FCM in plant biosystematics

The knowledge of nuclear DNA content (either in relative or absolute units) has been fruitfully utilized in various lines of plant biosystematics. Three principal areas of interest are: ploidy level estimation, genome size determination and reproduction mode detection.

Ploidy level estimation

Polyploidy has played a significant role in the evolution of vascular plants - up to 80 % of angiosperms and about 95 % of pteridophytes were estimated to be polyploid. Flow cytometry can be used for:

• reliable taxa discrimination in closely related polyploid groups
  (several angiosperm alliances encompass microspecies that differ only negligibly in morphological characters, however, are unambiguously defined by unique ploidy level)

FCM discrimination between diploid Anthoxanthum alpinum and tetraploid A. odoratum

• identification of heteroploid hybrids between closely related taxa

FCM profile of triploid hybrid of Empetrum together with its putative parents

• identification of rare cytotypes

Considerable cytotype variation in Lythrum salicaria revealed by flow cytometry

• cytotype distribution at various spatial scales

Empetrum cytotype distribution in Scandinavian countries

• investigation of population cytotype structure
  (new field allowing to raise numerous challenging questions, such as the overall distribution pattern (sympatry vs. parapatry), ecological preferences of individual cytotypes, inter- vs. intra-cytotype competition, mechanisms of reproductive isolation, or evolutionary forces governing the cytotype co-existence)

Sympatric occurrence of various Empetrum cytotypes

• investigation of reproduction behaviour
  (in heteroploid crosses, off-springs ploidy allows to discriminate between sexual and apomictic pathways and assess the frequency of non-reduced gametes)

Trihaploid and hexaploid progenies of Hieracium rubrum originating by haploid parthenogenesis and apomixis, respectively

• aneuploidy and sex detection
  (an ambitious task requiring analyses with very high resolution)

Presumably aneuploid plant analyzed together with diploid standard in Campanula patula

• endopolyploidy detection
  (the most convenient way for reliable detection of this type of genome organization)

Flow cytometric analysis of Kochia scoparia showing nuclei with 2C, 4C and 8C DNA amounts

Genome size determination

Genome size is the important feature of all living organisms. Two basic terms should be distinguished:

Holoploid genome size (C-value): DNA content of the unreplicated haploid nucleus (irrespective of the ploidy level)

Homoploid genome size (Cx-value): DNA content of the monoploid chromosome set (in polyploids averaged)

Holoploid and homoploid genome sizes equal in diploid species, however, the former exceeds the latter in polyploid species. Both values can be expressed either in DNA picograms or megabase pairs (1 pg = 978 Mbp).

Genome size data are useful for:

• identification of species with the same number of chromosomes
  (species-specific genome size occurs in several plant alliances)

Discrimination between species with the same number of chromosomes but different genome size in Dryopteris dilatata alliance

• detection of hybrids from homoploid crosses
  (providing the putative parents differ enough in genome size, FCM allows reliable detection of their homoploid crosses)
• assessment of genomic constitution in allopolyploid taxa or identification of putative parents
  (in some hybridogenous groups, identification of their progenitors is possible)
• wide-range utilization of genome size data
  (genome size allows to predict various phenotypic characters, or phenological and ecological behaviour)

Genome size determination in triploid Empetrum using Raphanus sativus as internal standard

• base composition
  (knowledge of AT/GC base proportion may provide further, more profound, insight into genome organization)

Simultaneous analysis of tetraploid Hieracium cymosum and Zea mays as internal standard stained with DAPI (fluorochrome with preferential binding to A-T base pairs)

The same species pair stained with propidium iodide (intercalating fluorochrome with no base preference). Note marked difference between peak positions in both analyses.

• agmatoploidy
  (non-lethal chromosome fragmentation owing to the presence of diffuse centromere leading to the increase of chromosome number under constant holoploid genome size)

Reproduction mode detection

A revolutionary advance for efficient reproduction pathway screening, based on different proportional DNA contents of embryo and endosperm in mature seeds. Considering the type of male and female gametes (reduced vs. unreduced), the embryo origin (zygotic vs. parthenogenetic), and the endosperm origin (pseudogamous vs. autonomous route), ten different pathways of seed formation can be reconstructed.

Flow cytometric acquisition of mature seeds in sexual Taraxacum with 2C embryo and 3C endosperm nuclei

Flow cytometric acquisition of mature seeds in apomictic Taraxacum with 2C embryo and 4C endosperm nuclei