Relationships between nuclear DNA content and seed and leaf size in soybean

 A correlation between genome size and agronomically important traits has been observed in many plant species. The goal of the present research was to determine the relationship between genome size, seed size, and leaf width and length in soybean [Glycine max (L.) Merr.] Twelve soybean strains, representing three distinct seed size groups, were analyzed. Flow cytometry was used to estimate their 2C nuclear DNA contents. Data on seed size and leaf size of the 12 strains were obtained from 1994 and 1995 field experiments. Variation of 2C nuclear DNA among the 12 soybean strains was 4.6%, ranging from 2.37 pg for a small-seed strain to 2.48 pg for a large-seed strain. Strain seed size was positively associated with leaf width (r=0.92) and leaf length (r=0.93). Genome size was highly correlated with seed size (r=0.97), leaf width (r=0.90) , and leaf length (r=0.93). The results of our study indicate that there is a significant correlation between genome size and leaf and seed size in soybean. It is possible that selection for greater seed size either leads to, or results from, greater genome size. If so, this relationship might be worth exploring at a more fundamental level. Received: 5 April 1997 / Accepted: 9 January 1998     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

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     

Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from seedlings of Brassica rapa L. and B. oleracea L. revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in Brassica rapa, while B. oleracea exhibited a mixture of cells with five ploidy levels, 2C, 4C, 8C, 16C and 32C. The distribution of cells with the different ploidy levels was tissue-specific and characteristic of the stage of development. Multiploidy was not found in the embryos of dry seeds. Rapid endoreduplication occurred during seedling development. It is most probable that multiploidy is, if not a general feature, at least very common in Brassica species. The physiological and genetic implications of this original feature are discussed. Received: 17 March 2000 / Accepted: 17 April 2000     

Flow cytometric evidence for multiple ploidy levels in the endosperm of some gymnosperm species

 Considered to be haploid tissue, the endosperm of coniferous trees has been extensively used by forest geneticists. Using laser flow cytometry, we show that endosperm ploidy level depends on the systematic position. The Abies, Cedrus and Pinus species tested exhibited uniform haploid endosperm compared to the diploid DNA content of the corresponding embryo. Endosperm of Cupressaceae contained multiple ploidy levels: Cupressus arizonica, Juniperus oxycedrus and Thuja orientalis endosperms exhibited a mixture of haploid–diploid nuclei, while C. atlantica and C. sempervirens endosperms contained six ploidy levels: 1C, 2C, 3C, 4C, 5C and 6C. Physiological and genetic implications of this original feature are discussed. Received: 17 August 1996 / Accepted: 18 October 1996     

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.     

Flow cytometric analysis of nuclear DNA inCrocus sativus and allies (Iridaceae)

The genome size and base composition of the triploidCrocus sativus and its two diploid most probable ancestors,C. cartwrightianus andC. thomasii, was investigated and compared inter- and intra-specifically by means of flow cytometry. There was little variation inC. sativus and little difference fromC. cartwrightianus. Crocus thomasii was significantly different from the others.Crocus cartwrightianus is the most probable diploid ancestor ofC. sativus.     

Flow cytometric evidence for endopolyploidization in cabbage (Brassica oleracea L.) flowers

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from flowers of cabbage (Brassica oleracea L.) revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in mature flowers. The distribution of cells with the differerent ploidy levels is tissue-specific and is characteristic of the stage of development. Nuclei of young flower buds exclusively gave 2C and 4C peaks, indicating that the tissues maintained diploid level. Endoreduplication was consistently detected during flower development. Endopolyploidy is probably common in differentiation of cabbage plants. Implications of this original feature are discussed. Received: 28 August 2000 / Revision accepted: 20 December 2000     

新鲜肺癌组织的DNA 含量分析

目的：研究新鲜肺癌组织的DNA含量。方法：采用流式细胞术对30例新鲜肺癌组织和5例正常对照组组织制成的单细胞悬液进行了DNA含量分析。结果：肺癌组G0／G1、S、G2／M各时相比率和细胞增殖指数以及DNA指数与对照组存在显著性差异（P〈0．01）。结论：肺病变组织细胞DNA的流式细胞术分析是判定肺部肿瘤恶性化的敏感指标。     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997     

Physical mapping of the 18S-25S and 5S ribosomal RNA genes in diploid bananas

Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

Comparative analysis of phenotypic and genotypic diversity among plantain landraces (Musa spp., AAB group)

Genetic diversity amongst 76 plantain landraces has been studied using RAPD analysis at two levels of intensity and compared with groupings based on phenotypic indices and morphotype. There was a good correlation (R²=0.78) between estimates of genetic diversity based on 76 RAPD bands and 164 RAPD bands. However, there was a poor correlation between RAPD-based estimates of genetic diversity and a phenotypic index based on agronomic characters. There was also a poor correlation between RAPD analyses and morphotype group (based on bunch type and stature). These results suggest that the traditional designations of plantain landraces based on morphotype do not provide a true reflection of overall genetic divergence. Similarly, classification systems using phenotypic indices based on agronomic characters may not provide accurate taxonomic differentiation. The level of genetic divergence within morphogroups based on bunch type suggests that True Horn plantains are derived from False Horn plantains which in turn are derived from French plantains. Genetic divergence was found to be generally quite low within the plantain landrace genepool, which is consistent with the proposed evolution of this germplasm through somatic mutation of a relatively small number of introductions. However, putative synonyms/duplicates have been shown to be genetically distinct. In contrast, a group of 12 landraces have been identified that are highly distinct from one another (showing 20–35% dissimilarity). Fertile members of this group may be useful for generating genetically diverse 2x and 4x breeding populations that can be used in breeding secondary triploid hybrid plantain varieties. Received: 8 January 2000 / Accepted: 2 March 2000     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum

A DAPI and ethidium bromide flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum was conducted. The material included 38 accessions of P. sativum of widely different geographic origin and altogether 14 samples of P. elatius, P. abyssinicum, P. humile and P. fulvum. The relative genome size values obtained with the three staining methods were strongly correlated. No evidence for genome size variation was found among P. sativum cultivars. In particular, certain Italian cultivars, for which strongly deviating C-values have been reported, proved to be invariant. The only occasion when ambiguous evidence for marginal genome size variation was found was when all 38 accessions taxonomically affiliated with P. sativum were considered. Pisum abyssinicum and P. fulvum differed from P. sativum by about 1.066-and 1.070-fold, respectively; 1 accession of P. humile differed by 1.089-fold, and 2 of P. elatius by 1.122- and 1.195-fold, respectively (ethidiumbromide comparison), while the other accessions of these taxa were not different from P. sativum. This variation may indicate taxonomic inhomogeneity and demands further investigation. Cultivated P. sativum has long been suspected of not being constant with respect to genome size. As shown here, these findings were not based on genuine differences, but rather were technical in origin.     

Flow cytometric analysis of benthic prokaryotes attached to sediment particles

Appropriate detachment treatments are required to analyze prokaryotes associated with streambed sediments by flow cytometry. Using our previously optimized protocol, two groups of cells exhibiting different nucleic acid contents were easily detectable. However, the Nucleic Acid Double Staining assay proved that detachment procedures negatively affect the cell membrane integrity.     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000     

Variation in nuclear DNA content across environmental gradients: a quantile regression analysis

The nuclear DNA content of angiosperms varies by several orders of magnitude. Previous studies suggest that variation in 2C DNA content (i.e. the amount of DNA in G1 phase nuclei, also referred to as the 2C-value) is correlated with environmental factors, but there are conflicting reports in the literature concerning the nature of these relationships. We examined variation in 2C DNA content for 401 species in the ecologically diverse California flora in relation to the mean July maximum temperature, January minimum temperature, and annual precipitation within the geographical ranges of these species. Species with small 2C-values predominate in all environments. Species with large 2C-values occur at intermediate July maximum temperatures, and decline in frequency at both extremes of the July temperature gradient, and with decreasing annual precipitation. Our analysis demonstrates the utility of quantile regression for statistical inference of complex distributions such as these. The method supports our observation that relationships between nuclear DNA content and environmental factors are stronger for species with large 2C-values.     

Unexpected DNA content in the endosperm of Cupressus dupreziana A. Camus seeds and its implications in the reproductive process

 Nuclear DNA content of embryo and endosperm from mature and immature Cupressus dupreziana A. Camus seeds was estimated using laser flow cytometry. Relative DNA content of endosperm nuclei corresponded to four ploidy levels: 2C, 4C, 6C and 8C. The embryo nuclei invariably exhibited a diploid pattern. In all endosperm tissue analyzed no haploid nucleus was found. This is problematic since, in gymnosperms, endosperm and female gametes originate from one functional haploid megaspore produced by meiosis. The possible origin and derivation of C. dupreziana endosperm are discussed in light of previous results concerning the two other Mediterranean cypresses, C. sempervirens and C. atlantica. Received: 15 January 1998 / Revision accepted: 27 March 1998     

Genetic transformation of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' via microprojectile bombardment

 An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into the banana genome. Received: 22 June 1998 / Revision received: 29 March 1999 / Accepted 1 May 1999