Evidence against Equimolarity of Large Repeat Arrangements and a Predominant Master Circle Structure of the Mitochondrial Genome from a Monkeyflower (Mimulus guttatus) Lineage with Cryptic CMS

Abstract

Despite intense investigation for over 25 years, the in vivo structure of plant mitochondrial genomes remains uncertain. Mapping studies and genome sequencing generally produce large circular chromosomes, whereas electrophoretic and microscopic studies typically reveal linear and multibranched molecules. To more fully assess the structure of plant mitochondrial genomes, the complete sequence of the monkeyflower (Mimulus guttatus DC. line IM62) mitochondrial DNA was constructed from a large (35 kb) paired-end shotgun sequencing library to a high depth of coverage (∼30×). The complete genome maps as a 525,671 bp circular molecule and exhibits a fairly conventional set of features including 62 genes (encoding 35 proteins, 24 transfer RNAs, and 3 ribosomal RNAs), 22 introns, 3 large repeats (2.7, 9.6, and 29 kb), and 96 small repeats (40–293 bp). Most paired-end reads (71%) mapped to the consensus sequence at the expected distance and orientation across the entire genome, validating the accuracy of assembly. Another 10% of reads provided clear evidence of alternative genomic conformations due to apparent rearrangements across large repeats. Quantitative assessment of these repeat-spanning read pairs revealed that all large repeat arrangements are present at appreciable frequencies in vivo, although not always in equimolar amounts. The observed stoichiometric differences for some arrangements are inconsistent with a predominant master circular structure for the mitochondrial genome of M. guttatus IM62. Finally, because IM62 contains a cryptic cytoplasmic male sterility (CMS) system, an in silico search for potential CMS genes was undertaken. The three chimeric open reading frames (ORFs) identified in this study, in addition to the previously identified ORFs upstream of the nad6 gene, are the most likely CMS candidate genes in this line.

Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes

“…As with the short repeats, these data are in striking contrast to the extremely low level of recombination detected for the great majority of comparably sized repeats in S. conica, S. noctiflora, and cucumber (9,60). Repeats of this size either are not present or were not analyzed in Vigna, Mimulus, and the four S. vulgaris mitogenomes (61)(62)(63).…”

“…2,400 short repeats in the 6.7-Mb genome of S. noctiflora genome (9, 60) and for only two of 110 short repeats in the 404-kb genome of Vigna angularis (61). AC frequency was not reported for the 526-kb Mimulus mitogenome (62), but the number of AC-consistent reads was given and indicates much lower AC frequency levels than in Viscum. As in Viscum, repeat length correlates with AC frequency in Mimulus.…”

Monilophyte mitochondrial rps1 genes carry a unique group II intron that likely originated from an ancient paralog in rpl2

“…Like in rps1i25g2, no traces indicating a previous existence of a maturase-like reading frame are discernible in any of the fern intron rpl2i846g2 homologs. Independent losses of intron rpl2i846g2 are hitherto only found in Equisetum bogotense (Knie et al 2015) and Mimulus guttatus (Mower et al 2012).…”

“…In hornworts (Li et al 2009;Xue et al 2010), lycophytes Isoetes engelmannii (Grewe et al 2009 and Selaginella moellendorffii (Hecht et al 2011) rpl2 genes are absent from the mtDNA, indicating transfer to the nucleus. Intron rpl2i846g2 is present in all tracheophytes where rpl2 is retained in the mtDNA with the exception of independent losses in Mimulus guttatus (Mower et al 2012) and Equisetum bogotense (Knie et al 2015). Liverworts carry the unrelated intron rpl2i28g2 in their mitochondrial rpl2 genes.…”

Organellar Genomes of Flowering Plants

“…Over the years, numerous attempts of recovering large populations of intact circular mtDNA have failed due to the predominance of linear and branched structures which show up in most of the electrophoretic and microscopic analyses of mitochondrial DNA (Bendich 1993 ). There is a suffi cient evidence to support both the circular and the linear, multibranched forms of mitochondrial DNA, yet the idea of mtDNA as circular DNA molecules has persisted (Mower et al 2012 ;Sloan 2013 ). Deep sequencing analyses in recent years have proven to be the myth busters with overwhelming evidences to prove that the linear forms not merely exist but exist in good percentage.…”