Mitochondrial Proteomics and Transcriptional Analysis of Cytoplasmic Male Sterility in Sugar Beet using iTRAQ and qRT–PCR

Sugar beet (Beta vulgaris L.) is an important raw material for the sugar industry, and its output is second only to sugar cane. Cytoplasmic male sterility (CMS) is a phenomenon of pollen abortion that has important implications in sugar beet hybrid breeding. Male plant sterility is usually considered to be associated with mitochondrial dysfunction. Although mitochondrial genes associated with male sterility have been well explored, the different mitochondrial proteomics of CMS in sugar beet are still poorly understood. In this study, differentially expressed mitochondrial proteomic analysis was performed on the flower buds of the male sterile line (DY5-CMS), its maintainer line (DY5-O) and a fertility restorer line (CL6), using an isobaric tag for relative and absolute quantitation (iTRAQ) technology. A total of 2260 proteins were identified by mass spectrometry, of which 538 were differentially expressed proteins. Most of them were involved in protein metabolism, carbohydrate and energy metabolism, and binding. More specifically, some cysteine and methionine metabolism proteins (A0A0J8BGE0, A0A0J8CZM6, A0A0J8D7W0 and A0A0J8BCR7) may play important roles during the formation of CMS. This study provided an in–depth understanding of the CMS molecular mechanism at the protein level in sugar beet.

Organelle genome assembly uncovers the dynamic genome reorganization and cytoplasmic male sterility associated genes in tomato

To identify cytoplasmic male sterility (CMS)-associated genes in tomato, we determined the genome sequences of mitochondria and chloroplasts in three CMS tomato lines derived from independent asymmetric cell fusions, their nuclear and cytoplasmic donors, and male fertile weedy cultivated tomato and wild relatives. The structures of the CMS mitochondrial genomes were highly divergent from those of the nuclear and cytoplasmic donors, and genes of the donors were mixed up in these genomes. On the other hand, the structures of CMS chloroplast genomes were moderately conserved across the donors, but CMS chloroplast genes were unexpectedly likely derived from the nuclear donors. Comparative analysis of the structures and contents of organelle genes and transcriptome analysis identified three genes that were uniquely present in the CMS lines, but not in the donor or fertile lines. RNA-sequencing analysis indicated that these three genes transcriptionally expressed in anther, and identified different RNA editing levels in one gene, orf265, that was partially similar to ATP synthase subunit 8, between fertile and sterile lines. The orf265 was a highly potential candidate for CMS-associated gene. This study suggests that organelle reorganization mechanisms after cell fusion events differ between mitochondria and chloroplasts, and provides insight into the development of new F1 hybrid breeding programs employing the CMS system in tomato.

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

<p class="042abstractstekst">The study quantified the effect of gibberellic acid (GA₃) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length<strong>, </strong>anther length, plant height and number of tiller hill^-1of male parent Giza 178R were significantly at 300 g GA₃ ha^-1 concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA₃ application for male parent and male to female ratio, while, number of fertile panicles hill^-1, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha^-1) was obtained by CMS line IR69625A using 300 g GA₃ ha^-1 of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.</p>

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

<p class="042abstractstekst">The study quantified the effect of gibberellic acid (GA₃) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length<strong>, </strong>anther length, plant height and number of tiller hill^-1of male parent Giza 178R were significantly at 300 g GA₃ ha^-1 concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA₃ application for male parent and male to female ratio, while, number of fertile panicles hill^-1, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha^-1) was obtained by CMS line IR69625A using 300 g GA₃ ha^-1 of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.</p>

Heterosis and Combining Ability For Floral and Yield Characters in Rice Using Cytoplasmic Male Sterility System

Developing high-yielding rice genotypes has become more urgent to ensure global food security with continuing population growth and the threat of environmental pressures. Cytoplasmic male sterility (CMS) system provides a valuable approach for commercial exploitation of heterosis and producing high-yielding and quality hybrid rice. Three CMS lines and ten diverse restorers were crossed using line × tester mating design. The obtained thirty F1 hybrids and their thirteen parents were evaluated. Yield traits as well as certain floral traits characters that influence the efficiency of crossing and hybrid seed production as duration of floret opening (min), stigma exsertion (mm), stigma length (mm), opening floret angle, and anther length (mm) were assessed. Highly significant variations were detected among parents, crosses, and parents vs. crosses for all the studied traits. The CMS line L2 and the restorer T5 were identified as good combiners for stigma exsertion, stigma length, opining floret angle, and duration of floret opening. Moreover, the hybrids L1×T1, L1×T3, L2×T2, L2×T5, L3×T4, L3×T5, and L3×T9 had positive SCA effects for most floral traits. Besides, the CMS lines L1 and L3 as well as the restorers T1, T2, T3, T6, and T9 proved to be the best general combiners for grain yield and certain contributing traits. The hybrids L1×T1, L1×T5, L1×T7, L2×T3, L2×T4, L2×T5, L2×T10, L3×T1, L3×T2, and L3×T6 displayed positive SCA effects for grain yield and one or more of its attributes. Both additive and non-additive gene effects are involved in the governing inheritance of all evaluated traits. The biochemical variations among the certain evaluated genotypes were further studied. The esterase and peroxidase isozymes were applied for verifying the genetic diversity at the protein level among the used CMS lines, restorers, and their crosses. All the applied isozymes displayed polymorphism for the parents and their crosses. The banding pattern and intensity differences provided accurate results on the reliable variability among the tested genotypes.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata)

Background Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. Results In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. Conclusions The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

Complete Mitochondrial Genome Sequence and Identification of a Candidate Gene Responsible for Cytoplasmic Male Sterility in Celery (Apium graveolens L.)

Celery (Apium graveolens L.) is an important leafy vegetable worldwide. The development of F1 hybrids in celery is highly dependent on cytoplasmic male sterility (CMS) because emasculation is difficult. In this study, we first report a celery CMS, which was found in a high-generation inbred line population of the Chinese celery “tanzhixiangqin”. Comparative analysis, following sequencing and assembly of the complete mitochondrial genome sequences for this celery CMS line and its maintainer line, revealed that there are 21 unique regions in the celery CMS line and these unique regions contain 15 ORFs. Among these ORFs, only orf768a is a chimeric gene, consisting of 1497 bp sequences of the cox1 gene and 810 bp unidentified sequences located in the unique region, and the predicted protein product of orf768a possesses 11 transmembrane domains. In summary, the results of this study indicate that orf768a is likely to be a strong candidate gene for CMS induction in celery. In addition, orf768a can be a co-segregate marker, which can be used to screen CMS in celery.