Determination of genome size variations among different date palm cultivars (Phoenix dactylifera L.) by flow cytometry.

Date palm is an important fruit crop and member of palm family, reported with varied ploidy levels, i.e., 14 (2n = 28) to 18 (2n = 36) pairs of chromosomes and genomic size due to the limited work done on its cytological aspect. The amount of nuclear DNA content is extremely important to understand the hereditary constituent of any species. Hence, the present study was conducted with the aim to estimate the ploidy level and especially the genomic size (C value) of date palm by studying fifty date palm cultivars with flow cytometry using propidium iodide (PI) as the fluorescent dye. The maize genome size (5.14 pg) was used as the internal reference. The results obtained regarding genomic size of date palm cultivars significantly varied and ranged from 1.59 to 1.84 pg 2C-1 or 780.11 to 903.06 Mbp when converted into base pairs of DNA. The average genome size of studied cultivars was observed to be 1.726 2C-1, while, that of nuclei significantly varied from cultivar to cultivar and appeared independent to the genomic size of the studied cultivars. Intraspecific variations were not detected among five exotic cultivars (Amber, Sugae, Medjool, Safawi and Ajwa) grown in Pakistan in relation to their place of origin. The cluster analysis exhibited two main groups of cultivars, the first group comprised 65.3% (33 cultivars) and the second group contained 35.7% (17 cultivars). In addition, no ploidy was observed among all the studied cultivars. The findings regarding the estimated genomic sizes of studied cultivars may be helpful in understanding date palm genetics, breeding and genome sequencing programs further.

Assessment of the potential for gene flow from transgenic maize (Zea mays L.) to eastern gamagrass (Tripsacum dactyloides L.).

Eastern gamagrass (Tripsacum dactyloides L.) belongs to the same tribe of the Poaceae family as maize (Zea mays L.) and grows naturally in the same region where maize is commercially produced in the USA. Although no evidence exists of gene flow from maize to eastern gamagrass in nature, experimental crosses between the two species were produced using specific techniques. As part of environmental risk assessment, the possibility of transgene flow from maize to eastern gamagrass populations in nature was evaluated with the objectives: (1) to assess the seeds of eastern gamagrass populations naturally growing near commercial maize fields for the presence of a transgenic glyphosate-tolerance gene (cp4 epsps) that would indicate cross-pollination between the two species, and (2) to evaluate the possibility of interspecific hybridization between transgenic maize used as male parent and eastern gamagrass used as female parent. A total of 46,643 seeds from 54 eastern gamagrass populations collected in proximity of maize fields in Illinois, USA were planted in a field in 2014 and 2015. Emerged seedlings were treated with glyphosate herbicide and assessed for survival. An additional 48,000 seeds from the same 54 eastern gamagrass populations were tested for the presence of the cp4 epsps transgene markers using TaqMan® PCR method. The results from these trials showed that no seedlings survived the herbicide treatment and no seed indicated presence of the herbicide tolerant cp4 epsps transgene, even though these eastern gamagrass populations were exposed to glyphosate-tolerant maize pollen for years. Furthermore, no interspecific hybrid seeds were produced from 135 hand-pollination attempts involving 1529 eastern gamagrass spikelets exposed to maize pollen. Together, these results indicate that there is no evidence of gene flow from maize to eastern gamagrass in natural habitats. The outcome of this study should be taken in consideration when assessing for environmental risks regarding the consequence of gene flow from transgenic maize to its wild relatives.

Meiotic pairing as an indicator of genome composition in polyploid prairie cordgrass (Spartina pectinata Link).

The existence of neopolyploidy in prairie cordgrass (Spartina pectinata Link) has been documented. The neohexaploid was discovered coexisting with tetraploids in central Illinois, and has been reported to exhibit competitiveness in the natural environment. It is hypothesized that the natural tetraploid cytotype produced the hexaploid cytotype via production of unreduced gametes. Meiosis I chromosome pairing was observed in tetraploid (2n = 4x = 40), hexaploid (2n = 6x = 60), and octoploid (2n = 8x = 80) accessions and the percentage of meiotic abnormality was determined. Significant differences in meiotic abnormality exist between tetraploid, hexaploid, and octoploid cytotypes. An elevated incidence of abnormal, predominantly trivalent pairing in the neohexaploid suggests that it may possess homologous chromosomes in sets of three, in contrast to the tetraploid and octoploid cytotypes, which likely possess homologous chromosomes in sets of two. Abnormal chromosome pairing in the hexaploid may result in unequal allocation of chromosomes to daughter cells during later stages of meiosis. Chromosome pairing patterns in tetraploid, hexaploid, and octoploid cytotypes indicate genome compositions of AABB, AAABBB, and AABBA'A'B'B', respectively.

Transcriptome Analysis of Spartina pectinata in Response to Freezing Stress.

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation.

Insight into genomic changes accompanying divergence: genetic linkage maps and synteny of Lucania goodei and L. parva reveal a Robertsonian fusion.

Linkage maps are important tools in evolutionary genetics and in studies of speciation. We performed a karyotyping study and constructed high-density linkage maps for two closely related killifish species, Lucania parva and L. goodei, that differ in salinity tolerance and still hybridize in their contact zone in Florida. Using SNPs from orthologous EST contigs, we compared synteny between the two species to determine how genomic architecture has shifted with divergence. Karyotyping revealed that L. goodei possesses 24 acrocentric chromosomes (1N) whereas L. parva possesses 23 chromosomes (1N), one of which is a large metacentric chromosome. Likewise, high-density single-nucleotide polymorphism-based linkage maps indicated 24 linkage groups for L. goodei and 23 linkage groups for L. parva. Synteny mapping revealed two linkage groups in L. goodei that were highly syntenic with the largest linkage group in L. parva. Together, this evidence points to the largest linkage group in L. parva being the result of a chromosomal fusion. We further compared synteny between Lucania with the genome of a more distant teleost relative medaka (Oryzias latipes) and found good conservation of synteny at the chromosomal level. Each Lucania LG had a single best match with each medaka chromosome. These results provide the groundwork for future studies on the genetic architecture of reproductive isolation and salinity tolerance in Lucania and other Fundulidae.

Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan.

PREMISE OF THE STUDY: Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus ×giganteus, a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus sacchariflorus and Miscanthus sinensis, for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. sacchariflorus and diploid M. sinensis in Japan. METHODS: Seeds were counted and DNA content was estimated by flow cytometry for plants of M. sacchariflorus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants. KEY RESULTS: Based on seed-set data, M. sacchariflorus has significantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. sacchariflorus plants in southern Japan. CONCLUSIONS: This is the first report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. ×giganteus, these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Out of the swamp: unidirectional hybridization with weedy species may explain the prevalence of Amaranthus tuberculatus as a weed.

*Amaranthus tuberculatus represents one of the most dramatic cases of weed invasion documented in the midwestern USA. The species is infamous for evolving resistance to multiple herbicides, and predicting whether these resistances may be transferred to widespread weeds of the Amaranthus hybridus aggregate is a matter of epidemiological concern. Here, we explore the patterns of genetic exchange between Amaranthus tuberculatus and A. hybridus in an effort to understand whether allele introgression occurs throughout the genome and if fecundity penalties are associated with genetic exchange. *We evaluated 192 homoploid BC(1)s at 197 amplified fragment length polymorphism (AFLP) loci, as well as two loci associated with herbicide resistance: ALS and PPO. We also assessed the fecundity of each genotype by evaluation of seed production or pollen development. *It was discovered that genetic exchange between the species is unidirectional. Whereas A. hybridus alleles transfer with little or no penalty to A. tuberculatus, the reciprocal exchange is significantly distorted and potentially of limited evolutionary consequence. *Our previous hypothesis suggesting unidirectional introgression at ALS owing to circumstantial linkage is now modified to account for the more generalized distortion of genetic exchange observed in this study.

Genome size and nucleotypic variation in Malus germplasm.

The genus Malus has anywhere between 25 and 33 species along with several subspecies. Malus species as well as clones within the same species have varying ploidy levels, as these are more than likely collected from different trees and (or) from different locations. In recent years, large numbers of Malus germplasm accessions have been collected and maintained at the United States National Germplasm Clonal Repository; however, genome sizes of this material have not yet been determined. In this study, leaf tissues from young grafted trees of 100 Malus species and hybrids growing in a nursery at the University of Illinois were collected and immediately used for extracting nuclei. Leaf tissues from apple and maize line W-22, used as an internal standard, were co-chopped and prepared for flow cytometric analysis. Apple nuclei were stained with propidium iodide, an intercalating dye, and a minimum of 8000 nuclei per sample were analyzed. Mean fluorescence of apple nuclei was then determined. A total of four replications per sample was used. Among 100 Malus accessions analyzed, one tetraploid, three triploid, and 96 diploid genotypes were identified. Significant differences in genome size were identified among the three ploidy types observed and also within diploid genotypes. The 2C mean value for tetraploids was 3.13 pg and ranged from 2.27 to 2.41 pg for triploids, whereas 2C values for diploids ranged between 1.44 and 1.72 pg. In addition, leaf impressions of young, fully expanded leaves were collected from young trees of 10 selected genotypes based on their ploidy and flow cytometric analysis and used to measure the nucleotypic parameter stomatal length. Ten stomata were measured per slide, three slides were analyzed per leaf, and three leaves were analyzed per accession. Overall, mean length of stomata ranged between 19.47 microm (diploid) and 27.6 microm (tetraploid), indicating that stomatal length in a tetraploid Malus genotype was 1.4-fold higher than that of a diploid genotype. A positive correlation between genome size and the nucleotypic parameter stomatal length was observed.

Saw palmetto extract induces nuclear heterogeneity in mice.

Saw palmetto (SW), a phytotherapeutic compound used in the treatment of prostate disease, was examined for potential nuclear effects. SW extract was incorporated into a complete casein-based semisynthetic rodent chow at 0%, 0.1% and 1% SW. SW was fed to mice for 6 weeks, after which the mice received a single i/p injection of either the known genotoxic agent methyl methanesulfonate (MMS) in saline or just saline. Forty-eight hours after injection, blood and bone marrow were collected for flow cytometric analysis. A significant effect of MMS was observed in both male and female mice with respect to: an increase in nuclear heterogeneity in bone marrow cells as measured by the coefficient of variation of the G1 peak in a flow histogram (6.32 versus 4.8 in male mice, 7.0 versus 4.9 in female mice) and an increase in the number of micronucleated blood cells (3.4% versus 0.56% male mice, 3.1% versus 0.6 in female mice) indicating a positive genotoxic response. SW also appears to increase the heterogeneity of bone marrow nuclei in a dose dependent manner (0-5.1%, 0.1-5.5% and 1-5.7% in male mice, 0-5.7%, 0.1-6.0% and 1-6.2% in female mice) without a concomitant increase in blood cell micronuclei. These results indicate that SW is not genotoxic with respect to physical DNA damage and that the changes observed in the bone marrow are due to chromatin conformation modifications in the nuclei of in vivo treated mouse cells.

PRINS-labeled knobs are not associated with increased chromosomal stickiness in the maize st1 mutant.

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.

Aquatic herbicides and herbicide contaminants: In vitro cytotoxicity and cell-cycle analysis.

Concerns have arisen about the possible effects of herbicide contamination in aquatic ecosystems. Crop herbicides are introduced into the aquatic environment both inadvertently through runoff events and intentionally through the use of those registered for use in waterways. Acetochlor and atrazine are two agricultural crop herbicides that have often been reported to contaminate waters. Diquat and fluridone are both registered aquatic management herbicides. In this study, a mammalian in vitro cell cytotoxicity assay was used to evaluate the cytotoxicity of these four commonly used herbicides. The ranked order of the cytotoxicity was: diquat (C(1/2) = 0.036 mM +/- 0.011) > acetochlor (C(1/2) = 0.060 mM +/- 0.010) > fluridone (C(1/2) = 0.172 mM +/- 0.029) atrazine (C(1/2) = 0.581 mM +/- 0.050). In addition, flow cytometric analysis was conducted on CHO cells to investigate the potential impact of these four herbicides on the cell cycle. Acetochlor and diquat had the greatest impact on the cell cycle. Acetochor exposure resulted in a decreased number of cells in the G1 phase of the cell cycle, whereas diquat exposure resulted in a decreased number of cells in both the G1 and G2 phases. Both atrazine and fluridone resulted in a decrease in cells in the G2 phase. The agricultural crop herbicides and aquatic management herbicides gave similar results in cytotoxicity and in the cell-cycle assay at the end points tested.

Differential metamorphosis alters the endocrine response in anuran larvae exposed to T3 and atrazine.

Pesticide chemical contamination is one of the suspected contributors of the amphibian population decline. The herbicide atrazine is one of the major surface water contaminants in the U.S. A previous study has shown that atrazine at concentrations as low as 100 parts per billion (ppb) increased the time to metamorphosis in Xenopus laevis tadpoles. However, questions remain as to the applicability of a study of a non-native species to a native organism. The possible effects of atrazine on developing Bufo americanus were explored. Atrazine at potentially (albeit high) environmental concentrations was found not to delay the metamorphosis of developing B. americanus tadpoles as observed in X. laevis. Several studies have indicated that atrazine affects thyroid hormones. Since thyroid hormones are critical in amphibian metamorphosis, B. americanus and X. laevis tadpoles were exposed to exogenous 3,5,3'-triiodothyronine (T3). X. laevis were found to be more responsive to the effects of exogenous T3 compared to B. americanus, indicating that X. laevis may be more sensitive to endocrine active chemicals than B. americanus. In X. laevis, nuclear heterogeneity has been associated with metamorphosis. Flow cytometric analysis of the nuclei of normal metamorphing B. americanus indicates a decrease in the amount of thyroid mediated chromatin alterations relative to the nuclei of metamorphing X. laevis. Indications are that the differential response to endocrine disruption is due to the differential role of chromatin associated gene expression during metamorphosis of B. americanus versus X. laevis. A second native species, Hyla versicolor, was observed to have the X. laevis nuclear pattern with respect to metamorphosis. As such, sensitivity to endocrine disruption is hypothesized not to be limited to laboratory non-native species.

Developmental impact of atrazine on metamorphing Xenopus laevis as revealed by nuclear analysis and morphology.

Atrazine is one of the major surface water contaminants in the midwestern United States. Speculations have arisen on the potential effects of atrazine contamination to anuran larvae developing in these surface waters. In this study, Xenopus laevis tadpoles were exposed to environmentally relevant concentrations of atrazine. Nuclear and morphological endpoints were used to assess the effects of atrazine on developing X. laevis. Atrazine significantly affected metamorphing X. laevis after three-weeks exposure compared to controls as revealed by flow cytometric nuclear DNA analysis. The flow cytometric analysis was reflective of developmental effects. The number of nuclei per organism also was analyzed. Nuclei number was found to be associated with X. laevis development. Nuclei counting showed significant effects of atrazine after five-weeks exposure. A third endpoint, Nieuwkoop and Faber (NF) morphological staging, also demonstrated that atrazine significantly affected development after four weeks. Atrazine was found to alter the timing of metamorphosis of X. laevis using both nuclear analysis and gross morphology. The NF staging was found to be a sensitive assay to measure effects of development, whereas flow cytometry provided an impartial quantitative measure.

Variation in nuclear DNA content in Malus species and cultivated apples.

The nuclear DNA content for a group of 40 Malus species and hybrids has been estimated using flow cytometry. Estimates of nuclear DNA content for this germplasm collection range from 1.45 pg for Malus fusca (diploid) to 2.57 pg for Malus ioensis (triploid). Among diploids, the nuclear (2C) DNA ranges from 1.45 pg for M. fusca to 1.68 pg for Malus transitoria. Among triploids, the nuclear (3C) DNA content ranges from 2.37 pg / 3C for Malus sikkimensis to 2.57 pg / 3C for M. ioensis. Given the complexity of the apple genome and its suggested allopolyploid origin, the results obtained in this study confirm earlier reports that polyploids can easily withstand the loss of a certain amount of DNA, and that there is a slight tendency towards diminished haploid nuclear DNA content with increased polyploidy.

Metamorphosis in Xenopus laevis is not associated with large-scale nuclear DNA content variation.

Amphibian metamorphosis is a complex process that has been speculated to involve DNA amplification and chromatin rearrangement. While recent studies have concentrated on chromatin rearrangement, only a few studies have dealt with variation in the amount of DNA during amphibian metamorphosis. In this study, nuclei were isolated from Xenopus laevis at various developmental stages. The nuclei were examined in both an unfixed state and a fixed state. The nuclei were stained with propidium iodide and analyzed by flow cytometry to determine their fluorescence intensity. The unfixed nuclei had higher fluorescence variation compared with the fixed nuclei. This increase in variation appeared due to the presence of nuclei of variable fluorescence intensity within the unfixed nuclei. Upon optimum fixation, which has been speculated to result in more homogeneous chromatin conformation and to reduce staining artifacts, the nuclei were observed to have less fluorescence intensity variation. The differential fluorescence observed in this study is consistent with the hypothesis that large-scale intra-individual DNA variation is not associated with amphibian metamorphosis.

Evaluation of EMS-induced DNA damage in the single cell gel electrophoresis (Comet) assay and with flow cytometric analysis of micronuclei.

Toxic agents in the environment pose serious threats to ecosystems and to the public health. The single cell gel electrophoresis (SCGE) or Comet assay quantitatively measures genomic damage as DNA strand breaks. The micronucleus (MCN) test is an established assay that measures chromosomal damage. Micronuclei are formed from chromosome fragments or from whole chromosomes that have not undergone mitosis properly. This test is usually conducted microscopically. However, micronuclei can also be analyzed using flow cytometry. Chinese hamster ovary (CHO) cells were exposed to ethylmethanesulfonate (EMS), for 4 h in a total volume of 25 microl. These cells were immediately analyzed for genomic DNA damage by SCGE. In concurrent parallel experiments, CHO cells were treated with EMS in 6-well plates for 4 h, the cells were washed and fresh medium was added. The cells were allowed to grow for 45 to 48 h to express micronuclei. The data demonstrated that both DNA strand breaks and micronuclei were induced in a significant and concentration-dependent manner. There was a significant and high correlation (r = 0.91; P < or = 0.001) between the acute induction of DNA strand breaks and the subsequent generation of micronuclei. These data indicate that using molecular and computer technologies, the genotoxic impact of toxic and environmental agents can be rapidly and comprehensively evaluated in mammalian cell systems.

Flow cytometric analyses of intraplant nuclear DNA content variation induced by sticky chromosomes.

BACKGROUND: In several plant species, sticky chromosomes are a consequence of genetic mutations or environmental effects on mitosis and meiosis. Sticky chromosomes result in an unequal distribution of genetic material in daughter cells. This unequal distribution is hypothesized to result in an increase in the coefficient of variation (CV) of the G1 peak of dividing cells. METHODS: The st1 mutant and a nonmutant line in the same genetic background of maize (Zea mays L.) were planted in a soilless mix. A wheat (Triticum aestivum L. em thell.) line was grown in both low and high aluminum-saturated soil. Both plant species were assessed for sticky chromosomes by Feulgen-stained mitotic analysis and flow cytometric analysis of propidium iodide (PI)-stained G1 nuclei. RESULTS: In the st1 mutant, a significant increase in the number of abnormal anaphase figures was observed. An increase in abnormal mitotic figures was observed in wheat plants grown in aluminum soil. Using flow cytometry, an increase in the CV of the G1/G0 peak was seen in the maize mutant and in wheat grown at high levels of aluminum saturation. This increase correlated with the number of abnormal anaphase cells observed. CONCLUSIONS: Flow cytometry was sensitive enough to detect the intraplant nuclear DNA variation associated with sticky chromosomes within a plant.