Plant Genome Size Research Articles

Does Large Genome Size Limit Speciation in Endemic Island Floras?

Genome sizes in plants vary by several orders of magnitude, and this diversity may have evolutionary consequences. Large genomes contain mainly noncoding DNA that may impose high energy and metabolic costs for their bearers. Here we test the large genome constraint hypothesis, which assumes that plant lineages with large genomes are diversifying more slowly Knight et al. (2005), using endemic floras of the oceanic archipelagos of the Canaries, Hawaii, and Marquesas Islands. In line with this hypothesis, the number of endemic species per genus is negatively correlated with genus-average genome size for island radiations on Hawaiian and Marquesas archipelagos. However, we do not find this correlation on the Canaries, which are close to the continent and therefore have higher immigration rate and lower endemism compared to Hawaii. Further work on a larger number of floras is required to test the generality of the large genome constraint hypothesis.

Exploring giant plant genomes with next-generation sequencing technology

Genome size in plants is characterised by its extraordinary range. Although it appears that the majority of plants have small genomes, in several lineages genome size has reached giant proportions. The recent advent of next-generation sequencing (NGS) methods has for the first time made detailed analysis of even the largest of plant genomes a possibility. In this review, we highlight investigations that have utilised NGS for the study of plants with large genomes, as well as describing ongoing work that aims to harness the power of these technologies to gain insights into their evolution. In addition, we emphasise some areas of research where the use of NGS has the potential to generate significant advances in our current understanding of how plant genomes evolve. Finally, we discuss some of the future developments in sequencing technology that may further improve our ability to explore the content and evolutionary dynamics of the very largest genomes.

Geometrical constraints in the scaling relationships between genome size, cell size and cell cycle length in herbaceous plants.

Plant nuclear genome size (GS) varies over three orders of magnitude and is correlated with cell size and growth rate. We explore whether these relationships can be owing to geometrical scaling constraints. These would produce an isometric GS-cell volume relationship, with the GS-cell diameter relationship with the exponent of 1/3. In the GS-cell division relationship, duration of processes limited by membrane transport would scale at the 1/3 exponent, whereas those limited by metabolism would show no relationship. We tested these predictions by estimating scaling exponents from 11 published datasets on differentiated and meristematic cells in diploid herbaceous plants. We found scaling of GS-cell size to almost perfectly match the prediction. The scaling exponent of the relationship between GS and cell cycle duration did not match the prediction. However, this relationship consists of two components: (i) S phase duration, which depends on GS, and has the predicted 1/3 exponent, and (ii) a GS-independent threshold reflecting the duration of the G1 and G2 phases. The matches we found for the relationships between GS and both cell size and S phase duration are signatures of geometrical scaling. We propose that a similar approach can be used to examine GS effects at tissue and whole plant levels.

C-value reassessment of plant standards: an image cytometry approach

Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max 'Polanka' as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.

The effects of rapid desiccation on estimates of plant genome size

Flow cytometry has become the dominant method for estimating nuclear DNA content in plants, either for ploidy determination or quantification of absolute genome size. Current best practices for flow cytometry involve the analysis of fresh tissue, however, this imposes significant limitations on the geographic scope and taxonomic diversity of plants that can be included in large-scale genome size studies. Dried tissue has been used increasingly in recent years, but largely in the context of ploidy analysis. Here we test rapid tissue drying with silica gel as a method for use in genome size studies, potentially enabling broader geographic sampling of plants when fresh tissue collection is not feasible. Our results indicate that rapid drying introduces comparatively minor error (<10%), which is similar to the error introduced by other common methodological variations such as instrument. Additionally, the relative effect of drying on genome size and data quality varied between species and buffers. Tissue desiccation provides a promising approach for expanding our knowledge of plant genome size diversity.

Organisation of the plant genome in chromosomes

The plant genome is organized into chromosomes that provide the structure for the genetic linkage groups and allow faithful replication, transcription and transmission of the hereditary information. Genome sizes in plants are remarkably diverse, with a 2350-fold range from 63 to 149,000 Mb, divided into n=2 to n= approximately 600 chromosomes. Despite this huge range, structural features of chromosomes like centromeres, telomeres and chromatin packaging are well-conserved. The smallest genomes consist of mostly coding and regulatory DNA sequences present in low copy, along with highly repeated rDNA (rRNA genes and intergenic spacers), centromeric and telomeric repetitive DNA and some transposable elements. The larger genomes have similar numbers of genes, with abundant tandemly repeated sequence motifs, and transposable elements alone represent more than half the DNA present. Chromosomes evolve by fission, fusion, duplication and insertion events, allowing evolution of chromosome size and chromosome number. A combination of sequence analysis, genetic mapping and molecular cytogenetic methods with comparative analysis, all only becoming widely available in the 21st century, is elucidating the exact nature of the chromosome evolution events at all timescales, from the base of the plant kingdom, to intraspecific or hybridization events associated with recent plant breeding. As well as being of fundamental interest, understanding and exploiting evolutionary mechanisms in plant genomes is likely to be a key to crop development for food production.

Comparative analysis of Gossypium and Vitis genomes indicates genome duplication specific to the Gossypium lineage

Genetic mapping studies have suggested that diploid cotton ( Gossypium) might be an ancient polyploid. However, further evidence is lacking due to the complexity of the genome and the lack of sequence resources. Here, we used the grape ( Vitis vinifera) genome as an out-group in two different approaches to further explore evidence regarding ancient genome duplication (WGD) event(s) in the diploid Gossypium lineage and its (their) effects: a genome-level alignment analysis and a local-level sequence component analysis. Both studies suggest that at least one round of genome duplication occurred in the Gossypium lineage. Also, gene densities in corresponding regions from Gossypium raimondii, V. vinifera, Arabidopsis thaliana and Carica papaya genomes are similar, despite the huge difference in their genome sizes and the different number of WGDs each genome has experienced. These observations fit the model that differences in plant genome sizes are largely explained by transposon insertions into heterochromatic regions.

Nuclear DNA amounts in angiosperms: targets, trends and tomorrow

The amount of DNA in an unreplicated gametic chromosome complement is known as the C-value and is a key biodiversity character of fundamental significance with many practical and predictive uses. Since 1976, Bennett and colleagues have assembled eight compilations of angiosperm C-values for reference purposes and subsequently these have been pooled into the Angiosperm DNA C-values Database (http://data.kew.org/cvalues/). Since the last compilation was published in 2005, a large amount of data on angiosperm genome size has been published. It is therefore timely to bring these data together into a ninth compilation of DNA amounts. Scope The present work lists DNA C-values for 2221 species from 151 original sources (including first values for 1860 species not listed in previous compilations). Combining these data with those published previously shows that C-values are now available for 6287 angiosperm species. Analysis of the dataset, which is by far the largest of the nine compilations published since 1976, shows that angiosperm C-values are now being generated at the highest rate since the first genome sizes were estimated in the 1950s. The compilation includes new record holders for the smallest (1C = 0·0648 pg in Genlisea margaretae) and largest (1C = 152·23 pg in Paris japonica) genome sizes so far reported, extending the range encountered in angiosperms to nearly 2400-fold. A review of progress in meeting targets set at the Plant Genome Size meetings shows that although representation for genera, geographical regions and some plant life forms (e.g. island floras and parasitic plants) has improved, progress to increase familial representation is still slow. In terms of technique it is now clear that flow cytometry is soon likely to become the only method available for plant genome size estimations. Fortunately, this has been accompanied by numerous careful studies to improve the quality of data generated using this technique (e.g. design of new buffers, increased awareness and understanding of problems caused by cytosolic inhibitors). It is also clear that although the speed of DNA sequencing continues to rise dramatically with the advent of next-generation and third-generation sequencing technologies, 'complete genome sequencing' projects are still unable to generate accurate plant genome size estimates.

Una herramienta eficaz en el estudio de la Botánica: la citometría de flujo

Over the last decade there has been a tremendous increase in the use of flow cytometry (FCM) in studies on the biosystematics, ecology and population biology of vascular plants. Most studies, address questions related to spatial distribution and evolutionary significance of genome duplication (polyploidy), chromosomal variation (aneuploidy) and variation in genome size. The unsurpassed speed and reliability of estimating differences in nuclear DNA content by FCM paves the way for large-scale surveys at the landscape, population, individual and tissue levels. Another attractive feature of FCM is the possibility of reformulating former taxonomic concepts to propose robust classifications based on a detailed understanding of population structure and phenotypic variation of plant groups under investigation. In this review, special attention is paid to FCM as applied to Botany studies, and some new and less wellknown uses of it for plants will be discussed. It is likely that in the future the use of FCM in studies on taxonomy, ecology and population biology of plants will increase both in scope and frequency. Flow cytometry alone, but especially in combination with other molecular and phenotypic approaches, promises advances in our understanding of the functional significance of variation in genome size in plants.

The right stuff: evidence for an ‘optimal’ genome size in a wild grass population

The immense variation exhibited in plant genome size is remarkable. In flowering plants alone, the observed variation in genome size (i.e. the total amount of DNA in an unreplicated nucleus) is huge, ranging from 0.630 Gbp (billion base pairs) to almost 125 Gbp – a difference of nearly 2000-fold. However, despite this immense variation, the cumulative distribution of genome sizes in plants is significantly skewed towards smaller sizes. A natural corollary is that the skewed distribution is an indication of large genomes being disadvantageous (Knight et al., 2005). It has long been proposed that genome size has measurable phenotypic and ecological consequences which are mediated through its effect on cellular parameters (such as nuclear volume, meiotic and mitotic duration, and cell size), leading some to suggest that genome size can have a nongenic role in the functioning of an organism (Bennett, 1972).

The quest for suitable reference standards in genome size research

in the molecular genomics era, as the flood gates opento deliver a deluge of DNA sequence data, knowledge of thetotal amount of DNA that comprises an organism’s genome(5 genome size) remains vital for many diverse fields of biol-ogy. Indeed, it may be considered to be a key biodiversitycharacter with both practical and biological consequences forthe organism. Knowledge of genome size is important, forexample, in ongoing and planned sequencing programs toassess costs and to know how much DNA to sequence, or fordetermining how many large insert clones (e.g., bacterial arti-ficial chromosomes [BACs] or fosmids) are needed for con-structing genomic libraries. In addition, various genetic finger-printing tools (e.g., microsatellites and AFLPs) have beenshown to be sensitive to genome size so knowledge of thischaracter is important before embarking on such studies.From a biological perspective variation in genome size hasbeen shown to have diverse yet predictable consequences foran organism, influencing, for example, how it will respondto changes in carbon dioxide, rising temperatures, andpollution (1).While people have been estimating genome sizes in plantsand animals for over 50 years, the last decade has seen a hugegrowth in the number of estimates published, not only to pro-vide data for molecular studies but also for large scale compar-ative analyses seeking to understand the biological and evolu-tionary significance of the 40,000-fold range of genome sizesencountered across eukaryotes (2). Indeed, data are now avail-able for over 10,000 species, accessible through the internetin three databases (Animal Genome Size Database—www.genomesize.com; Fungal Genome Size Database—www.zbi.ee/fungal-genomesize; and the Plant DNA C-values Database—http://data.kew.org/cvalues).

Methodology significantly affects genome size estimates: Quantitative evidence using bryophytes

Flow cytometry (FCM) is commonly used to determine plant genome size estimates. Methodology has improved and changed during the past three decades, and researchers are encouraged to optimize protocols for their specific application. However, this step is typically omitted or undescribed in the current plant genome size literature, and this omission could have serious consequences for the genome size estimates obtained. Using four bryophyte species (Brachythecium velutinum, Fissidens taxifolius, Hedwigia ciliata, and Thuidium minutulum), three methodological approaches to the use of FCM in plant genome size estimation were tested. These included nine different buffers (Baranyi's, de Laat's, Galbraith's, General Purpose, LB01, MgSO(4), Otto's, Tris.MgCl(2), and Woody Plant), seven propidium iodide (PI) staining periods (5, 10, 15, 20, 45, 60, and 120 min), and six PI concentrations (10, 25, 50, 100, 150, and 200 microg ml(-1)). Buffer, staining period and staining concentration all had a statistically significant effect (P = 0.05) on the genome size estimates obtained for all four species. Buffer choice and PI concentration had the greatest effect, altering the 1C-values by as much as 8% and 14%, respectively. As well, the quality of the data varied with the different methodology used. Using the methodology determined to be the most accurate in this study (LB01 buffer and PI staining for 20 min at 150 microg ml(-1)), three new genome size estimates were obtained: B. velutinum: 0.46 pg, H. ciliata: 0.30 pg, and T. minutulum: 0.46 pg. While the peak quality of flow cytometry histograms is important, researchers must consider that changes in methodology can also affect the relative peak positions and therefore the genome size estimates obtained for plants using FCM.

Flow cytometric analysis using SYBR Green I for genome size estimation in coffee

Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G 0/ G 1 peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57–2.85%) compared to those stained with propidium iodide (2.75–4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations

Studying the spatial distribution of cytotypes and genome size in plants can provide valuable information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated. Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored. Extensive variation in chromosomes numbers (2n = 2x = 30, 2n = 4x = 60, 2n = 6x = 90 and 2n = 12x =180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n = 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also extensively within diploid (up to 1.17-fold) and tetraploid (1.22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution. Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.

Recent progress in plant genome size evolution

It has been known that eukaryotic genomes span a wide range of sizes regardless of organism complexity. The observed differences in genome size are primarily due to polyploidy level and abundance of non-coding DNA, especially the contribution of transposable elements (TEs). Here we reviewed the current progress in genome size variation of plant species and the underlying evolutionary forces that contribute to genome expansion or contraction. Polyploidization and the accumulation of transposable element are the primary contributors to genome expansion. As to the mechanisms of DNA loss, unequal homologous recombination and illegitimate recombination are thought to be the counterbalances to the unlimited expansion of a genome. The evolutionary direction of plant genome size is also discussed, which tends to favor larger genomes with deletion mechanisms acting to only attenuate genome expansion but not reverse.

Extremely small genomes in two unrelated dipteran insects with shared early developmental traits

We discovered extremely small genomes (1C ~100 Mb) in the dipteran insects Coboldia fuscipes (Scatopsidae) and Psychoda cinerea (Psychodidae). The small genomes of these species cannot be explained by a fast developmental rate, which has been shown to correlate with small genome sizes in animals and plants but might accommodate the combined effects of other developmental traits, including small egg size, thin blastoderm layer, and long-germ development.

Genome sequence comparison of Col and Ler lines reveals the dynamic nature of Arabidopsis chromosomes

Large differences in plant genome sizes are mainly due to numerous events of insertions or deletions (indels). The balance between these events determines the evolutionary direction of genome changes. To address the question of what phenomena trigger these alterations, we compared the genomic sequences of two Arabidopsis thaliana lines, Columbia (Col) and Landsberg erecta (Ler). Based on the resulting alignments large indels (>100 bp) within these two genomes were analysed. There are ∼8500 large indels accounting for the differences between the two genomes. The genetic basis of their origin was distinguished as three main categories: unequal recombination (Urec)-derived, illegitimate recombination (Illrec)-derived and transposable elements (TE)-derived. A detailed study of their distribution and size variation along chromosomes, together with a correlation analyses, allowed us to demonstrate the impact of particular recombination-based mechanisms on the plant genome evolution. The results show that unequal recombination is not efficient in the removal of TEs within the pericentromeric regions. Moreover, we discovered an unexpectedly high influence of large indels on gene evolution pointing out significant differences between the various gene families. For the first time, we present convincing evidence that somatic events do play an important role in plant genome evolution.

Measuring genome size of desert plants using dry seeds

Use of seeds instead of leaves for the flow cytometric measurement of DNA content is of particular interest to botanists and plant ecologists, since it allows estimation of genome sizes for species having reduced leaves or that accumulate staining inhibitors within leaves, and also for species growing in regions where cytometers are not readily available. The seeds of 24 desert species, including wildflowers, cacti, shrubs, and trees were analyzed by flow cytometry. Nuclei were used from either total seeds or seed tissues, following dissection to determine the seed parts that were most suitable for genome size measurement. In addition, the mass of 100 seeds was established. The seeds of 14 species contained only cells occupying a mitotic cell cycle. For 10 other species, endoreplicated nuclei (up to 32C) were detected. Using entire seeds or their parts, it was possible to estimate genome sizes for all of the species, which ranged from 0.79 pg per 2C in Parkinsonia aculeata L. to 26.96 pg per 2C in Agave parryi Engelm., thus this kind of plant material can be used for the cytometric measuring of nuclear DNA content. However, a detailed understanding of seed biology is needed to interpret the results correctly. The relationships among genome size, seed mass, and desert growing conditions are also discussed.

Interspecific hybridisation and genome size analysis in Buddleja

Interspecific hybrids Buddlejadavidii × Buddlejaweyeriana, Buddlejaweyeriana × Buddlejadavidii and Buddlejadavidii × Buddlejalindleyana were generated using in vitro embryo rescue 10–11 weeks after manual pollination. The morphological variation within the F1 populations was limited. The F1 progeny of B. davidii × B. lindleyana was almost sterile and no F2 generation was obtained. From the other hybrids, F2 generations were made by self pollinations and back crosses. Hybrid nature of all F1 and F2 seedlings was confirmed by AFLP. Chromosome counting and genome size measurement for B. weyeriana (F2 selection of (diploid) B. globosa × (tetraploid) B. davidii) revealed a higher chromosome number (76 chromosomes) and genome size than expected, indicating 2n-gametes formation occurred during meiosis of B. globosa. The F1 hybrids B. weyeriana × B. davidii (76 chromosomes) had an intermediate genome size compared with the genome size of the parent plants, proving their hybrid nature. However, the F1 and F2 hybrids of B. davidii × B. weyeriana all had 76 chromosomes but had a lower genome size than expected, suggesting the occurrence of chromosome rearrangements in the genome of the hybrids. B. lindleyana had 38 chromosomes, while the F1 hybrids of B. davidii × B. lindleyana had 76 chromosomes. Also genome size measurements revealed that the F1 seedlings B. davidii × B. lindleyana had higher genome sizes than expected. Both the results of chromosome counting and genome size measurement indicate that 2n-gametes formation took place during meiosis of B. lindleyana.

Book review: Flow cytometry with plant cells: Analysis of genes, chromosomes and genomes

Book review: Flow cytometry with plant cells: Analysis of genes, chromosomes and genomes