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Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

ABSTRACT: BACKGROUND:Nsa cytoplasmic male sterility (CMS) is a novel alloplasmic male sterility system derived from somatic hybridization between Brassica napus and Sinapis arvensis. Identification of the CMS-associated gene is a prerequisite for a better understanding of the origin and molecular mechanism of this CMS. With the development of genome sequencing technology, organelle genomes of Nsa CMS line and its maintainer line were sequenced by pyro-sequencing technology, and comparative analysis of the organelle genomes was carried out to characterize the organelle genome composition of Nsa CMS as well as to identify the candidate Nsa CMS-associated genes. RESULTS:Nsa CMS mitochondrial genome showed a higher collinearity with that of S. arvensis than B. napus, indicating that Nsa CMS mitochondrial genome was mainly derived from S. arvensis. However, mitochondrial genome recombination of parental lines was clearly detected. In contrast, the chloroplast genome of Nsa CMS was highly collinear with its B. napus parent, without any evidence of recombination of the two parental chloroplast genomes or integration from S. arvensis. There were 16 open reading frames (ORFs) specifically existed in Nsa CMS mitochondrial genome, which could not be identified in the maintainer line. Among them, three ORFs (orf224, orf309, orf346) possessing chimeric and transmembrane structure are most likely to be the candidate CMS genes. Sequences of all three candidate CMS genes in Nsa CMS line were found to be 100% identical with those from S. arvensis mitochondrial genome. Phylogenetic and homologous analysis showed that all the mitochondrial genes were highly conserved during evolution. CONCLUSIONS:Nsa CMS contains a recombined mitochondrial genome of its two parental species with the majority form S. arvensis. Three candidate Nsa CMS genes were identified and proven to be derived from S. arvensis other than recombination of its two parental species. Further functional study of the candidate genes will help to identify the gene responsible for the CMS and the underlying molecular mechanism.

SUBMITTER: Sang SF

PROVIDER: S-EPMC6836354 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Project description:BACKGROUND: The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility. RESULTS: It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the ? subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the ? subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered. CONCLUSION: The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through retrograde regulation of nuclear gene expression in Brassica juncea.

Project description:BackgroundBrassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few.ResultsIn this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, "6251AB" and "6284AB". At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, "4001AB" and "4006AB". Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between "4001A" and "4001B", two differentially expressed miRNAs between "4006A" and "4006B", four differentially expressed miRNAs between "6251A" and "6251B", and ten differentially expressed miRNAs between "6284A" and "6284B". The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5' modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed.ConclusionsOur findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

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Organelle genome composition and candidate gene identification for Nsa cytoplasmic male sterility in Brassica napus.

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