Plant Genome Size Research Articles

Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment

Flow cytometry has become the method of choice to measure the DNA content (genome size) in plants. Ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics and biotechnology. Although the cell wall is a problem when isolating plant cells, cytometry remains a powerful tool in plant sciences. Based on our 30-years’ experience in this field, this review will focus at first on genome size measurement using simply isolated nuclei: the good practice for acquisition, nuclei isolation, appropriate buffers, kind of tissues to use. The second part will briefly review what kind of measurements it is possible to make in plant cytometry, and for what purpose: base composition, ploidy level, cell cycle, endoreplication, seed screening, and nuclei/chromosomes sorting. We will address troubleshooting. The commonly-used mathematic tools will be discussed.

Morphology and genome size of Epipactis helleborine (L.) Crantz (Orchidaceae) growing in anthropogenic and natural habitats.

BackgroundThe process of apophytism or spreading native species to human-made habitats is one of the main elements in the creation of plant cover in anthropogenic areas. Lately, an increase of anthropogenic localities with valuable flora has been observed. Apophytes are also members of the family Orchidaceae, especially from the genus Epipactis. The aim of the study was to (i) determine and compare the phenotypic variation of E. helleborine (L.) Crantz plants in anthropogenic and natural habitats, (ii) compare the genome size of plants growing in natural and anthropogenic habitats. The results reported in this study may indicate that a habitat influences morphological characteristics of plant species.MethodsField studies were conducted on four native stands and four stands in anthropogenic areas of E. helleborine in Poland in years 2011–2013. Biometrical analyses were performed on shoots and flowers. The flowers were characterised by 25 biometric features and measured using a Nikon SMZ 800 binocular, microscopic Moticam-1SP cameras and the MIPlus07 programme (Conbest Co.). The nuclear DNA content was determined in fresh and young leaves of E. helleborine, collected from four natural and four anthropogenic populations.ResultsWe observed that in anthropogenic populations: (i) shoots were higher than shoots from natural populations, (ii) flowers differed significantly in terms of ten biometric features between habitats, (iii) the genome size of some population differed significantly between plants growing in natural and anthropogenic habitats.DiscussionAccording to some researchers, the presence of phenotypic variability and the occurrence of ecotypes are adaptation strategies of plants to environmental changes. In our opinion, in the case of the studied anthropogenic habitats (roadside) in which the E. helleborine populations grew, we can talk about ecofen due to the often repeated set of characteristic features, i.e., high shoots, long inflorescence and long, broad leaves. We agree, however, that it is difficult to isolate a taxonomic unit for ecofen due to the lack of experimental research.

Estimation of Nuclear DNA Content and Determination of Relationship Between Altitude and Genome Size of USDA Turkish Oat (Avena spp.) Collection

Crop improvement is an important approach to overcome challenges raised from future uncertainties of agricultural systems and growing human population. The fundamental need for such improvement efforts is the availability of well characterized plant germplasm with sufficient genetic diversity. The 2C DNA is defined as the nuclear DNA content of an unreplicated diploid cell (in G1 phase) and is used both to get an estimate of genome size and ploidy level. Flow cytometry provides accurate and fast estimation of the genome size of plants. The genus Avena belongs to Poaceae (Gramineae) family and includes approximately 30 species including common oat (A. sativa). There are 837 Avena accessions in the USDA (United States Department of Agriculture) Germplasm Resources Information Network (USDA-GRIN) collected from Turkey. This also is the largest ex situ Avena collection from Turkey. However, initial genomic characterization of the collection has not yet been conducted. We estimated genome sizes and determined ploidy levels of Turkish oat collection. Nuclear DNA content of accessions ranged from 25.66 to 26.56 pg for A. sativa, from 25.48 to 26.88 pg for A. sterilis and from 24.85 to 26.41 pg for A. fatua. The average and range values for all three hexaploid species were in a similar close range. The range for putative tetraploid accessions belonging to A. barbata was from 12.79 to 16.90 pg. We found a number of aggregates. Furthermore, there was a negative correlation found between altitude and genome size. Obtained results will help to better utilize Avena collection in breeding efforts.

FlowPloidy: An R package for genome size and ploidy assessment of flow cytometry data.

Premise of the StudyDespite advantages in terms of reproducibility, histogram analysis based on nonlinear regression is rarely used in genome size assessments in plant biology. This is due in part to the lack of a freely available program to implement the procedure. We have developed such a program, the R package flowPloidy.Methods and ResultsflowPloidy builds on the existing statistical tools provided with the R environment. This base provides tools for importing flow cytometry data, fitting nonlinear regressions, and interactively visualizing data. flowPloidy adds tools for building flow cytometry models, fitting the models to histogram data, and producing visual and tabular summaries of the results.ConclusionsflowPloidy fills an important gap in the study of plant genome size. This package will enable plant scientists to apply a more powerful statistical technique for assessing genome size. flowPloidy improves on existing software options by providing a no‐cost workflow streamlined for genome size and ploidy determination.

Genome-Wide Survey and Comparative Analysis of Long Terminal Repeat (LTR) Retrotransposon Families in Four Gossypium Species

Long terminal repeat (LTR) retrotransposon is the most abundant DNA component and is largely responsible for plant genome size variation. Although it has been studied in plant species, very limited data is available for cotton, the most important fiber and texture crop. In this study, we performed a comprehensive analysis of LTR retrotransposon families across four cotton species. In tetraploid Gossypium species, LTR retrotransposon families from the progenitor D genome had more copies in D-subgenome, and families from the progenitor A genome had more copies in A-subgenome. Some LTR retrotransposon families that insert after polyploid formation may still distribute the majority of its copies in one of the subgenomes. The data also shows that families of 10~200 copies are abundant and they have a great influence on the Gossypium genome size; on the contrary, a small number of high copy LTR retrotransposon families have less contribution to the genome size. Kimura distance distribution indicates that high copy number family is not a recent outbreak, and there is no obvious relationship between family copy number and the period of evolution. Further analysis reveals that each LTR retrotransposon family may have their own distribution characteristics in cotton.

Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability.

A reassessment of the genome size-invasiveness relationship in reed canarygrass (Phalaris arundinacea).

Genome size is hypothesized to affect invasiveness in plants. Key evidence comes from a previous study of invasive eastern North American populations of the grass Phalaris arundinacea: invasive genotypes with smaller genomes had higher growth rates, and genome sizes were smaller in the invasive vs. native range. This study aimed to re-investigate those patterns by examining a broader range of North American populations and by employing the modern best-practice protocol for plant genome size estimation in addition to the previously used protocol. Genome sizes were measured using both internal and pseudo-internal standardization protocols for 20 invasive and nine native range accessions of P. arundinacea. After a round of vegetative propagation to reduce maternal environmental effects, growth (stem elongation) rates of these accessions were measured in the greenhouse. Using the best-practice protocol, there was no evidence of a correlation between genome size and growth rates (P = 0.704), and no evidence for differences in genome sizes of invasive and native range accessions (P > 0.353). However, using the older genome size estimation protocol, both relationships were significant (reproducing the results of the previous study). Genome size reduction has not driven increased invasiveness in a broad sample of North American P. arundinacea. Further, inappropriate genome size estimation techniques can create spurious correlations between genome size and plant traits such as growth rate. Valid estimation is vital to progress in understanding the potentially widespread effects of genome size on biological processes and patterns.

Extreme haplotype variation in the desiccation-tolerant clubmoss Selaginella lepidophylla

Plant genome size varies by four orders of magnitude, and most of this variation stems from dynamic changes in repetitive DNA content. Here we report the small 109 Mb genome of Selaginella lepidophylla, a clubmoss with extreme desiccation tolerance. Single-molecule sequencing enables accurate haplotype assembly of a single heterozygous S. lepidophylla plant, revealing extensive structural variation. We observe numerous haplotype-specific deletions consisting of largely repetitive and heavily methylated sequences, with enrichment in young Gypsy LTR retrotransposons. Such elements are active but rapidly deleted, suggesting “bloat and purge” to maintain a small genome size. Unlike all other land plant lineages, Selaginella has no evidence of a whole-genome duplication event in its evolutionary history, but instead shows unique tandem gene duplication patterns reflecting adaptation to extreme drying. Gene expression changes during desiccation in S. lepidophylla mirror patterns observed across angiosperm resurrection plants.

Polyploidy and genome size variation in Phlox nana (Polemoniaceae) from the Pecos plains of New Mexico and the Davis Mountains of West Texas

Polyploidy is conspicuous in the genus Phlox, and some species exhibit variation in ploidy levels, or cytotypic variation. Diploid, tetraploid and hexaploid popula-tions of P. nana occur across parts of the species distribution in the southwestern United States and northern Mexico. A recent study highlighted two areas for which ploidy level inferences were challenging: a population on the Pecos Plains of New Mexico (“Caprock”) and the Davis Mountains region of West Texas. Plants in these areas were sampled and chromosome counts and flow cytometry methods were used to assess ploidy levels and genome sizes. Homoploid variation in ge-nome size was unambiguously documented: the genome size of tetraploid plants from the Davis Mountains was significantly larger than that of plants from Caprock. The general condition of larger genome sizes for plants in the Davis Mountains explains previous difficulty in determining ploidy levels within the region. Most plants at the Caprock population appeared to be tetraploid (2n=28), but chromosome counts revealed variants, including some putative pentaploids. Within the Davis Mountains region, both diploid (2n=14) and tetraploid (2n=28) cytotypes were documented, with a parapatric distribution. Overall, this study clarifies patterns of cytotypic diversity in P. nana, highlights an example of infraspecific, homoploid genome size variation, and contributes to a framework for ongoing evolutionary investigation in this study system.

Genome size of Alexandrium catenella and Gracilariopsis lemaneiformis estimated by flow cytometry

Flow cytometry (FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb (1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb (1C) and 112.73 ± 14.00 Mb (1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

The Small Nuclear Genomes of Selaginella Are Associated with a Low Rate of Genome Size Evolution.

The haploid nuclear genome size (1C DNA) of vascular land plants varies over several orders of magnitude. Much of this observed diversity in genome size is due to the proliferation and deletion of transposable elements. To date, all vascular land plant lineages with extremely small nuclear genomes represent recently derived states, having ancestors with much larger genome sizes. The Selaginellaceae represent an ancient lineage with extremely small genomes. It is unclear how small nuclear genomes evolved in Selaginella. We compared the rates of nuclear genome size evolution in Selaginella and major vascular plant clades in a comparative phylogenetic framework. For the analyses, we collected 29 new flow cytometry estimates of haploid genome size in Selaginella to augment publicly available data. Selaginella possess some of the smallest known haploid nuclear genome sizes, as well as the lowest rate of genome size evolution observed across all vascular land plants included in our analyses. Additionally, our analyses provide strong support for a history of haploid nuclear genome size stasis in Selaginella. Our results indicate that Selaginella, similar to other early diverging lineages of vascular land plants, has relatively low rates of genome size evolution. Further, our analyses highlight that a rapid transition to a small genome size is only one route to an extremely small genome.

Genome size and ploidy influence angiosperm species' biomass under nitrogen and phosphorus limitation.

Summary Angiosperm genome sizes (GS) range c. 2400‐fold, and as nucleic acids are amongst the most phosphorus‐ (P) and nitrogen (N)‐demanding cellular biomolecules, we test the hypothesis that a key influence on plant biomass and species composition is the interaction between N and P availability and plant GS.We analysed the impact of different nutrient regimes on above‐ground biomass of angiosperm species with different GS, ploidy level and Grime's C‐S‐R (competitive, stress‐tolerant, ruderal) plant strategies growing at the Park Grass Experiment (Rothamsted, UK), established in 1856.The biomass‐weighted mean GS of species growing on plots with the addition of both N and P fertilizer were significantly higher than that of plants growing on control plots and plots with either N or P. The plants on these N + P plots are dominated by polyploids with large GS and a competitive plant strategy.The results are consistent with our hypothesis that large genomes are costly to build and maintain under N and P limitation. Hence GS and ploidy are significant traits affecting biomass growth under different nutrient regimes, influencing plant community composition and ecosystem dynamics. We propose that GS is a critical factor needed in models that bridge the knowledge gap between biodiversity and ecosystem functioning.

Differential heterochromatin distribution, flow cytometric genome size and meiotic behavior of chromosomes in three Cucurbitaceae species

Basic cytogenetic investigation of economically important members of Cucurbitaceae is a prerequisite to understand their genetic architecture. The study reports detailed chromosome morphometric analyses with fluorescence banding technique, differential distribution of heterochromatin, flow cytometric nuclear DNA contents and meiotic behavior of the native populations of Benincasa hispida, Luffa cylindrica and Trichosanthes dioica in India. In spite of frequent vegetative propagation, different populations of these cultivated cucurbits show regular genetic processes evident from stable chromosome counts, nuclear DNA sizes, ploidy levels and regular meiotic behavior. Distal CMA+ve signals have been scored in two pairs of chromosomes in B. hispida and four pairs of chromosomes in L. cylindrica, representing the GC rich heterochromatic portions in genomes. The presence of terminal DAPI+ve bands in the chromosomes of male and female populations of T. dioica indicates the predominance of AT rich heterochromatin. In addition to DAPI bands, terminal CMA+ve bands in the female plants of T. dioica depict the chromosomal signs of sexual differentiation in this species. The existence of rod bivalent in meiosis of male plants facilitates further approach to characterize the genetics of chromosomal evolution since gross similarity in genome size and karyotypic features of male and female plants advocates genic control of sexual dimorphism in T. dioica.

The rising world of flow cytometric analysis of pollen grains.

Key terms flow cytometry; genome size; plant sciences; pollen grains; pulse analysis FLOW cytometry is nowadays recognized as a vital tool in plant sciences, with more and more applications in both basic and applied research (1). Due to the unique characteristics of plant material, i.e., complex three-dimensional tissues with the presence of rigid cell walls, specific methods had to be developed to produce suspensions of single particles of interest (2). These particles of interest include, nuclei, mitochondria, chloroplasts, and chromosomes, with most of the applications being focused around the estimation of nuclear DNA content (either in absolute or relative amounts), leading to increasing impacts in the fields of plant breeding and population biology. One of the few single particles produced by plants is the pollen grain. Still, most works focused on studying pollen grains have involved the extraction of nuclei or sperm cells, rather than using the intact pollen. This is mostly due to the autofluorescence and/or specific staining of the pollen exine, and to the particle size limitations of some flow cytometers (3). To add to this, isolation of high-quality nuclei from pollen grains is often difficult, at least in comparison with somatic tissues. Together, these difficulties have led to a very minute number of publications involving flow cytometric analysis of pollen grains in the last 25 years.

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species.

Genome size variation in gymnosperms under different growth conditions

The genome is structurally and functionally influenced by ecological factors during adaptive processes. Several natural factors can cause this, and here we present the effects of different growth conditions on the genome size. Genome size assessments were carried out by flow cytometry for a set of 19 taxa for each of three growing conditions: (i) wild plants growing in their natural habitats used as a control group; (ii) potted plants; and (iii) bonsai plants. Our results show a large variation in the genome size of bonsai and potted plants compared to their wild representatives. The most important conclusion is that 1Cx values measured in potted plants can be up to 8.48% lower and in bonsai plants up to 26.83% higher than the values assessed for the respective wild individuals. In the case of Juniperus thurifera, this divergence largely exceeded the genome size variation previously estimated along the natural geographical range of the species. Such deviation from expected values could be interpreted as genuine genome size variation, or to result from biochemical or/and DNA compactness changes triggered by growth conditions. The present results provide evidence for plant response to human-induced environmental changes, thus making the current approach potentially interesting for the prediction of the influence of climate change on plants and for other applications.

Emerging trends in polyploidy research

The Key Word ‘Polyploidy’ (i.e., the presence of more than two chromosome sets/more than two complete genomes in a cell) coined by Winkler in 1916 [25] has lived nearly a Century. Genome sizes in plants are remarkably diverse, with a 2350-fold range from the smallest 63 Mb (in two species of carnivorous Genlisea, G. aurea (2n=approximately 52) and G. margaretae (2n=approximately 40)), to the largest 149 000 MB (in Paris japonica, 2n=8x=40), divided into n=2 to n=aprox. 600 chromosomes [1]. The polyploidy phenomenon has intrigued biologists over the years and many attractive hypotheses have been proposed in an attempt to assign functionality to the increased content of a duplicated genome [14, 19–21].With the advent of newer tools of genetic analysis there has been tremendous resurgence of interest in polyploidy research, and several publishers have brought out special issues of the journals / reference books [4, 6, 12, 17, 18, 22, 24]. Ch romosome doub l i n g i s b r ough t abou t by endoreduplication/2n gametes/inter-genomic hybridization— all constraining genetic stability. That polyploidy can be induced with chemicals such as Colchicine, was first demonstrated by OJ Eigsti in 1935 cited in Blakeslee and Avery [2]. Genome duplication impedes sexual reproduction and may impact growth and development. A change to tetraploid state nearly doubles the cell volume and enhances cell surface area by about 1.5 times, often facilitating development of larger organs in plants. But in animals there is a tendency to preserve the same body size by reducing the overall number of cells, thus indicating differential effect to polyploidy change across the taxonomic groups [13].

Genome size in aquatic and wetland plants: fitting with the large genome constraint hypothesis with a few relevant exceptions

Genome size was assessed by flow cytometry in 55 populations of 53 taxa with different kinds of relationship with freshwater environments (25 populations of aquatic plants, 24 of wetland plants and six occurring both in aquatic and wetland habitats). Nuclear DNA content is provided for the first time for one family, five genera, 28 species, four subspecies and one variety. Around three quarters of the results obtained belong to the lowest levels of nuclear DNA content (very small and small values), which agrees with the large genome constraint hypothesis. Nevertheless, some remarkably high values were also found. Further studies would still be required to increase the set of water-dependent plants with known nuclear DNA amount in view of clearly establishing any relationship between genome size and aquatic habitat. A synthesis of the current knowledge on genome size in water-dependent angiosperms shows a distribution of C-values biased towards very small genomes, a tendency especially strong in wetland plants, where species of 2C < 1 pg are four times more frequent than in angiosperms as a whole.

PAV markers in Sorghum bicolour: genome pattern, affected genes and pathways, and genetic linkage map construction.

Key message 5,511 genic small-size PAVs in sorghum were identified and examined, including the pattern and the function enrichment of PAV genes. 325 PAV markers were developed to construct a genetic map. Presence/absence variants (PAVs) correlate closely to the phenotypic variation, by impacting plant genome sizes and the adaption to the environment. To shed more light on their genome-wide patterns, functions and the possibility of using them as molecular markers, we generated next generation genome sequencing data for four sorghum inbred lines and used associated bioinformatic pipelines to identify small-size PAVs (40–10 kb). Five thousand five hundreds and eleven genic PAVs (40–10 kb) were identified and found to affect 3,238 genes. These PAVs were mainly distributed on the sub-telomeric regions, but the highest proportions occurred in the vicinity of the centromeric regions. One of the prominent features of the PAVs is the high occurrence of long terminal repeats retrotransposons and DNA transposons. PAVs caused various alterations to gene structure, primarily including the coding sequence variants, intron variants, transcript ablation, and initiator codon changes. The genes affected by PAVs were significantly enriched in those involved in stress responses and protein modification. We used 325 PAVs polymorphic between two sorghum inbred lines Ji2731 and E-Tian, together with 49 SSR markers, and constructed a genetic map, which consisted of 10 linkage groups corresponding to the 10 chromosomes of sorghum and spanned 1,430.3 cM in length covering 97 % of the physical genome. The resources reported here should be useful for genetic study and breeding of sorghum and related species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2458-4) contains supplementary material, which is available to authorized users.

The hidden side of plant invasions: the role of genome size.

The ecological role of genome size in plant biology, biogeography, and morphology has garnered increasing attention as the methods and technology associated with measuring cytological characteristics have become more reliable and accessible. However, how plant genome size influences plant invasions and at what stage in the invasion this influence occurs have been little explored. Several large-scale analyses of published data have yielded valuable interspecific comparisons, but experimental studies that manipulate environmental factors are needed, particularly below the species level, to fully understand the role that genome size plays in plant invasion. In this review, we summarize the available knowledge, discuss the integration of genome size data into invasion research, and suggest how it can be applied to detect and manage invasive species. We also explore how global climate change could exert selective pressures on plant populations with varying genome sizes, thereby increasing the distribution range and invasiveness of some populations while decreasing others. Finally, we outline avenues for future research, including considerations of large-scale studies of intraspecific variation in genome size of invasive populations, testing the interaction of genome size with other factors in macroecological analyses of invasions, as well as the role this trait may play in plant-enemy interactions.