The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions

Abstract

Cytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6^-, the putative mutant copy with a 300 bp truncation at the 5'-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6⁺) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6⁺ copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6⁺ is expressed only in MF samples, while apt6^- mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6^- could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility.

Keywords: ATP synthase; F1 hybrid breeding; energy deficiency model; male sterility; marker-assisted selection; mtDNA; organelle assembly; pollen.

Figure 1

Annotation of Foeniculum vulgare mitochondrial genome assembled from a cytoplasmic male sterile (CMS) accession. The annotation was created with GeSeq [34] and visualized with OGDRAW. Genes marked with an asterisk (*) contain introns.

Figure 2

Nucleotide acid alignment of atp6 genes sequences retrieved from the mtDNA of male fertile (MF, atp6⁺) and cytoplasmic male sterile (CMS, atp6⁻) accessions of Foeniculum vulgare.

Figure 3

(A) Nucleotide alignment between the two atp6 copies retrieved within the mitochondrial genomes of Foeniculum vulgare. atp6⁺ indicates the wild type gene (1167 bp) found in the male fertile (MF) mtDNA while atp6⁻ is an atp6-like sequence lacking the first 300 bp (864 bp) retrieved in the cytoplasmic male sterile (CMS) mtDNA. Raw sequencing reads from MF and CMS accessions were mapped to atp6⁺ and atp6⁻. (B) PCR profiles generated in two CMS commercial lines and their MF maintainers with specific primers for atp6⁺(left) and atp6⁻ (right). (C) Protein alignment of atp6⁺, atp6⁻, and the atp6 protein from Daucus carota (Dc_atp6).

Figure 4

Evaluation of atp6⁺ (A), atp6⁻ (B), atp1 (C), and atp9 (D) mRNA expression by qPCR analysis. Expression analysis were conducted on male fertile (MF, n = 4, red triangles) and male sterile samples (CMS, n = 4, blue squares). Four different tissues were evaluated for each gene: leaf (L), flower before anthesis (0% flowering, FBA), flower in half anthesis (50% flowering, FHA) and flower in full anthesis (100% flowering, FFA). Ct values were normalized for gapdh. Statistical significance was evaluated with unpaired Student’s t-test (* p < 0.05). All data are shown as mean ± SD.

Figure 5

Involvement of atp6 in cytoplasmic male sterility in fennel. The atp6⁺ gene duplication and transfer from the mtDNA to the nuclear genome is a recurrent and consistent feature of eukaryotic genome evolution that seems to have characterized also the evolutionary path of Foeniculum vulgare. Subsequently, a mitochondrial rearrangement event produced a truncation of 300 bp in the mtDNA atp6 copy (atp6⁻) leading to a cytoplasmic male sterility phenotype. Expression analyses revealed that (i) atp6⁺ gene is constantly expressed in male fertile plants (MF), where it is available both in the mtDNA and, probably, in the nuclear DNA (ii) atp6⁺ gene is never expressed in male sterile plants, where is available only in the nuclear DNA (probably as pseudogene) (iii) atp6⁻ is constantly expressed in male sterile plants (MF), where is available in the mtDNA.