new orfs .
after excluding any partially overlapped orfs with known genes in the same translational frame,  <dig> orfs were identified as novel genes .
the total length of these novel orfs was  <dig>  bp, which occupied  <dig>  % of the barley mitochondrial genome.
identification of transposable fragments
censor  <cit>  was used to identify integrated transposable elements showing similarities to the sequences in the poaceae , oryza , panicoideae  and triticum  databases.
a total of  <dig>  dna transposons, ltr retrotransposons and non-ltr retrotransposons were found in poaceae,  <dig>  were found in oryza and  <dig>  were found in panicoideae .
by contrast, only  <dig> ltr retrotransposon and two non-ltr retrotransposons  were found in triticum.table  <dig> transposable elements in the barley mitochondrial genome
poaceae
a
oryza
a
panicoideae
a
triticum
a
dna transposon
13
837
9
522
8
406
ltr retrotransposon
57
<dig>
41
572
49
8591
16
2043
non-ltr
18
4155
9
978
16
3685
2
103
total
88
<dig>
59
9586
73
<dig>
18
2146
asequence sources  for transposable element analysis in censor
repeat sequences and chloroplast-derived region in the mitochondrial genome
six major repeat sequences  and  <dig> repeat sequences  were found in the barley mitochondrial genome .
there were four repeats in no.
<dig> and three in nos.
<dig>   <dig>   <dig>   <dig> and  <dig>  the largest repeat was  <dig>  bp, corresponding to the contig_h602_0022-0039-0169- <dig> .fig.
<dig> repeat sequences and integrated chloroplast genome in the map of the barley mitochondrial genome of accession h <dig>  repeat sequences in different colors are mapped onto the barley mitochondrial genome .
detailed information about these repeats is shown in additional file 6: table s <dig>  the predicted sequences integrated from the chloroplast genome are shown inside the circle
according to previous reports  <cit> , several trnas that function in the mitochondria originated in the chloroplast.
in the barley mitochondrial genome, trnc, trnf, trns <dig> and trnw could represent trna genes of chloroplast origin .
we analyzed the sequence similarity between the mitochondrial genome of h <dig> and the chloroplast genomes  <cit>  by blastn analysis of barley cvs.
barke  and morex  and found three homologous regions  .
homologous region  <dig>  harboring trnfm, trnr, rps <dig> and psaa, contained 6307 bp in the r <dig> repeat region , suggesting that the mitochondrial genome contained two copies of homologous region  <dig>  among these genes, trnfm and trnr were identified using the mitochondrial genome annotation database, whereas rps <dig> and psaa were not identified.
homologous region  <dig> was 2265 bp in length and harbored trnv.
homologous region  <dig> contained 1110 bp of sequences specific to the chloroplast sequence of barke.
although homologous regions  <dig> and  <dig> were found in the chloroplast genomes of several monocot species such as rice and maize, region  <dig> was only found in barke.
fifteen other homologous regions, ranging from  <dig> to 558 bp in length, were also found by blastn analysis .
transfer of mitochondrial dna into the barley nuclear genome
we analyzed nucleotide sequence similarities between the mitochondrial genome of h <dig> and the published barley genome sequences , comprising pseudomolecules of seven chromosomes and un-anchored scaffolds and contigs published in ensembl plants hordeum vulgare , using blastn analysis .
using the threshold e-value =  <dig>  aligned sequence length > 400 bp, we detected  <dig> barley genome sequences showing similarities to the barley mitochondrial genome sequence.
of these, many of the longest un-anchored genome sequences appeared to be mitochondrial sequences, since the lengths of un-anchored sequences and aligned sequences were quite similar.
for example, the sequence of morex_contig_ <dig>  which was  <dig>  bp in length, aligned completely with the mitochondrial sequence without mismatches or gaps.
of the genome sequences within the threshold, seven were derived from the chloroplast genome and  <dig> were un-anchored contigs that were potentially derived from the mitochondrial genome in the published barley genome sequence obtained by whole-genome shotgun sequencing.
apart from the misidentified mitochondrial sequence, nuclear sequences of mitochondrial origin  were observed.
of the  <dig> possible numts anchored to pseudomolecules,  <dig>  bp of sequence on chromosome 6h was the longest that aligned with the mitochondrial genome.
the total amounts of aligned sequences varied among chromosomes, ranging from  <dig>  bp  to  <dig>  bp , indicating that the mitochondrial sequences were not evenly transferred to the nuclear genome  <cit> .
snp analysis of barley germplasm
barley germplasm comprising  <dig> cultivars from  <dig> regions  and four wild accessions were genotyped at three snps that were identified between the h <dig> and haruna nijo mitochondrial genomes.
of the  <dig> cultivars showing a nucleotide call on snp <dig> ,  <dig> and  <dig> contained an a and t, respectively .
of the four wild barley accessions, two contained a and two contained t. for snp <dig> , all of the cultivars and three wild barley accessions contained g, but one wild barley, h <dig>  contained t. for snp <dig> ,  <dig> of  <dig> cultivars and one wild barley  contained a .
most ethiopian cultivars contained c in snp <dig>  although other regional differences in the allele distribution were not obvious.
comparative genomics of barley mitochondria with those of wheat and other monocot species
we compared the genes/proteins in the barley mitochondrial genome with those in other grass species including common wheat  and wheat ancestors , perennial ryegrass , sorghum , maize  and rice .
barley nad <dig> encoded nad <dig>  a  <dig> aa polypeptide .
the sequence comprising aa  <dig> to  <dig> in nad <dig> was conserved among grass species, whereas the c-terminus is highly variable.
barley nad <dig> was conserved among grass species except wheat, which contained a longer nad <dig> due to frame-shifting caused by a 4 nt insertion/deletion .
the rps <dig> gene had 39-nt imperfect repeats corresponding to  <dig> aa .
barley rps <dig> had three repeats, whereas wheat has one and rice, maize and perennial ryegrass each have partial one .
the genes cox <dig>  atp <dig>  rps <dig>  rpl <dig> and rrn <dig> also varied between barley and wheat .table  <dig> differences in mitochondrial genes encoding proteins between barley and wheat
nad6
nad9
cox1
atp6
rps2
rps4
rpl5
rrn18
abonen and bird  <cit>  indicated that the n-terminus of atp <dig> in the mitochondrial genome had interspecies variation caused by processing
bkubo et al.
<cit>  reported rps <dig> in barley  and interspecies variation in triticeae at its c-terminus
fig.
<dig> structures of the encoded protein sequences in various grass species.
the structures of a nad <dig>  b nad <dig> and c rps <dig> in the barley mitochondrial genome were compared with those of seven other grass species and subspecies .
light blue bars show the primary structures of proteins, and orange lines indicate the start codon.
the numbers above the light blue bars indicate the position from the start codons of barley proteins.
a
green box i indicates the non-conserved amino acid sequences in nad <dig> among grass species.
b nad <dig> of t. aestivum, ae.
speltoides and t. timopheevii have an additional  <dig> aa on their n-terminal side compared with those of other grass species including barley.
c
red bars indicate  <dig> aa repeats.
green boxes ii and iii indicate species-specific amino acid sequences in rps4
we analyzed the evolutionary relationships among grass species by examining the dna sequences of protein-coding genes in the mitochondrial genome.
a phylogenetic tree constructed in mega <dig> is shown in fig.
<dig>  barley belonged to the triticeae clade, which included t. aestivum, t. timopheevii and ae.
speltoides.
l. perenne was placed closer to barley than to s. bicolor, z. mays and o. sativa, which belonged to another clade.fig.
<dig> phylogenetic tree constructed using the dna sequences of  <dig> protein-coding genes comprising a total of  <dig>  bp in the mitochondrial genomes of eight grass species and subspecies by the maximum likelihood method.
the protein-coding genes include all electron transport chain genes, ccmb, ccmc, ccmfn, matr and mttb.
the sequences of these genes can be found under the following accession numbers: ap <dig> , ap <dig> , jx <dig> , dq <dig> , ay <dig> , ba <dig>  and dq <dig> .
the percentage of trees in which the associated taxa clustered together in  <dig> replicates of the bootstrap test is shown next to the branches.
scale bar indicates the number of base substitutions per site
discussion
features of the barley mitochondrial genome
in this study, we determined the complete nucleotide sequences of mitochondrial genomes from the wild barley accession h <dig> and the japanese malting cultivar haruna nijo.
these two haplotypes differ in origin and have sufficient polymorphisms in the nuclear dna sequences for a proper comparison  <cit> .
however, we detected a high level of sequence identity  between the two mitochondrial genomes; the mitochondrial genomes of these wild and cultivated barley lines were almost identical in terms of both nucleotide sequence and genome structure.
our annotation indicated that the barley mitochondrial genome contains a total of  <dig> genes .
these genes share similar components with genes in the wheat mitochondrial genome  <cit> , as described in the results and additional file 4: table s <dig>  orf prediction also suggested that the genome contains  <dig> orfs  comprising 16 % of the total genome .
six types of repeated sequences  larger than  <dig> kbp were found in the barley mitochondrial genome .
the repeated sequences were 100 % identical to each other and are located in direct orientation.
there were two copies of r <dig> to r <dig> and three copies of r <dig> .
using censor analysis, we identified a series of transposon types .
the total sequence lengths of copia, gypsy and non-ltr retrotransposons in the mitochondrial genome were  <dig>   <dig> and 4155 bp, respectively.
in the nuclear genome of barley , ca.
<dig>  % is occupied by mobile elements, with a retroelement/dna transposon ratio of  <dig>  %.
of these, the ratios of copia, gypsy and non-ltr retrotransposons are  <dig> ,  <dig>  and  <dig>  %, respectively.
we found the retroelement/dna transposon ratio in the mitochondrial genome to be  <dig>  %, which is much higher than that of the nuclear genome.
the barley mitochondrial genome appears to have more gypsy elements and many more non-ltr retrotransposons than the nuclear genome.
the barley mitochondrial genome contained three chloroplast-homologous sequences  larger than  <dig> kbp .
of these, two larger sequences were homologous to the chloroplast sequences of five barley cultivars  <cit>  and even to those in wheat and rye.
this result indicates that these homologous sequences were transferred from the chloroplast genome to the mitochondrial genome before the divergence of barley from a wheat/rye lineage.
another region  showed sequence similarity only to the chloroplast genome of barley cv.
barke.
this 1110 bp sequence could be cultivar specific, and its transfer from the mitochondrial genome to the chloroplast genome might have occurred quite recently only in this cultivar.
however, gene transfer between genomes is usually restricted to transfer from the chloroplast and mitochondria to the nucleus, known as nupt and numt, respectively, and from the chloroplast to mitochondria.
it is highly improbable that we encountered a rare transfer event from the mitochondria to chloroplasts in this cultivar.
it is likely, therefore, that this 1110 bp mitochondrial sequence was mis-assembled with the chloroplast genome of cv.
barke.
the chloroplast-homologous sequence of 6307 bp in the barley mitochondrial genome was longer than the corresponding sequence in the wheat mitochondrial genome, and annotation information  revealed that the trnas trnr and trnfm were included in this fragment.
in addition, the 2265 bp sequence contained trnv.
perhaps these chloroplast-derived trnas function in the barley mitochondria, although experimental evidence is needed to support this notion.
diversity of barley mitochondrial sequences
in the present study, only a few nucleotide polymorphisms were found between wild barley and cultivated barley mitochondrial haplotypes.
barley cultivation has a history of ca.
<dig>  years after domesticated from its wild progenitor.
according to the estimation of domestication mutation events on the btr <dig>  and btr <dig> loci, which are responsible for the reproductive barrier via seed dispersal  <cit> , there are two main sources of cultivated barley ancestors.
it is surprising that the organellar genome of the wild haplotype used in this study was quite similar to that of the cultivated barley haruna nijo.
of the snps detected in four wild and  <dig> cultivated barleys, only one wild barley showed an identical haplotype to h <dig> that was not found in the cultivated barleys examined .
interestingly, the haplotype in haruna nijo was also detected in wild barley accession h <dig>  among the three snps, the h <dig> allele of snp <dig> was abundant in north african and ethiopian landraces, indicating that snp <dig> can be traced to the germplasm distributed in north africa, with an origin related to wild barley accession h <dig>  the haplotype with the h <dig> alleles in snp <dig> and snp <dig> was also present in both wild and cultivated barley.
since there is no crossing barrier between the wild ancestral form and cultivated barley, it is possible that the mitochondrial genome of h <dig> resulted from hybridization between wild and cultivated barley.
more extensive sequencing of the mitochondrial genome is needed to elucidate mitochondrial genome evolution in wild and cultivated barley.
genome integration
wicker et al.
<cit>  reported that the mitochondrial genome was included in the nuclear genome, as revealed by low-redundancy shotgun genome sequencing.
according to blastn sequence similarity analysis  in the present study, many of the published genome sequences shared similarity with the mitochondrial genome .
the length of total integration of the mitochondrial genome into the chromosome-anchored nuclear genome sequence was  <dig>  bp, which is estimated to represent  <dig>  % of the total nuclear genome size in barley .
however, these mitochondrial sequences were not distributed evenly on the chromosomes, indicating that there are hot spots of chromosomal regions where mitochondrial sequences had transferred .
the mitochondrial genome shared similarity with many of the long un-anchored sequences, which could represent sequences corresponding to the mitochondrial genome itself generated from whole-genome shotgun sequencing.
the results also suggested that the plastid genome shares  <dig>  bp of sequence with the mitochondrial genome, which had three duplicated regions corresponding to  <dig>  %  of the total plastid genome .
thus, the current complete barley mitochondrial genome sequence represents an important tool for annotating the nuclear genome sequence, as well as the mitochondrial sequence itself spuriously included in the previously reported nuclear genome sequence.
comparative genomics with other crops
the size of the barley mitochondrial genome was  <dig> and 7 % larger than that of wheat  and rice , respectively, but 30 % smaller than that of perennial ryegrass .
the barley mitochondrial genome was comparable in size to those of maize .
multiple genome alignment analysis using progressivemauve  <cit>  revealed the presence of more than  <dig> short syntenic regions between barley and wheat .
this complex pattern of fragmented synteny prevented us from reconstructing the evolutionary process by which extensive rearrangements occurred between the two mitochondrial genomes.
by contrast, a high proportion of genes in the mitochondrial genome of barley were almost the same as those of other grass species.
in particular, the nucleotide sequences of protein-coding genes of the electron transport chain were highly conserved among grasses, although the presence of a few variations allowed us to construct a phylogenetic tree showing clear differentiation between triticeae and other grass species .
figure  <dig> shows a phylogenetic tree reconstructed from the nucleotide sequences of protein coding genes in the electron transport chain and those that function in cytochrome c biogenesis, as well as matr and mttb.
the tree indicates that barley is relatively closely related to wheat, as expected.
the analysis using the program mitofy also shows the sequences of individual genes of barely are closely related to those of corresponding genes of wheat.
therefore, there is no indication that any genes of barely have derived from other unrelated plant species by horizontal gene transfer.
based on the analysis of the sequences of nuclear genes encoding multi-domain plastid acetyl-coa carboxylase  and plastid 3-phosphoglycerate kinase , it was estimated that hordeum diverged from the triticum/aegilops lineage ca.
<dig> million years ago   <cit> .
on the other hand, comparative chloroplast genome analysis led to the estimate that barley diverged from rye and wheat approximately 8– <dig> mya  <cit> .
since barley shares a homoeologous chromosome system with wheat, only translocations differentiate the barley and wheat genomes.
although the gene contents in the barley mitochondrial genome were quite similar to those of wheat  <cit> , three genes  differed considerably between barley and wheat in terms of structure .
compared to wheat nad <dig>  barley nad <dig> contained a 198 nt extension in the 3′ coding region.
this extension may produce a nad <dig> polypeptide that is  <dig> aa longer in barley than in wheat.
although the length of nad <dig> deduced from the genome sequence of perennial ryegrass was nearly identical to that of barley, significant variations in both length and sequence were observed in the c-terminal half of nad <dig> among grass species .
nad <dig> is a hydrophobic membrane protein and a component of the nadh dehydrogenase complex .
complex i consists of approximately  <dig> proteins, seven subunits of which are encoded by the mitochondrial genome  <cit> ; it is difficult to imagine that nad <dig> variants are properly integrated into such a sophisticated molecular machinery.
to investigate the possibility that all grasses actually contain a short conserved nad <dig>  we looked for a potential stop codon before an in-frame stop codon in each nad <dig> gene that might have been created by rna editing.
sequence comparison, however, showed no clear indication that a new stop codon was created and that premature termination helped increase the similarity among nad <dig> polypeptides.
it should be noted, however, that nad <dig> could be expressed from mrnas lacking a stop codon in arabidopsis  <cit> .
in arabidopsis , nad <dig> mrna was processed upstream of the in-frame stop codons, and the resulting mrnas lacking stop codons were translatable  <cit> .
this observation suggests that mrna processing occurs at some points before an in-frame stop codon and that processing cancels apparent length variations in nad <dig> genes among grass species.
if this occurs, highly similar nad <dig> polypeptides will be translated and integrated into complex i of each grass species.
determining the amino acid sequence constituting the c-termini of nad <dig> from barley and other grasses will help test this notion.
the nad <dig> gene from barley shared identical sequences with those of most grass species.
an exception was wheat nad <dig>  which is 291 nt longer than nad <dig> genes from barley and other species.
two possible initiation codons  are present in the 5′ region of wheat nad <dig>  and if the second atg is used for translation, as suggested by lamattina et al.
<cit> , a similar polypeptide to that of barley will be produced in wheat.
the rps <dig> gene contains variable numbers of a 39 nt repeat, depending on the species; this repeat is categorized as a minisatellite.
some plant mtdnas contain minisatellites, most of which are located in intergenic regions, although a few occur in gene-coding and intronic regions  <cit> .
the size of the abovementioned minisatellite  is a multiple of three, which may explain why this minisatellite has survived in the coding region of rps <dig> even after the divergence of the barley/wheat lineages.
since the variability of minisatellites is generally high, such minisatellites can be used to find polymorphisms among the mitochondrial genomes of wild and cultivated barley.
CONCLUSIONS
the mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was quite different from that of wheat, which was used as a reference.
our data show that mitochondrial genome information is essential for correct annotation of the barley nuclear genome.
analysis of our newly generated mitochondrial sequence identified a significant number of fragmented mitochondrial sequences in the nuclear genome, plastid genomes and  un-anchored genomic sequences.
the small polymorphisms in the mitochondrial genome sequences between the wild and cultivated barley lines examined in this study should be further explored to help confirm that the diversity of barley mitochondrial genome sequences is indeed small.
alternatively, we may have chosen samples with similar gene pools in terms of mitochondrial variation.
additional files
additional file 1: table s <dig>  a primers used for gap filling in the h <dig> mitochondrial genome, b primers used for gap filling in the haruna nijo mitochondrial genome.
additional file 2: table s <dig>  primer pairs used for snp analysis.
additional file 3: table s <dig>  a contigs of the mitochondrial genome of h <dig> generated by  <dig> sequencing, b contigs of the mitochondrial genome of haruna nijo generated by  <dig> sequencing.
additional file 4: table s <dig>  genes identified in the mitochondrial genome of barley.
additional file 5: table s <dig>  novel orfs in the barley mitochondrial genome.
additional file 6: table s <dig>  repeated sequences in the mitochondrial genome of h <dig>
additional file 7: table s <dig>  syntenic region between the mitochondrial genome and chloroplast genome in barley.
additional file 8: table s <dig>  blastn analysis between the mitochondrial sequences of h <dig> and pseudomolecules of seven chromosomes and un-anchored scaffolds and contigs published in ensembl plants hordeum vulgare .
additional file 9: table s <dig>  snp genotypes of  <dig> wild and  <dig> cultivated barleys.
additional file 10: figure s <dig>  alignment of protein sequences encoded in the mitochondrial genomes of grass species.
abbreviations
bacbacterial artificial chromosome
orfopen reading frame
pcrpolymerase chain reaction
snpsingle nucleotide polymorphism
we thank dr. tsuyoshi tanaka for repeat sequence analysis and ms. yuka motoi, ms. hiromi ando and ms. nami yamaji for technical assistance.
barley seeds were provided by the national bioresource project of barley, mext of japan.
funding
ks is supported by the scientific technique research promotion program for agriculture, forestry fisheries and food industry of japan  and jsps kakenhi .
tt is supported by jsps kakenhi .
availability of data and materials
sequence data from this article have been submitted to the ddbj/embl/genbank data libraries under accession numbers ap <dig> and ap <dig> for h <dig> and haruna nijo, respectively.
phylogenetic data in fig.
<dig> are available from treebase .
authors’ contributions
tt and ks designed the research.
mt, hh and hy conducted experiments.
hh, tt and ks wrote the paper.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval and consent to participate
not applicable.

 cultivated barley was domesticated from its wild ancestral form ca.  years ago. cultivated and wild barley share closely related genomes that do not exhibit crossing or recombination barriers. barley is a self-pollinated diploid with seven pairs of chromosomes comprising a large haploid genome of gbp. the barley genome has been analyzed by bac fingerprinting, bac-end sequencing, whole-genome shotgun sequencing and transcriptome analysis. the genome data also include haplotype sequence information for several cultivated and wild barley lines, which provides million snps among haplotypes. the possibility of multiple domestication events for barley has been discussed based on phylogenic analyses of cloned genes. the wild ancestral form of barley is widely distributed in the near east to central asia, with a center in the fertile crescent. during the evolution of wild barley, its mitochondrial genomes might have diversified. for example, ahokas detected cytoplasmic male sterility, one of the main traits derived from the mitochondrial genome, in a wild barley germplasm. this finding indicates that the organellar genomes have differentiated in wild barley during its long evolutionary process. although the gene contents of the mitochondrial genomes of flowering plants are nearly identical, gene order and genome configuration are highly variable even within a species due to genome rearrangement. tsunewaki et al. explored the evolutionary dynamics of the wheat mitochondrial genome with respect to its structural differentiation during cereal evolution by comparing the nucleotide sequences of wheat genes to those of rice and maize. the nuclear genome of barley is closely related to those of wheat and its relatives, i.e., members of the triticeae tribe. the mitochondrial genome structure of common wheat cv. chinese spring is almost identical to that of the chinese wheat cultivar yumai, with differences of seven snps and indels in non-coding regions. noyszewski et al. sequenced the triticeae mitochondrial genomes of aegilops longissima, t. turgidum and an alloplasmic line with the ae. longissima cytoplasm that carries the t. turgidum nucleus, finding evidence suggesting that the alloplasmic condition accelerates evolution towards forming new mitochondrial genomes. however, to date, little information about the mitochondrial genome in barley has been published. in a study investigating the chloroplast genome, which together with the mitochondrial genome composes the plasmon, middleton et al. found that the chloroplast sequences of cultivated and wild barley are closely related. the divergence time of the two barley sequences was estimated to be  ±   years using semi-penalized likelihood. a comparison of the chloroplast genome sequences of cultivated barley and common wheat identified four deletions and five insertions greater than 50 bp in the chloroplast genome sequence of common wheat. this similarity in chloroplast sequence indicates that cultivated and wild barley are more closely related to each other than cultivated barley and cultivated wheat. the haplotypes used in the current study were the japanese malting barley cultivar ‘haruna nijo’ and the wild barley accession ‘h602’. haruna nijo has been extensively used as a crossing parent for developing high-quality malting barley cultivars in japan. the origin of this cultivar can be traced to the haplotype of a malting cultivar introduced more than 100 years ago from europe. haruna nijo has also been used to generate genomic resources, e.g., a bac library, full-length cdnas, and whole genome shotgun sequence analysis. wild barley accession h has been used as a haplotype that is distantly related to haruna nijo and thus, a high-resolution transcript map of haruna nijo/h has been developed. both haplotypes may represent cultivated and wild barley gene pools, suggesting that they are appropriate materials for revealing the diversity of mitochondrial genomes in barley. in this study, we determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley and compared their structures. we then analyzed the gene contents and unique regions of each genome in detail at the sequence level. since no mitochondrial genome sequence has been published for barley, we compared the resulting sequences to the published mitochondrial sequences of related species, i.e., wheat and its relatives, to clarify the evolutionary process of barley mitochondria. methods plant materials and mitochondrial dna extraction mitochondrial dna samples were extracted from etiolated seedlings barley cultivar ‘haruna nijo’ and wild barley accession ‘h602’ according to the method of bonen and gray, with minor modifications. barley seeds were obtained from the national bioresource project of barley, mext of japan. using discontinuous sucrose density gradient centrifugation, dnase-i treated mitochondria were collected from the interface between and  m sucrose. the mitochondrial dna samples were purified by etbr/cscl density-gradient ultracentrifugation. for diversity analysis of snps, seed samples from barley cultivars and four wild accessions preserved at the institute of plant science and resources, okayama university, japan were used. sequencing, assembly and completion both sequencing with the gs-flx titanium platform, and illumina sequencing with the miseq platform were performed to generate longer reads by gs-flx system and correct read errors with high redundant shorter reads by miseq system. the libraries for sequencing were prepared with gs titanium rapid library preparation kit and sequenced using gs flx titanium picotiterplate kit 70x and gs flx titanium sequencing kit xl+ following the manufacturer’s instructions. gs flx titanium rapid library mid adapter kit, gs flx titanium lv empcr kit v and gs flx titanium empcr breaking kit lv/mv 12pc were used for adapter ligation and emulsion pcr during library and sequencing preparation. the libraries for illumina sequencing were prepared with nextera xt dna sample preparation kit and nextera xt index kit and sequenced with miseq reagent kit v 500 cycles following the manufacturer’s instructions. the reads from sequencing were assembled with newbler. the generated contigs were filtered by average depth. blastn analysis was performed to exclude contamination of chloroplast genome sequences. filtered contigs were temporally ordered by hooking shared terminator reads in each contig. modified touchdown pcr was performed to connect the contigs and to validate the snps with the following program: initial denaturation at 98 °c for 2 min, 10 cycles of a melting step at 98 °c for 10 s, an annealing step at 65 °c decreased 1 °c/cycle for 15 s and an extension step at 72 °c for 1 min, followed by 20 cycles of denaturation at 95 °c for 10 s, annealing at 55 °c for 20 s and extension at 72 °c for 1 min, with a final extension at 72 °c for 10 min. the 10 μl reaction mixture for pcr included 1 ng mitochondrial dna as template, 1× primestar® max dna polymerase and  μm specific primers listed in additional file 1: tables s1a, b and additional file 2: tables s the pcr amplicons were checked by electrophoresis in  % agarose gels in tbe running buffer. direct sequencing of the pcr amplicons was performed on a 3130xl genetic analyzer. to increase the accuracy of genome sequencing, illumina-reads from h and haruna nijo were mapped and aligned onto the reference mitochondrial draft genome of h genome annotation and comparative analysis the genes encoding mitochondrial proteins and rrnas were identified using blastn and mitofy based on the known annotation of the mitochondrial genes of wheat cv. chinese spring. the trnascan-se server was used to identify trnas. repeat sequences were identified by align sequences nucleotide blast using mitochondrial sequences from haruna nijo. partial sequences sharing more than 10 % of nucleotides with the original genes were identified as pseudogenes. for transposable element analysis, censor was used with four sequence sources including poaceae, oryza, panicoideae and triticum. to identify novel orfs, ‘getorf’ analysis in emboss was conducted with the following parameters: predicted protein more than aa in length; methionine as the start codon; circular dna as the query sequence. after getorf analysis, the orfs overlapping with known genes were eliminated. alignments and phylogenetic analysis phylogenetic analysis was carried out using concatenated nucleotide sequences of the protein-coding genes extracted from the complete mitochondrial genome sequences deposited in the public databases. all positions containing gaps and missing data were eliminated, yielding a total of positions in the final dataset. alignments and phylogenetic tree construction were performed by the maximum likelihood method with the mega program. snp analysis a modified touchdown pcr procedure, as described previously, was performed for snp analysis with specific primer pairs using 50 ng genomic dna from cultivated and wild accessions of barley. pcr amplicons were sequenced with a 3130xl genetic analyzer.  sequencing and assembly of the mitochondrial genome in barley the mitochondrial genomes of wild and cultivated barley were analyzed by sequencing. a total of and reads corresponding to mbp and mbp were generated from h and haruna nijo, respectively. these reads were independently de novo assembled using newbler with average contig sizes of and 1624 bp, n contig sizes of and 1789 bp and largest contig sizes of and  bp for h and haruna nijo respectively. after trimming of duplicated sequences, these contigs were aligned in contigs for h and contigs for haruna nijo. the contig sequences were aligned, followed by sequencing of the pcr products with a 3130xl genome analyzer. draft master circular maps of the mitochondrial genome were generated for both h and haruna nijo. the illumina miseq reads were mapped onto the master circles and re-aligned to develop high quality mitochondrial sequences. from the mitochondrial sequencing libraries of h and haruna nijo,, and, paired reads were generated, respectively. reads of h were trimmed with clc genomic workbench, and the resulting pairs of reads were mapped onto the draft master circle of h a  bp circular molecule with a g/c content of  % was developed for h using the same methods, another  bp circular molecule was developed for haruna nijo with pairs of, trimmed reads. the alignment of two mitochondrial circular molecules identified three snps between h and haruna nijo at the positions of nt, and. snp was located in a genic region, and snp and snp were located in intergenic regions. these snps were validated by sanger sequencing of pcr products, including the alleles amplified by specific primers. annotation of the mitochondrial genome the master circles were annotated by blast analysis against the mitochondrial genome annotation databases mitofy and trnascan-se, and protein-coding genes, three ribosomal rnas and transfer rnas were identified. of the protein-coding genes, encoded members of the electron transport chain and atp synthase, including the following: nine subunits of complex i including nad nad nad nad nad4l, nad nad nad and nad9; an apocytochrome b subunit of complex iii; three subunits of complex iv including cox cox and cox3; and five subunits of complex v including atp atp atp atp and atp four additional protein-coding genes were involved in cytochrome c biogenesis, including ccmb, ccmc, ccmfc and ccmfn. there were genes for ribosomal proteins, namely rps rps rps rps rps rps rps rpl rpl and pseudogene rpl2p, as well as two other protein-coding genes, matr and mttb. among the protein-coding genes, there were two copies of nad nad ccmc, rps and rpl among rrna and trna genes, there were three copies of rrm rrn trnd, trnfm trnk and trnq and two copies of rrn and trnr. of the two copies of cox2b, the one nearest to cox2a was regarded as active cox2b, whereas the other, which is located far from cox2a, was regarded as a pseudogene due to the general rule of cis-splicing between cox2a and cox2b.fig. positions of genes on the barley mitochondrial genome map. genes encoding proteins, rrna and trna are shown outside and inside the circle. arrowhead indicates gene or exon, p indicates pseudogene, a, b, c, d and e indicate cis- and trans-spliced genic exon and underscores followed by numbers indicate gene copies prediction of new genes we used the ‘getorf’ command in embos to identify unique genes in the barley mitochondrial genome, leading to the prediction of new orfs. after excluding any partially overlapped orfs with known genes in the same translational frame, orfs were identified as novel genes. the total length of these novel orfs was  bp, which occupied  % of the barley mitochondrial genome. identification of transposable fragments censor was used to identify integrated transposable elements showing similarities to the sequences in the poaceae, oryza, panicoideae and triticum databases. a total of dna transposons, ltr retrotransposons and non-ltr retrotransposons were found in poaceae, were found in oryza and were found in panicoideae. by contrast, only ltr retrotransposon and two non-ltr retrotransposons were found in triticum.table transposable elements in the barley mitochondrial genome poaceae a oryza a panicoideae a triticum a dna transposon 13 837 9 522 8 406 ltr retrotransposon 57 41 572 49 8591 16 2043 non-ltr 18 4155 9 978 16 3685 2 103 total 88 59 9586 73 18 2146 asequence sources for transposable element analysis in censor repeat sequences and chloroplast-derived region in the mitochondrial genome six major repeat sequences and repeat sequences were found in the barley mitochondrial genome. there were four repeats in no. and three in nos. and the largest repeat was  bp, corresponding to the contig_h602_0022-0039-0169-.fig. repeat sequences and integrated chloroplast genome in the map of the barley mitochondrial genome of accession h repeat sequences in different colors are mapped onto the barley mitochondrial genome. detailed information about these repeats is shown in additional file 6: table s the predicted sequences integrated from the chloroplast genome are shown inside the circle according to previous reports, several trnas that function in the mitochondria originated in the chloroplast. in the barley mitochondrial genome, trnc, trnf, trns and trnw could represent trna genes of chloroplast origin. we analyzed the sequence similarity between the mitochondrial genome of h and the chloroplast genomes by blastn analysis of barley cvs. barke and morex and found three homologous regions. homologous region harboring trnfm, trnr, rps and psaa, contained 6307 bp in the r repeat region, suggesting that the mitochondrial genome contained two copies of homologous region among these genes, trnfm and trnr were identified using the mitochondrial genome annotation database, whereas rps and psaa were not identified. homologous region was 2265 bp in length and harbored trnv. homologous region contained 1110 bp of sequences specific to the chloroplast sequence of barke. although homologous regions and were found in the chloroplast genomes of several monocot species such as rice and maize, region was only found in barke. fifteen other homologous regions, ranging from to 558 bp in length, were also found by blastn analysis. transfer of mitochondrial dna into the barley nuclear genome we analyzed nucleotide sequence similarities between the mitochondrial genome of h and the published barley genome sequences, comprising pseudomolecules of seven chromosomes and un-anchored scaffolds and contigs published in ensembl plants hordeum vulgare, using blastn analysis. using the threshold e-value =  aligned sequence length > 400 bp, we detected barley genome sequences showing similarities to the barley mitochondrial genome sequence. of these, many of the longest un-anchored genome sequences appeared to be mitochondrial sequences, since the lengths of un-anchored sequences and aligned sequences were quite similar. for example, the sequence of morex_contig_ which was  bp in length, aligned completely with the mitochondrial sequence without mismatches or gaps. of the genome sequences within the threshold, seven were derived from the chloroplast genome and were un-anchored contigs that were potentially derived from the mitochondrial genome in the published barley genome sequence obtained by whole-genome shotgun sequencing. apart from the misidentified mitochondrial sequence, nuclear sequences of mitochondrial origin were observed. of the possible numts anchored to pseudomolecules,  bp of sequence on chromosome 6h was the longest that aligned with the mitochondrial genome. the total amounts of aligned sequences varied among chromosomes, ranging from  bp to  bp, indicating that the mitochondrial sequences were not evenly transferred to the nuclear genome. snp analysis of barley germplasm barley germplasm comprising cultivars from regions and four wild accessions were genotyped at three snps that were identified between the h and haruna nijo mitochondrial genomes. of the cultivars showing a nucleotide call on snp, and contained an a and t, respectively. of the four wild barley accessions, two contained a and two contained t. for snp, all of the cultivars and three wild barley accessions contained g, but one wild barley, h contained t. for snp, of cultivars and one wild barley contained a. most ethiopian cultivars contained c in snp although other regional differences in the allele distribution were not obvious. comparative genomics of barley mitochondria with those of wheat and other monocot species we compared the genes/proteins in the barley mitochondrial genome with those in other grass species including common wheat and wheat ancestors, perennial ryegrass, sorghum, maize and rice. barley nad encoded nad a aa polypeptide. the sequence comprising aa to in nad was conserved among grass species, whereas the c-terminus is highly variable. barley nad was conserved among grass species except wheat, which contained a longer nad due to frame-shifting caused by a 4 nt insertion/deletion. the rps gene had 39-nt imperfect repeats corresponding to aa. barley rps had three repeats, whereas wheat has one and rice, maize and perennial ryegrass each have partial one. the genes cox atp rps rpl and rrn also varied between barley and wheat.table differences in mitochondrial genes encoding proteins between barley and wheat nad6 nad9 cox1 atp6 rps2 rps4 rpl5 rrn18 abonen and bird indicated that the n-terminus of atp in the mitochondrial genome had interspecies variation caused by processing bkubo et al. reported rps in barley and interspecies variation in triticeae at its c-terminus fig. structures of the encoded protein sequences in various grass species. the structures of a nad b nad and c rps in the barley mitochondrial genome were compared with those of seven other grass species and subspecies. light blue bars show the primary structures of proteins, and orange lines indicate the start codon. the numbers above the light blue bars indicate the position from the start codons of barley proteins. a green box i indicates the non-conserved amino acid sequences in nad among grass species. b nad of t. aestivum, ae. speltoides and t. timopheevii have an additional aa on their n-terminal side compared with those of other grass species including barley. c red bars indicate aa repeats. green boxes ii and iii indicate species-specific amino acid sequences in rps4 we analyzed the evolutionary relationships among grass species by examining the dna sequences of protein-coding genes in the mitochondrial genome. a phylogenetic tree constructed in mega is shown in fig.  barley belonged to the triticeae clade, which included t. aestivum, t. timopheevii and ae. speltoides. l. perenne was placed closer to barley than to s. bicolor, z. mays and o. sativa, which belonged to another clade.fig. phylogenetic tree constructed using the dna sequences of protein-coding genes comprising a total of  bp in the mitochondrial genomes of eight grass species and subspecies by the maximum likelihood method. the protein-coding genes include all electron transport chain genes, ccmb, ccmc, ccmfn, matr and mttb. the sequences of these genes can be found under the following accession numbers: ap, ap, jx, dq, ay, ba and dq. the percentage of trees in which the associated taxa clustered together in replicates of the bootstrap test is shown next to the branches. scale bar indicates the number of base substitutions per site discussion features of the barley mitochondrial genome in this study, we determined the complete nucleotide sequences of mitochondrial genomes from the wild barley accession h and the japanese malting cultivar haruna nijo. these two haplotypes differ in origin and have sufficient polymorphisms in the nuclear dna sequences for a proper comparison. however, we detected a high level of sequence identity between the two mitochondrial genomes; the mitochondrial genomes of these wild and cultivated barley lines were almost identical in terms of both nucleotide sequence and genome structure. our annotation indicated that the barley mitochondrial genome contains a total of genes. these genes share similar components with genes in the wheat mitochondrial genome, as described in the results and additional file 4: table s orf prediction also suggested that the genome contains orfs comprising 16 % of the total genome. six types of repeated sequences larger than kbp were found in the barley mitochondrial genome. the repeated sequences were 100 % identical to each other and are located in direct orientation. there were two copies of r to r and three copies of r. using censor analysis, we identified a series of transposon types. the total sequence lengths of copia, gypsy and non-ltr retrotransposons in the mitochondrial genome were and 4155 bp, respectively. in the nuclear genome of barley, ca.  % is occupied by mobile elements, with a retroelement/dna transposon ratio of  %. of these, the ratios of copia, gypsy and non-ltr retrotransposons are, and  %, respectively. we found the retroelement/dna transposon ratio in the mitochondrial genome to be  %, which is much higher than that of the nuclear genome. the barley mitochondrial genome appears to have more gypsy elements and many more non-ltr retrotransposons than the nuclear genome. the barley mitochondrial genome contained three chloroplast-homologous sequences larger than kbp. of these, two larger sequences were homologous to the chloroplast sequences of five barley cultivars and even to those in wheat and rye. this result indicates that these homologous sequences were transferred from the chloroplast genome to the mitochondrial genome before the divergence of barley from a wheat/rye lineage. another region showed sequence similarity only to the chloroplast genome of barley cv. barke. this 1110 bp sequence could be cultivar specific, and its transfer from the mitochondrial genome to the chloroplast genome might have occurred quite recently only in this cultivar. however, gene transfer between genomes is usually restricted to transfer from the chloroplast and mitochondria to the nucleus, known as nupt and numt, respectively, and from the chloroplast to mitochondria. it is highly improbable that we encountered a rare transfer event from the mitochondria to chloroplasts in this cultivar. it is likely, therefore, that this 1110 bp mitochondrial sequence was mis-assembled with the chloroplast genome of cv. barke. the chloroplast-homologous sequence of 6307 bp in the barley mitochondrial genome was longer than the corresponding sequence in the wheat mitochondrial genome, and annotation information revealed that the trnas trnr and trnfm were included in this fragment. in addition, the 2265 bp sequence contained trnv. perhaps these chloroplast-derived trnas function in the barley mitochondria, although experimental evidence is needed to support this notion. diversity of barley mitochondrial sequences in the present study, only a few nucleotide polymorphisms were found between wild barley and cultivated barley mitochondrial haplotypes. barley cultivation has a history of ca.  years after domesticated from its wild progenitor. according to the estimation of domestication mutation events on the btr and btr loci, which are responsible for the reproductive barrier via seed dispersal, there are two main sources of cultivated barley ancestors. it is surprising that the organellar genome of the wild haplotype used in this study was quite similar to that of the cultivated barley haruna nijo. of the snps detected in four wild and cultivated barleys, only one wild barley showed an identical haplotype to h that was not found in the cultivated barleys examined. interestingly, the haplotype in haruna nijo was also detected in wild barley accession h among the three snps, the h allele of snp was abundant in north african and ethiopian landraces, indicating that snp can be traced to the germplasm distributed in north africa, with an origin related to wild barley accession h the haplotype with the h alleles in snp and snp was also present in both wild and cultivated barley. since there is no crossing barrier between the wild ancestral form and cultivated barley, it is possible that the mitochondrial genome of h resulted from hybridization between wild and cultivated barley. more extensive sequencing of the mitochondrial genome is needed to elucidate mitochondrial genome evolution in wild and cultivated barley. genome integration wicker et al. reported that the mitochondrial genome was included in the nuclear genome, as revealed by low-redundancy shotgun genome sequencing. according to blastn sequence similarity analysis in the present study, many of the published genome sequences shared similarity with the mitochondrial genome. the length of total integration of the mitochondrial genome into the chromosome-anchored nuclear genome sequence was  bp, which is estimated to represent  % of the total nuclear genome size in barley. however, these mitochondrial sequences were not distributed evenly on the chromosomes, indicating that there are hot spots of chromosomal regions where mitochondrial sequences had transferred. the mitochondrial genome shared similarity with many of the long un-anchored sequences, which could represent sequences corresponding to the mitochondrial genome itself generated from whole-genome shotgun sequencing. the results also suggested that the plastid genome shares  bp of sequence with the mitochondrial genome, which had three duplicated regions corresponding to  % of the total plastid genome. thus, the current complete barley mitochondrial genome sequence represents an important tool for annotating the nuclear genome sequence, as well as the mitochondrial sequence itself spuriously included in the previously reported nuclear genome sequence. comparative genomics with other crops the size of the barley mitochondrial genome was and 7 % larger than that of wheat and rice, respectively, but 30 % smaller than that of perennial ryegrass. the barley mitochondrial genome was comparable in size to those of maize. multiple genome alignment analysis using progressivemauve revealed the presence of more than short syntenic regions between barley and wheat. this complex pattern of fragmented synteny prevented us from reconstructing the evolutionary process by which extensive rearrangements occurred between the two mitochondrial genomes. by contrast, a high proportion of genes in the mitochondrial genome of barley were almost the same as those of other grass species. in particular, the nucleotide sequences of protein-coding genes of the electron transport chain were highly conserved among grasses, although the presence of a few variations allowed us to construct a phylogenetic tree showing clear differentiation between triticeae and other grass species. figure  shows a phylogenetic tree reconstructed from the nucleotide sequences of protein coding genes in the electron transport chain and those that function in cytochrome c biogenesis, as well as matr and mttb. the tree indicates that barley is relatively closely related to wheat, as expected. the analysis using the program mitofy also shows the sequences of individual genes of barely are closely related to those of corresponding genes of wheat. therefore, there is no indication that any genes of barely have derived from other unrelated plant species by horizontal gene transfer. based on the analysis of the sequences of nuclear genes encoding multi-domain plastid acetyl-coa carboxylase and plastid 3-phosphoglycerate kinase, it was estimated that hordeum diverged from the triticum/aegilops lineage ca. million years ago. on the other hand, comparative chloroplast genome analysis led to the estimate that barley diverged from rye and wheat approximately 8– mya. since barley shares a homoeologous chromosome system with wheat, only translocations differentiate the barley and wheat genomes. although the gene contents in the barley mitochondrial genome were quite similar to those of wheat, three genes differed considerably between barley and wheat in terms of structure. compared to wheat nad barley nad contained a 198 nt extension in the 3′ coding region. this extension may produce a nad polypeptide that is aa longer in barley than in wheat. although the length of nad deduced from the genome sequence of perennial ryegrass was nearly identical to that of barley, significant variations in both length and sequence were observed in the c-terminal half of nad among grass species. nad is a hydrophobic membrane protein and a component of the nadh dehydrogenase complex. complex i consists of approximately proteins, seven subunits of which are encoded by the mitochondrial genome; it is difficult to imagine that nad variants are properly integrated into such a sophisticated molecular machinery. to investigate the possibility that all grasses actually contain a short conserved nad we looked for a potential stop codon before an in-frame stop codon in each nad gene that might have been created by rna editing. sequence comparison, however, showed no clear indication that a new stop codon was created and that premature termination helped increase the similarity among nad polypeptides. it should be noted, however, that nad could be expressed from mrnas lacking a stop codon in arabidopsis. in arabidopsis, nad mrna was processed upstream of the in-frame stop codons, and the resulting mrnas lacking stop codons were translatable. this observation suggests that mrna processing occurs at some points before an in-frame stop codon and that processing cancels apparent length variations in nad genes among grass species. if this occurs, highly similar nad polypeptides will be translated and integrated into complex i of each grass species. determining the amino acid sequence constituting the c-termini of nad from barley and other grasses will help test this notion. the nad gene from barley shared identical sequences with those of most grass species. an exception was wheat nad which is 291 nt longer than nad genes from barley and other species. two possible initiation codons are present in the 5′ region of wheat nad and if the second atg is used for translation, as suggested by lamattina et al., a similar polypeptide to that of barley will be produced in wheat. the rps gene contains variable numbers of a 39 nt repeat, depending on the species; this repeat is categorized as a minisatellite. some plant mtdnas contain minisatellites, most of which are located in intergenic regions, although a few occur in gene-coding and intronic regions. the size of the abovementioned minisatellite is a multiple of three, which may explain why this minisatellite has survived in the coding region of rps even after the divergence of the barley/wheat lineages. since the variability of minisatellites is generally high, such minisatellites can be used to find polymorphisms among the mitochondrial genomes of wild and cultivated barley.  the mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was quite different from that of wheat, which was used as a reference. our data show that mitochondrial genome information is essential for correct annotation of the barley nuclear genome. analysis of our newly generated mitochondrial sequence identified a significant number of fragmented mitochondrial sequences in the nuclear genome, plastid genomes and un-anchored genomic sequences. the small polymorphisms in the mitochondrial genome sequences between the wild and cultivated barley lines examined in this study should be further explored to help confirm that the diversity of barley mitochondrial genome sequences is indeed small. alternatively, we may have chosen samples with similar gene pools in terms of mitochondrial variation. additional files additional file 1: table s a primers used for gap filling in the h mitochondrial genome, b primers used for gap filling in the haruna nijo mitochondrial genome. additional file 2: table s primer pairs used for snp analysis. additional file 3: table s a contigs of the mitochondrial genome of h generated by sequencing, b contigs of the mitochondrial genome of haruna nijo generated by sequencing. additional file 4: table s genes identified in the mitochondrial genome of barley. additional file 5: table s novel orfs in the barley mitochondrial genome. additional file 6: table s repeated sequences in the mitochondrial genome of h additional file 7: table s syntenic region between the mitochondrial genome and chloroplast genome in barley. additional file 8: table s blastn analysis between the mitochondrial sequences of h and pseudomolecules of seven chromosomes and un-anchored scaffolds and contigs published in ensembl plants hordeum vulgare. additional file 9: table s snp genotypes of wild and cultivated barleys. additional file 10: figure s alignment of protein sequences encoded in the mitochondrial genomes of grass species. abbreviations bacbacterial artificial chromosome orfopen reading frame pcrpolymerase chain reaction snpsingle nucleotide polymorphism we thank dr. tsuyoshi tanaka for repeat sequence analysis and ms. yuka motoi, ms. hiromi ando and ms. nami yamaji for technical assistance. barley seeds were provided by the national bioresource project of barley, mext of japan. funding ks is supported by the scientific technique research promotion program for agriculture, forestry fisheries and food industry of japan and jsps kakenhi. tt is supported by jsps kakenhi. availability of data and materials sequence data from this article have been submitted to the ddbj/embl/genbank data libraries under accession numbers ap and ap for h and haruna nijo, respectively. phylogenetic data in fig.  are available from treebase. authors’ contributions tt and ks designed the research. mt, hh and hy conducted experiments. hh, tt and ks wrote the paper. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate not applicable.
these mitochondrial genomes contained protein-coding genes, three ribosomal rnas, transfer rnas, new orfs, six major repeat sequences and several types of transposable elements. mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. two independent circular maps of the  bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines h and haruna nijo, with only three snps detected between haplotypes. sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. we determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. wild barley and cultivated barley have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes. little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley. of the barley mitochondrial genome-encoded proteins, nad nad and rps had unique structures among grass species. the mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. hordeum vulgaremitochondrial genomede novo assemblycomparative genomicskakehhijp15k07257sato kazuhiro the scientific technique research promotion program for agriculture, forestry fisheries and food industry of japan25013asato kazuhiro kakenhijp26292007terachi toru issue-copyright-statement© the author 2016 barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of gbp.
 sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of gbp. wild barley and cultivated barley have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes.  we determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. two independent circular maps of the  bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines h and haruna nijo, with only three snps detected between haplotypes. these mitochondrial genomes contained protein-coding genes, three ribosomal rnas, transfer rnas, new orfs, six major repeat sequences and several types of transposable elements. of the barley mitochondrial genome-encoded proteins, nad nad and rps had unique structures among grass species.  the mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hordeum vulgaremitochondrial genomede novo assemblycomparative genomicskakehhijp15k07257sato kazuhiro the scientific technique research promotion program for agriculture, forestry fisheries and food industry of japan25013asato kazuhiro kakenhijp26292007terachi toru issue-copyright-statement© the author 2016 