The relationship between nuclear DNA content and leaf strategy in seed plants

BACKGROUND AND AIMS: Species' 2C-values (mass of DNA in G(1) phase 2n nuclei) vary by at least four orders of magnitude among seed plants. The 2C-value has been shown to be co-ordinated with a number of other species traits, and with environmental variables. A prediction that species 2C-values are negatively related to leaf life span (LL) and leaf mass per area (LMA) is tested. These leaf traits are components of a major dimension of ecological variation among plant species. METHODS: Flow cytometry was used to measure the 2C-values for 41 Australian seed plant species, 40 of which were new to the literature. Where possible, LL and LMA data from the global literature were combined with 2C-values from our data set and online C-value databases. KEY RESULTS: Across all species, weak positive relationships were found between 2C-values and both LL and LMA; however, these did not reflect the relationships within either angiosperms or gymnosperms. Across 59 angiosperm species, there were weak negative relationships between 2C-values and both LL (r2 = 0.13, P = 0.005) and LMA (r2 = 0.15, P = 0.002). These relationships were the result of shifts to longer LL and greater LMA in woody compared with herbaceous growth forms, with no relationships present within growth forms. It was not possible to explain a positive relationship between 2C-values and LMA (r2 = 0.30, P = 0.024) across 17 gymnosperm species. The 2C-value was not related to LL or LMA either across species within orders (except for LMA among Pinales), or as radiation divergences in a model phylogeny. CONCLUSIONS: Gymnosperms appear to vary along a spectrum different from angiosperms. Among angiosperms, weak negative cross-species relationships were associated with growth form differences, and traced to a few divergences deep in the model phylogeny. These results suggest that among angiosperms, nuclear DNA content and leaf strategy are unrelated.     

Evolution of DNA amounts across land plants (embryophyta)

BACKGROUND AND AIMS: DNA C-values in land plants (comprising bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms) vary approximately 1000-fold from approx. 0.11 to 127.4 pg. To understand the evolutionary significance of this huge variation it is essential to evaluate the phylogenetic component. Recent increases in C-value data (e.g. Plant DNA C-values database; release 2.0, January 2003; http://www.rbgkew.org.uk/cval/homepage.html) together with improved consensus of relationships between and within land plant groups makes such an analysis timely. METHODS: Insights into the distribution of C-values in each group of land plants were gained by superimposing available C-value data (4119 angiosperms, 181 gymnosperms, 63 monilophytes, 4 lycophytes and 171 bryophytes) onto phylogenetic trees. To enable ancestral C-values to be reconstructed for clades within land plants, character-state mapping with parsimony and MacClade was also applied. KEY RESULTS AND CONCLUSIONS: Different land plant groups are characterized by different C-value profiles, distribution of C-values and ancestral C-values. For example, the large ( approximately 1000-fold) range yet strongly skewed distribution of C-values in angiosperms contrasts with the very narrow 12-fold range in bryophytes. Further, character-state mapping showed that the ancestral genome sizes of both angiosperms and bryophytes were reconstructed as very small (i.e. < or =1.4 pg) whereas gymnosperms and most branches of monilophytes were reconstructed with intermediate C-values (i.e. >3.5, <14.0 pg). More in-depth analyses provided evidence for several independent increases and decreases in C-values; for example, decreases in Gnetaceae (Gymnosperms) and heterosperous water ferns (monilophytes); increases in Santalales and some monocots (both angiosperms), Pinaceae, Sciadopityaceae and Cephalotaxaceae (Gymnosperms) and possibly in the Psilotaceae + Ophioglossaceae clade (monilophytes). Thus, in agreement with several focused studies within angiosperm families and genera showing that C-values may both increase and decrease, it is apparent that this dynamic pattern of genome size evolution is repeated on a broad scale across land plants.     

Nuclear DNA content of some important plant species

Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and the horticultural plants should facilitate molecular studies of these crops.     

Estimation of nuclear DNA content of plants by flow cytometry

A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO₄ buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA     

Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species

Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious.     

Flow cytometric analysis of nuclear DNA content in Musa

 Nuclear genome size variation was studied in Musa acuminata (A genome), Musa balbisiana (B genome) and a range of triploid clones differing in genomic constitution (i.e. the relative number of A and B genomes). Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. The A and B genomes of Musa differ in size, the B genome being smaller by 12% on average. No variation in genome size was found among the accessions of M. balbisiana (average genome size 537 Mbp). Small, but statistically significant, variation was found among the subspecies and clones of M. acuminata (ranging from 591 to 615 Mbp). This difference may relate to the geographical origin of the individual accessions. Larger variation in genome size (8.8%) was found among the triploid Musa accessions (ranging from 559 to 613 Mbp). This variation may be due to different genomic constitutions as well as to differences in the size of their A genomes. It is proposed that a comparative analysis of genome size in diploids and triploids may be helpful in identifying putative diploid progenitors of cultivated triploid Musa clones. Statistical analysis of data on genome size resulted in a grouping which agreed fairly well with the generally accepted taxonomic classification of Musa. Received: 11 May 1998 / Accepted: 29 September 1998     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints

BACKGROUND AND AIMS: Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used. METHODS: AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used. KEY RESULTS: Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid. CONCLUSIONS: We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.     

Comparative analysis of DNA nuclear content by flow cytometry on strawberry plants propagated via runners and regenerated from meristem and callus cultures

ABSTRACT

DNA variation may occur in plant species grown either in vivo or in vitro. In this study flow cytometric analyses were undertaken on Fragaria x ananassa Duch. runner plants, and on plants regenerated from callus cultures of leaf explants and from meristem cultures. Our aims were to investigate DNA variation in runner plants of different cultivars, and to compare DNA content in plants of the same cultivar obtained by different propagation procedures (i.e. from meristems or callus cultures). Plants growing in vitro and in the greenhouse were also compared. A good regeneration ability was observed in all the cultivars, with different percentages of shoot formation. No significant differences were detected in multiplication rate and rooting percentage within cultivars. This work documents the occurrence of DNA variations in strawberry plants in vivo and in vitro. Flow cytometric measurements of DNA content showed the presence of 4C nuclei, besides 2C nuclei, in runner plants of cultivar Pajaro. DNA content variations (2C/4C nuclei) were observed in plants regenerated from callus cultures. These variations were lost after transfer of the plants to the greenhouse, except for cultivar Don. The extent of such DNA variations was influenced by genotype. Our study confirms earlier reports indicating that DNA variation induced by in vitro culture could be lost or retained after transfer of the plants to the greenhouse.     

基于流式细胞术的乌饭树核DNA含量(2C-值)测定

核DNA含量(2C-值)是描述植物生物多样性的一个重要特征参数。该研究利用流式细胞仪检测越橘属植物乌饭树核DNA含量,建立了适合乌饭树的流式细胞术测定方法:以野生乌饭树的嫩叶为材料,以已知核DNA含量的水稻品种‘日本晴’为内标,采用GPB解离液,细胞核悬液加入50μL獉mL-1碘化丙啶染色5 min即可上机检测。结果表明:(1) 9个乌饭树单株的核DNA含量平均值为(1.22±0.03) pg,最小值为1.18 pg,最大值为1.27 pg。(2)检测结果与已知的越橘属二倍体植株的2C-值含量相似,且不同地理来源的单株DNA含量没有显著差异(P>0.05),推测这9个单株为二倍体植株。(3)测定的乌饭树核DNA含量(2C-值)可丰富越橘属植物的C-值库;基于流式细胞术建立的乌饭树核DNA含量测定方法可为该属其他植物的相关研究提供借鉴。     

Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.     

Comparison of four nuclear isolation buffers for plant DNA flow cytometry

• Background and Aims DNA flow cytometry requires preparationof suspensions of intact nuclei, which are stained using a DNA-specificfluorochrome prior to analysis. Various buffer formulas weredeveloped to preserve nuclear integrity, protect DNA from degradationand facilitate its stoichiometric staining. Although nuclearisolation buffers differ considerably in chemical composition,no systematic comparison of their performance has been madeuntil now. This knowledge is required to select the appropriatebuffer for a given species and tissue. • Methods Four common lysis buffers (Galbraith's, LB01,Otto's and Tris.MgCl₂) were used to prepare samples from leaftissues of seven plant species (Sedum burrito, Oxalis pes-caprae,Lycopersicon esculentum, Celtis australis, Pisum sativum, Festucarothmaleri and Vicia faba). The species were selected to covera wide range of genome sizes (1·30–26·90pg per 2C DNA) and a variety of leaf tissue types. The followingparameters were assessed: forward (FS) and side (SS) light scatters,fluorescence of propidium iodide-stained nuclei, coefficientof variation of DNA peaks, presence of debris background andthe number of nuclei released from sample tissue. The experimentswere performed independently by two operators and repeated onthree different days. • Key Results Clear differences among buffers were observed.With the exception of O. pes-caprae, any buffer provided acceptableresults for all species. LB01 and Otto's were generally thebest buffers, with Otto's buffer providing better results inspecies with low DNA content. Galbraith's buffer led to satisfactoryresults and Tris.MgCl₂ was generally the worst, although ityielded the best histograms in C. australis. A combined analysisof FS and SS provided a ‘fingerprint’ for each buffer.The variation between days was more significant than the variationbetween operators. • Conclusions Each lysis buffer tested responded to a specificproblem differently and none of the buffers worked best withall species. These results expand our knowledge on nuclear isolationbuffers and will facilitate selection of the most appropriatebuffer depending on species, tissue type and the presence ofcytosolic compounds interfering with DNA staining.     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents

BACKGROUND: Perusing the literature on nuclear 'genome size' shows that the term is not stabilized, but applied with different meanings. It is used for the DNA content of the complete chromosome complement (with chromosome number n), for which others use 'C-value', but also for the DNA content of the monoploid chromosome set only (with chromosome number x). Reconsideration of the terminology is required. AIM: Our purpose is to discuss the currently unstable usage of the terms 'genome size' and 'C-value', and to propose a new unified terminology which can describe nuclear DNA contents with ease and without ambiguity. PROPOSALS: We argue that there is a need to maintain the term genome size in a broad sense as a covering term, because it is widely understood, short and phonetically pleasing. Proposals are made for a unified and consensual terminology. In this, 'genome size' should mean the DNA content based on chromosome number x and n, and should be used mainly in a general sense. The necessary distinction of the kinds of genome sizes is made by the adjectives 'monoploid' and the neology 'holoploid'. 'Holoploid genome size' is a shortcut for the DNA content of the whole chromosome complement characteristic for the individual (and by generalization for the population, species, etc.) irrespective of the degree of generative polyploidy, aneuploidies, etc. This term was lacking in the terminology and is for reasons of linguistic consistency indispensable. The abbreviated terms for monoploid and holoploid genome size are, respectively, Cx-value and C-value. Quantitative data on genome size should always indicate the C-level by a numerical prefix, such as 1C, 1Cx, 2C, etc. The proposed conventions cover general fundamental aspects relating to genome size in plants and animals, but do not treat in detail cytogenetic particularities (e.g. haploids, hybrids, etc.) which will need minor extensions of the present scheme in a future paper.     

Historical relationships of the Mexican cloud forests: a preliminary vicariance model applying Parsimony Analysis of Endemicity to vascular plant taxa

Mexican cloud forests, situated between 600 and 3000 m of elevation, exhibit a remarkable high biotic diversity. They follow a fragmented pattern, similar to that of an archipelago, that makes them suitable to vicariance modelling. A Parsimony Analysis of Endemicity (PAE) was applied to the presence/absence of 1267 species of vascular plants (gymnosperms, angiosperms, and pteridophytes) from twenty-four patches of Mexican cloud forests, in order to postulate a preliminary hypothesis of relationships. The single cladogram obtained grouped the twenty-four cloud forests into five clades. These results indicate that the Sierra Madre Oriental, Sierra Madre del Sur, and Serranías Meridionales floristic provinces do not represent natural units. A preliminary vicariance model is presented to explain the sequence of fragmentation of the Mexican cloud forests.     

Estimation of the nuclear DNA content of gossypium species

BACKGROUND AND AIMS: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices. METHODS: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates. KEY RESULTS: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes. CONCLUSIONS: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.