Combining genome size and pollen morphology data to study species relationships in the genus Daucus (Apiaceae).

BACKGROUND: The genus Daucus (Apiaceae) comprises about 40 wild species and the cultivated carrot, a crop of great economic and nutritional importance. The rich genetic diversity of wild Daucus species makes them a valuable gene pool for carrot improvement breeding programs. Therefore, it is essential to have good knowledge of the genome structure and relationships among wild Daucus species. To broaden such knowledge, in this research, the nuclear DNA content for 14 Daucus accessions and four closely related species was estimated by flow cytometry and their pollen morphology was analyzed by light and scanning electron microscopy (SEM). RESULTS: The flow cytometric analysis showed a 3.2-fold variation in the mean 2C values among Daucus taxa, ranging from 0.999 (D. carota subsp. sativus) to 3.228 pg (D. littoralis). Among the outgroup species, the mean 2C values were 1.775-2.882 pg. The pollen grains of Daucus were tricolporate, mainly prolate or perprolate (rarely) in shape, and mainly medium or small (rarely) in size (21.19-40.38 µm), whereas the outgroup species had tricolporate, perprolate-shaped, and medium-sized (26.01-49.86 µm) pollen grains. In the studied taxa, SEM analysis revealed that exine ornamentation was striate, rugulate, perforate, or the ornamentation pattern was mixed. At the time of shedding, all pollen grains were three-celled, as evidenced by DAPI staining. We also found high positive correlations between the length of the polar axis (P) and the length of the equatorial diameter (E) of pollen grains, as well as between P and P/E. However, when comparing cytogenetic information with palynological data, no significant correlations were observed. CONCLUSIONS: This study complements the information on the nuclear DNA content in Daucus and provides comprehensive knowledge of the pollen morphology of its taxa. These findings may be important in elucidating the taxonomic relationships among Daucus species and can help in the correct identification of gene bank accessions. In a broader view, they could also be meaningful for the interpretation of evolutionary trends in the genus.

Using carrot centromeric repeats to study karyotype relationships in the genus Daucus (Apiaceae).

BACKGROUND: In the course of evolution, chromosomes undergo evolutionary changes; thus, karyotypes may differ considerably among groups of organisms, even within closely related taxa. The genus Daucus seems to be a promising model for exploring the dynamics of karyotype evolution. It comprises some 40 wild species and the cultivated carrot, a crop of great economic significance. However, Daucus species are very diverse morphologically and genetically, and despite extensive research, the taxonomic and phylogenetic relationships between them have still not been fully resolved. Although several molecular cytogenetic studies have been conducted to investigate the chromosomal structure and karyotype evolution of carrot and other Daucus species, detailed karyomorphological research has been limited to carrot and only a few wild species. Therefore, to better understand the karyotype relationships within Daucus, we (1) explored the chromosomal distribution of carrot centromeric repeats (CentDc) in 34 accessions of Daucus and related species by means of fluorescence in situ hybridization (FISH) and (2) performed detailed karyomorphological analysis in 16 of them. RESULTS: We determined the genomic organization of CentDc in 26 accessions of Daucus (belonging to both Daucus I and II subclades) and one accession of closely related species. The CentDc repeats were present in the centromeric regions of all chromosomes of 20 accessions (representing 11 taxa). In the other Daucus taxa, the number of chromosome pairs with CentDc signals varied depending on the species, yet their centromeric localization was conserved. In addition, precise chromosome measurements performed in 16 accessions showed the inter- and intraspecific karyological relationships among them. CONCLUSIONS: The presence of the CentDc repeats in the genomes of taxa belonging to both Daucus subclades and one outgroup species indicated the ancestral status of the repeat. The results of our study provide useful information for further evolutionary, cytotaxonomic, and phylogenetic research on the genus Daucus and may contribute to a better understanding of the dynamic evolution of centromeric satellites in plants.

Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae).

BACKGROUND: Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet (Beta vulgaris L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages. RESULTS: We established a methyl salicylate-based protocol that significantly improved the transparency of the B. vulgaris ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat. CONCLUSIONS: The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species' embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.