DNA Ploidy Estimation Research Articles

Guide to the genus Rubus L. (Rosaceae) in South Africa – Disentangling a taxonomic Gordian knot with the help of ploidy and reproductive data

Rubus is notorious for its taxonomic complexity. Although some light has been recently shed on the taxonomy of the species from the Greater Cape Floristic Region in South Africa, many questions remain unanswered. This contribution is based on extensive field and herbarium studies across South Africa and aims at clarifying Rubus taxonomy in the region with the help of DNA-ploidy estimation and assessment of reproductive mode. We distinguish a total of 18 taxa from the region, of which six are native. Furthermore, the two native species R. pinnatus and R. ludwigii exhibit geographic variation that can be treated at the level of subspecies, and the new combination R. pinnatus subsp. pappei is provided. We do not recognise any infraspecific taxa in the widely distributed sexual hexaploid R. rigidus. All of the taxa are described and are photographically documented. Several other taxa were found only as solitary inadequate specimens and need further study. Moreover, 12 primary hybrids were identified, of which seven were derived from hybridization between native and alien species. Contrary to previous reports, each of the species exhibited a single ploidy level, and hybrids were, with one or two exceptions, intermediate between the parents in their ploidy. Whereas native taxa are strictly sexual, most polyploid aliens reproduce by facultative apomixis and hybrids are mostly sterile or sexual. However, apomixis was detected in R. sect. Arguti × R. pinnatus and R. bergii × R. thaumasius.

DNA ploidy in Salix retusa agg. only partly in line with its morphology and taxonomy

Salix retusa agg. encompasses small, prostrate willows occurring in the subalpine and alpine zones of European mountains. Karyological relationships among taxa in this group remain unclear. In this study, DNA ploidy was estimated using flow cytometry of silica‐dried specimens, covering the entire distribution area of the species. As a result, the presence of two main lineages was revealed: the exclusively diploid S. serpillifolia and the predominantly octoploid S. retusa s.l. Only at the western and eastern limits of the distributional range of the latter species different levels of DNA ploidy occurred: hexaploidy in the western Pyrenees, and up to decaploidy in the southern Carpathians. These results were compared with leaf morphology analyses presented in the work of Kosiński et al. (2017). In the western Pyrenean populations, the lower ploidy corresponded to smaller leaf sizes as compared to other populations of S. retusa s.l. The elevated ploidy in the southern Carpathian populations did not translate into bigger dimensions of plants in this area. However, the populations from the northern part of the Carpathian arc, distinguished by larger leaves and sometimes considered as a distinct taxon (S. kitaibeliana), consisted mainly of octoploid individuals. Thus, DNA ploidy estimations did not confirm the separate taxonomic status of S. kitaibeliana.

Pollen precedence in sexual Potentilla puberula and its role as a protective reproductive barrier against apomictic cytotypes.

Cross-pollination is a major factor determining the demographic dynamics of mixed-ploidy populations. Typically, rare cytotypes are suppressed due to reduced female fertility by losing gametes in heteroploid crosses (i.e., through minority cytotype exclusion). In species with reproductive differentiation into sexual and apomictic cytotypes, sexuals might be reproductively suppressed by apomicts (or transformed due to introgression of apomixis genes). Pollen precedence potentially acts as a post-pollination pre-fertilization barrier protecting sexuals against their apomictic counterparts. We estimated the role of pollen precedence as a barrier against cross-fertilization of tetraploid sexuals by penta- and heptaploid gametophytic apomicts in Potentilla puberula (Rosaceae) by means of controlled crosses, and inference of the paternity through DNA ploidy estimation of embryos. Individuals from five regions spanning an elevational and biogeographic gradient were used to account for the variation in the relative frequencies of reproductive modes across the study area. We tested (1) whether the application of heteroploid pollen (sexual × apomictic) causes a reduction of seed yield compared to homoploid crosses (sexual × sexual), and (2) if so, whether pollen precedence recovers the seed yield in simultaneous applications of pollen from sexuals and apomicts (mixed-ploidy). Seed yield was significantly lower in hetero- than in homoploid crosses. We found clear evidence for precedence of homoploid pollen, despite a 13% to 15% of embryos experienced a change in ploidy due to heteroploid fertilizations. Thus, our study indicates that pollen precedence operates as a barrier against intercytotype fertilization in P. puberula, promoting the integrity of the sexual cytotype and their co-existence with apomictic individuals.

Abstract 4502: DNA ploidy in tumor correlates with age, and explains some of the poor prognostic impact of age in endometrial carcinomas

Abstract Background: Endometrial cancer incidence is increasing in industrialized countries, and the demand for better prognostic markers in order to individualize treatment is emerging. DNA ploidy has previously shown independent prognostic impact in smaller studies. In this study, we wanted to investigate the prognostic impact of ploidy in primary endometrial tumors, and the link between DNA ploidy and patient age at diagnosis in relation to traditional clinicopathologic prognostic variables. Study design: 663 patients treated for endometrial carcinoma at Haukeland University Hospital, Norway, from 1981 through 2010 were included. DNA ploidy in fresh tumor specimens was measured by flow-cytometry. Comprehensive clinical and histopathologic data, treatment and complete follow-up were collected. The software PASWStatistics18 was used for statistical analysis. Results: DNA aneuploidy was significantly correlated to high patient age at diagnosis (p<0.001). It was also correlated to non-endometrioid histology and high-grade tumors (p<0.001) and high FIGO stage (p=0.012). In univariate survival analysis (Kaplan Meier), the 5-year disease specific survival of patients with diploid tumors was 89%, versus 65% for aneuploid tumors (p<0.001). In Cox regression analysis, aneuploidy was significantly associated with poor survival (HR=2.4, 95%CI 1.6 - 3.8, p<0.001), adjusted for FIGO stage, age, grade, and histologic subtype. Interestingly, the prognostic impact of patient age diminished when ploidy status was included in the Cox analysis. Conclusion: DNA ploidy estimation in endometrial carcinoma tumor tissue is highly associated with patient age at diagnosis, as well as prognosis. The prognostic impact of age decreases when adjusting for ploidy status in tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4502. doi:1538-7445.AM2012-4502

Apoptotic protein expression, glycogen content, DNA ploidy and cell proliferation in hepatoblastoma subtyping and their role in prognostication

To evaluate Bcl-xL, Bax, PCNA, cytokeratin 19 (CK-19), glycogen content and DNA ploidy expression in hepatoblastoma (HB) and their prognostic value. This retrospective study on 26 cases of HB involved DNA ploidy estimations separately for various subtypes on histological sections using image cytometry of Feulgen-stained smears. Glycogen content, PCNA, CK-19, Bax and Bcl-xL expression on tissue sections were evaluated. THE outcome on follow-up (mean 86 months) and Kaplan-Meier survival analysis were performed. Fetal areas were diploid (84%); embryonal areas were aneuploid (89.47%) (p < 0.001). PCNA labeling index was low in fetal (10.82%) and high in embryonal areas (59.85%) (p = 0.03). CK-19 was negative in fetal, but focally positive in embryonal areas. Bax was negative in fetal (80%) and positive in embryonal areas (88.23%) (p < 0.001); Bcl-xL was more frequently positive in fetal (90%) than embryonal areas (52.94%) (p = 0.02). Fetal cells were rich in glycogen. Kaplan-Meier survival analysis showed good initial radiological response to chemotherapy (p = 0.009), glycogen content (p = 0.0137) and DNA diploid cases (p = 0.0429) were associated with good outcome on univariate analysis. Histology typing (p = 0.085), Bcl-xL (p = 0.689), Bax (p = 0.27), CK-19 (p = 0.281), PCNA (p = 0.689), age (p = 0.24), sex (p = 0.5661), stage (p = 0.24) and α-fetoprotein levels (p = 0.49) were unrelated to outcome. Multivariate analysis showed glycogen content to be the most statistically significant variable (0.024). Fetal and embryonal areas show different staining patterns for PCNA, Bax and Bcl-xL. DNA ploidy and glycogen content are significant prognostic variables.

Genome size in Dipsacaceae and Morina longifolia (Morinaceae)

Dipsacaceae and Morinaceae have for a long time been regarded as separate but related families, whereas according to APG III they are included within the larger family Caprifoliaceae. Although genome size studies seldom provide conclusive characters for higher level systematics, they can yield useful information at a lower taxonomy level. In this study, we used DNA flow cytometry (supplemented by Feulgen densitometry) for measurement of genome size variation in the Dipsacaceae genera Cephalaria, Dipsacus, Knautia, Lomelosia, Pterocephalus, Scabiosa, Sixalix, Succisa, and Succisella, and Morina of the Morinaceae. At the monoploid level the Dipsacaceae genera (x = 7–10) vary 5.94-fold between 0.902 and 5.362 pg DNA (1Cx-value), whereas Morina longifolia (x = 17) has only 0.670 pg DNA. At the holoploid level 11.58-fold variation occurs between 0.902 and 10.446 pg DNA (1C-value). In Knautia sect. Trichera ploidy levels 2x, 4x, 6x are accompanied by corresponding increments of C-values, but genome downsizing is observed. In Knautia sect. Tricheroides the only investigated species K. integrifolia (2n = 20) has only 0.60-fold the mean genome size of sect. Trichera. Scabiosa canescens (2n = 2x = 16) has approximately double the C-value of all other Austrian Scabiosa species at the diploid level (pseudopolyploidy). These values raise concern against DNA-ploidy estimations at the interspecific level when chromosome numbers are unknown. The species sorted into two major clades of an existing phylogenetic tree of Dipsacaceae differ characteristically in their range of Cx-values. The Knautia–Cephalaria–Dipsacus–Succisella clade has the great majority of its Cx-values larger than those of the Scabiosa–Pterocephalus–Lomelosia clade.

Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry—new prospects for plant research

Only fresh plant material is generally used for rapid DNA ploidy estimation by flow cytometry (FCM). This requirement, however, substantially limits convenient FCM application in plant biosystematics, population biology, and ecology. As desiccation is a routine way for sample preservation in field botany, potential utilization of dehydrated tissues of vascular plants in FCM research was examined. Standard DAPI protocol was employed to evaluate the performance of 60 air-dried species, spanning more than 100-fold range of nuclear DNA amounts. Multiploid Vaccinium subg. Oxycoccus was selected as model taxon for detailed investigation and cytotype comparison. A majority of analyzed plants yielded distinct peaks with reasonable coefficients of variation after several months of storage at room temperature. Fluorescence intensity of nuclei isolated from desiccated tissues was highly comparable with that for fresh material, allowing reliable DNA ploidy estimation. Deep-freezer preservation substantially extended Vaccinium samples lifetime (at least to 3 years) and maintained high histogram resolution. The introduced approach eliminates the need for fresh material in many vascular plants and thus opens new prospects for plant FCM. Convenient cytotype investigation in field research and retrospective ploidy determination in already herbarized samples are among the principal advantages.