Whole Mitochondrial Genome Sequencing and Transcriptional Analysis to Uncover an RT102-Type Cytoplasmic Male Sterility-Associated Candidate Gene Derived from Oryza rufipogon

Abstract

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants fail to produce functional pollen and is associated with the expression of a novel open reading frame (orf) gene encoded by the mitochondrial genome. An RT102A CMS line and an RT102C fertility restorer line were obtained by successive backcrossing between Oryza rufipogon W1125 and O. sativa Taichung 65. Using next-generation pyrosequencing, we determined whole-genome sequences of the mitochondria in RT102-CMS cytoplasm. To identify candidates for the CMS-associated gene in RT102 mitochondria, we screened the mitochondrial genome for the presence of specific orf genes that were chimeric or whose products carried predicted transmembrane domains. One of these orf genes, orf352, which showed different transcript sizes depending on whether the restorer of fertility (Rf) gene was present or not, was identified. The orf352 gene was co-transcribed with the ribosomal protein gene rpl5, and the 2.8 kb rpl5-orf352 transcripts were processed into 2.6 kb transcripts with a cleavage at the inside of the orf352 coding region in the presence of the Rf gene. The orf352 gene is an excellent candidate for the CMS-associated gene for RT102-CMS.

Multi-step formation, evolution, and functionalization of new cytoplasmic male sterility genes in the plant mitochondrial genomes

“…These recombinant structures including the ORFs and their flanking sequences, of which cs1 to cs10 are conserved in the mitochondrial genomes of Oryza and other plant species, were named S###. The ORFs that were verified as functional CMS genes in this study (see below) and a previous report [14] were named "WA###", while the putative ORFs with no CMS function (see below) and those functionally uncharacterized were named "orf###", where "###" indicates the number of amino acids (aa) encoded by the ORFs. For the homologous ORFs of the same aa number but with different flanking sequences, lower case letters (a, b, c, etc) were added at the end.…”

“…The five ORFs (WA352a, WA352b, WA352c, orf367, and orf356) in these structures share the same segments (284s, cs3, cs2, and cs1). WA352a is the same as orf352 of the functional CMS gene reported in CMS-RT102 rice [14]. WA352b and WA352c have identical sequences, but differ from WA352a by five single-nucleotide polymorphisms (SNPs).…”

“…Previous studies proved that WA352c and WA352a have CMS function [10,14]. As orf356, orf367 and WA314 have structures similar to WA352a and WA352c, we analyzed whether they also have CMS function.…”

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

“…Novel and chimeric mitochondrial sequences are a frequent result of this recombination (Wise et al, 1987;Kennell and Pring, 1989;Wen and Chase, 1999;Gallagher et al, 2002;Okazaki et al, 2013;Yamagishi and Bhat, 2014;Tang et al, 2017), in which recombination sometimes leads to the creation of transcripts that interfere with normal male gametophyte development (Kitazaki and Kubo, 2010) via the generation of toxic and/or disruptive transmembrane proteins (Korth et al, 1991;Kim et al, 2007;Wan et al, 2007;Zhang et al, 2007;Yang et al, 2009;Gulyas et al, 2010;Jing et al, 2012;Flores-Renteria et al, 2013;Ji et al, 2013;Luo et al, 2013;Okazaki et al, 2013;Park et al, 2013;Hu et al, 2014). Surprisingly, such genes are not only abundant in many fertile plants, such as Arabidopsis thaliana Unseld et al, 1997), Beta vulgaris (Kubo et al, 2000), Oryza sativa (Notsu et al, 2002), Brassica napus (Handa, 2003), Zea mays (Clifton et al, 2004), Triticum aestivum (Ogihara et al, 2005), and Nicotiana tabacum (Sugiyama et al, 2005), but are also constitutively expressed.…”

“…Many examples of CMS stem from these consequences of recombination Chen and Liu, 2014;Yamagishi and Bhat, 2014;Charlesworth, 2017;Tang et al, 2017), and include chimeric gene fusions (Rathburn and Hedgcoth, 1991;Tuteja et al, 2013;Wang et al, 2013b;Szklarczyk et al, 2014;Tang et al, 2017), partial/orphan ORFs (Shearman et al, 2014), and disruptions in gene orientation/promoter association (Horn et al, 2014). Often, these chimeric CMS genes exhibit co-transcription with upstream or downstream functional genes, such as Turf13 in CMS-T maize (Wise et al, 1987;Kennell and Pring, 1989), orf352 in RT102-CMS rice (Okazaki et al, 2013), and orf355 and orf77 in CMS-S maize (Wen and Chase, 1999;Gallagher et al, 2002). CMS genes typically affect the mitochondrial electron transfer chain pathways, and are commonly classified into four categories according to the components they affect:…”