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

Abstract

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.

Keywords: CMS-associated gene; celery; cytoplasmic male sterility (CMS); mitochondrial genome.

Figure 1

Phenotype of flowers from the celery CMS line W99A and its maintainer line W99B. (a) Flower of maintainer line W99B; (b) anther of maintainer line W99B; (c) flower of completely stamen degeneration type; (d) anther of completely stamen degeneration type; (e) flower of incomplete stamen degeneration type; (f) anther of incomplete stamen degeneration type; (g,h) TTC staining for pollen viability of the celery CMS line W99A and its maintainer line W99B. (a,c,e) scale = 5 cm; (b,f) scale = 0.4 mm; (d) scale = 0.8 mm; (g,h) = 200 µm.

Figure 2

Microspore development of anthers from the celery CMS line W99A and its maintainer line W99B. (a–f) Celery CMS line W99A; (g–l) maintainer line W99B. (a,g) stamen primordium stage; (b,h) sporogonia and sporogenetic cell stage; (c,i) pollen mother cell stage; (d,j) tetrad stage, (e,k), uninucleate stage; (f,l) pollen mature stage. MMC: microspore mother cell, Msp: microspore, MC: meiotic cells, T: tapetum, Tds: tetrad microspore, PG: pollen grain. E: Extexine, En: endodermis, ML: middle lamella. Scale bars = 50 µm.

Figure 3

Mitochondrial genome maps of the celery CMS line W99A (a) and its maintainer line W99B (b). Genes names inside and outside of the circle indicate clockwise and counterclockwise transcription, respectively. Different colours indicate different functions of gene products. The inner circles indicates syntenic sequence blocks.

Figure 4

The structural variation map of W99A and W99B mitochondrial genomes.

Figure 5

Gene structure of orf768a. (a) The orf768a sequence has a chimeric structure including cox1, arrow indicates the 5′-to-3′ direction. (b) Transmembrane domains of the orf768a gene products. (c) Tertiary structure analysis of cox1. (d) Tertiary structure analysis of orf768a.

Figure 6

PCR confirmation of the specificity of ORFs for CMS celery. (a) RT-PCR detection of orf768a, orf340a-1/2, orf340a-1/2, orf241a, orf115b, orf163a, orf254a; (b) CMS maker orf768a tests in 41 pepper inbred lines.