Quantity Of Nuclear DNA Research Articles

Assessment of mitochondrial DNA copy number variation relative to nuclear DNA quantity between different tissues

Mitochondrial DNA is a widely tested genetic marker in various fields of research and diagnostics. Nonetheless, there is still little understanding on its abundance and quality within different tissues.Aiming to obtain deeper knowledge about the content and quality of mtDNA, we investigated nine tissues including blood, bone, brain, hair (root and shaft), cardiac muscle, liver, lung, skeletal muscle, and buccal mucosa of 32 deceased individuals using two real-time quantitative PCR-based assays with differently sized mtDNA and nDNA targets.The results revealed that the quantity of nDNA is a weak surrogate to estimate mtDNA quantities among tissues of an individual, as well as tissues across individuals. Especially hair showed extreme variation, depicting a range of multiple magnitudes of mtDNA molecules per hair fragment. Furthermore, degradation can lead to fewer fragments being available for PCR. The results call for parallel determination of the quantity and quality of mtDNA prior to downstream genotyping assays.

Assessing the usefulness of Raman spectroscopy and lipid analysis of decomposed human bones in forensic genetics and molecular taphonomy

Bones are among the structures most likely to be recovered after death. However, the low quantity of preserved DNA and complex processing from sample to DNA profile make forensic DNA analysis of bones a challenging task. Raman spectroscopy and gas chromatography-mass spectrometry (GC/MS), have the potential to be useful as screening tools for DNA analysis and in decomposition studies. The objective of this research was to assess the usefulness of such molecular investigations. Femur samples collected from 50 decomposing human bodies were subjected to Raman spectroscopy and GC/MS. Assessment of nuclear DNA quantity and short tandem repeat (STR) genotyping efficiency were also performed. Raman parameters (crystallinity, carbonate-to-phosphate ratio, mineral-to-matrix ratio) and detected lipids were recorded. Background fluorescence proved problematic for Raman analysis of forensic bones. Regardless, it was not associated with less preserved DNA or less detected STR alleles. Fatty acids, hydrocarbons, and five types of fatty acid methyl esters (FAMEs) were detected. The main phosphate peak position in Raman spectra was significantly correlated with preserved DNA (p = 0.03713), while significantly more STR alleles were detected in bones containing methyl hexadecenoate (p = 0.04236). Detection of FAMEs in the bone matrix suggests a reaction between methanol produced by bacteria and free fatty acids, which are not associated with the level of preservation of endogenous DNA. The techniques assessed have shown to be useful in molecular taphonomy studies and forensic genetics.

Estimation of Nuclear DNA Content in Some Aegilops Species: Best Analyzed Using Flow Cytometry.

The genera Triticum and Aegilops have been considered as the main gene pool of wheat due to their features, such as tolerance of all types of abiotic and biotic stresses. This study was conducted to evaluate the cytogenetic analyses in 115 native and wild populations from eleven Aegilops species using their nuclear DNA quantification. Mean 2C nuclear DNA contents of different ploidy levels in the wild wheat of Turkey and Iran were measured using the flow cytometry technique. The obtained results showed that the mean nuclear DNA content in diploid species varied from 10.09 pg/2C (Ae. umbellulata) to 10.95 pg/2C (Ae. speltoides var. ligustica) in Turkey. In Iranian diploids, the mean nuclear DNA content varied from 10.20 pg/2C (Ae. taushii) to 11.56 pg/2C (Ae. speltoides var. ligustica). This index in the tetraploid species of Turkey varied from 18.09 pg/2C (Ae. cylindrica) to 21.65 pg/2C (Ae. triaristata), and in Iranian species, it was from 18.61 pg/2C (Ae. cylindrica) to 21.75 pg/2C (Ae. columnaris). On the other hand, in the hexaploid species of Turkey, this index varied from 31.59 pg/2C (Ae. crassa) to 31.81 pg/2C (Ae. cylindrica); in the Iranian species, it varied from 32.58 pg/2C (Ae. cylindrica) to 33.97 pg/2C (Ae. crassa). There was a significant difference in the DNA content of Turkey and Iran diploid as well as tetraploid species; however, in hexaploid species, the difference was not significant. It was concluded that the variation in intraspecific genome size was very low in diploid and tetraploid populations; this means that the low variation is not dependent on geographic and climatic parameters. On the other hand, the interspecific variation is significant at the diploid and tetraploid populations. It is generally very difficult to distinguish Aegilops species from each other in natural conditions; meanwhile, in this study, all species could be, easily, quickly and unambiguously, distinguished and separated using the FCM technique.

Comparison of DNA preservation between ribs and vertebrae.

The choice of skeletal element types and their intra-bone parts is important because of differences in DNA preservation, and this must be considered when sampling bones for DNA testing. When incomplete skeletons are found, ribs and vertebrae have been shown to be the most suitable for genetic identification of bones from the torso. This study compares the preservation of DNA between 12th thoracic vertebrae and first ribs to determine which bone type is more suitable for genetic typing. The study analyzed 35 12th thoracic vertebrae and 29 first ribs from one mass grave from the Second World War with commingled skeletal remains excavated. Bone DNA preservation was estimated by measuring nuclear DNA concentration and its degradation and through short tandem repeat (STR) typing success. Previous studies performed on aged skeletal remains have shown that the DNA content of the first ribs and 12th thoracic vertebrae has high intra-bone variability, and this was considered when sampling the bones. After full demineralization extraction, the PowerQuant System (Promega) was used to measure the quantity and quality of DNA, and the GlobalFiler kit (Applied Biosystems) was used for STR typing. The results showed that DNA yield and degradation and STR typing success exhibited no statistically significant difference between first ribs and 12th thoracic vertebrae, and there was no intra-individual difference when comparing only paired bones from the same individuals. Consequently, with intra-bone DNA variability considered, the first ribs or the 12th thoracic vertebrae can be selected when sampling to genetically identify the skeletal remains of highly degraded torsos. HIGHLIGHTS: The first ribs and thoracic vertebrae are the most suitable bones for sampling from the torso. The proximal part of first rib and posterior vertebral column of the 12th thoracic vertebrae yielded the most DNA. The first ribs were compared with the 12th thoracic vertebrae, and the sampling process considered intra-bone DNA variability. The quality and quantity of nuclear DNA and success of STR typing were measured. The first ribs yielded the same DNA yields as well as STR typing success as the 12th thoracic vertebrae. When only the torso is present, it is not of high importance whether the first ribs or the 12th thoracic vertebrae are collected.

Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190–16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140–16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000–0.1 pg/µL (r2 ≥ 0.999). The accuracy was examined using NIST SRM 2372a, and its components A, B, and C were quantified with differences of −29.4%, −35.0%, and −22.0%, respectively, against the mtDNA concentrations calculated from published NIST data. We also examined the specificity of the primers, stability of the reaction mix, precision, tolerance against PCR inhibitors, and cross-reactivity against DNA from various animal taxa. Our newly developed mtDNA quantification method is expected to be useful for forensic mtDNA analysis.

The use of flow cytometry for fungal nuclear DNA quantification.

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscope-based cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement.

Intra-bone nuclear DNA variability in Second World War metatarsal and metacarpal bones.

DNA analysis of Second World War skeletal remains is challenging because of the limited yield of DNA that is usually recovered. Recent forensic research has focused on determining which skeletal elements are superior in their preservation of DNA, and little focus has been placed on measuring intra-bone variability. Metatarsals and metacarpals outperformed all the other bones in DNA yield when analyzing all representative skeletal elements of three Second World War victims, and intra-bone variability was not studied. Soft-tissue remnants were found to contribute to higher DNA yield in trabecular bone tissue. Because metatarsals and metacarpals are composed of trabecular epiphyses and a dense diaphysis, the goal of this study was to explore intra-bone variability in DNA content by measuring nuclear DNA quantity and quality using the PowerQuant System (Promega). A total of 193 bones from a single Second World War mass grave were examined. From each bone, DNA was extracted from the compact diaphysis and from both spongy epiphyses combined. This study confirms higher DNA quantity in epiphyses than diaphyses among all the bones analyzed, and more DNA was obtained from metacarpal epiphyses than from metatarsal epiphyses. Therefore, whenever the possibility for sampling both metacarpals and metatarsals from skeletal remains exists, collecting metacarpals is recommended. In cases in which the hands are missing, metatarsals should be sampled. In any case, epiphyses are a richer source of DNA than diaphyses.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains

DNA yield varies by anatomical region, and the selection of bone types that yield maximum recovery of DNA is important to maximize the success of human identification of skeletal remains. The goal of our study was to explore inter- and intra-individual variation in DNA content by measuring nuclear DNA quantity and quality and autosomal STR typing success to determine the most promising skeletal elements for bone sampling. To exclude the influence of taphonomic issues as much as possible, three complete male skeletons from a single Second World War mass grave were examined and all representative skeletal element types of the human body were analyzed. Forty-eight different types of bones from the head, torso, arm, leg, hand, and foot were sampled from each skeleton, 144 bones altogether. The samples were cleaned, and half a gram of bone powder was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen). DNA content and rates of DNA degradation were determined with the PowerQuant (Promega), and the Investigator ESSplex SE QS (Qiagen) was used for STR typing. The highest-yielding bones mostly produced the most complete STR profiles. Among the skeletal elements containing on average the most DNA and producing the most complete profiles in all three skeletons examined were metacarpals, metatarsals, and the petrous portion of the temporal bone. Metatarsals and metacarpals can easily be sampled without using a saw, thus reducing potential DNA contamination. Skeletons from the Second World War can be used as a model for poorly preserved skeletal remains, and the results of the investigation can be applied for genetic identification of highly degraded skeletal remains in routine forensic casework. Although the research was limited to only three skeletons found in a unique mass grave, the data obtained could contribute to sampling strategies for identifying old skeletal remains. More Second World War skeletons will be analyzed in the future to investigate inter-bone variation in the preservation of DNA.

Karyotype and leaf epidermis histology traits of Digitaria abyssinica (Hochst. Ex A. Rich.) (Poaceae)

Digitaria abyssinica (African couchgrass) has economic potential as forage. This species is taxonomically complex, with a high number of synonyms, basic chromosome number x = 9, and diploid and tetraploid cytotypes. Anatomical, histological, and cytotaxonomic studies may contribute to the taxonomic description of this species and reveal its polyploidization and hybridization events. To this end, this study aimed to characterize the karyotype and leaf epidermis histology of two D. abyssinica genotypes. Chromosome characterization was performed based on chromosome banding using chromomycin A (CMA) and 4,6-diamidino-2-phenylindole (DAPI) and identification of 35S and 5S rDNA sites by fluorescent in situ hybridization. Nuclear DNA quantification was performed by flow cytometry. Paradermal sections were obtained for the leaf epidermis study by light and electron microscopy. The results confirmed that both genotypes are tetraploid (2n = 36). The number and distribution of 35S and 5S rDNA sites suggest the occurrence of postpolyploidization structural chromosomal changes or hybridization processes. Despite intraspecific variation in the number of 5S rDNA sites and CMA+/DAPI+ bands, no changes were identified in karyotypical symmetry or genome size. Leaf epidermis histology traits and cytogenetic data can support breeding programs and germplasm banks in identifying species or cultivars.

Copy numbers of mitochondrial genes change during melon leaf development and are lower than the numbers of mitochondria

Melon is a useful plant species for studying mitochondrial genetics because it contains one of the largest and structurally diverse mitochondrial genomes among all plant species and undergoes paternal transmission of mitochondria. We used droplet digital (dd) PCR in combination with flow cytometric determination of nuclear DNA quantities to determine the absolute per-cell copy numbers of four mitochondrial genes (nad9, rps1, matR, and atp6) across four stages of melon leaf development. The copy numbers of these mitochondrial genes not only varied during leaf development but also differed among each other, and there was no correlation between the copy numbers of the mitochondrial genes and their transcript levels. The gene copy numbers varied from approximately 36.8 ± 4.5 (atp6 copies in the 15th leaf) to approximately 82.9 ± 5.7 (nad9 copies in the 9th leaf), while the mean number of mitochondria was approximately 416.6 ± 182.7 in the 15th leaf and 459.1 ± 228.2 in the 9th leaf. These observations indicate that the leaf cells of melon do not contain sufficient copies of mitochondrial genes to ensure that every mitochondrion possesses the entire mitochondrial genome. Given this cytological evidence, our results indicate that mtDNA in melon exists as a sub-genomic molecule rather than as a single-master circle and that the copy numbers of individual mitochondrial genes may vary greatly. An improved understanding of the molecular mechanism(s) controlling the relative prevalence and transmission of sub-genomic mtDNA molecules should provide insights into the continuity of the mitochondrial genome across generations.

Unconventional vegetables collected in Brazil: chromosome number and description of nuclear DNA content

Abstract The aim of this study was to perform chromosome counts and nuclear DNA quantification of the unconventional vegetables species: bertalha (Basella alba L.), vinagreira verde (Hibiscus sabdariffa L.), azedinha (Rumex acetosa L.), peixinho (Stachys byzantina K. Koch), and capuchinha (Tropaeolum majus L.). Metaphase chromosomes were obtained from the pre-treatment of root meristem with 8-hydroxyquinoline or colchicine and lides were prepared by the flame-drying technique and stained with 5% Giemsa. DNA quantification was performed by flow cytometry. Chromosome number and DNA content (pg) estimated for each species were: Rumex acetosa: 2n=2x=14 and 7.04 pg; Basella alba: 2n=2x=44 and 7.05 pg; Tropaeolum majus: 2n=2x=28 and 2.08 pg; Stachys byzantine: 2n=2x=30 and 1.54 pg, and Hibiscus sabdariffa: 2n=4x=72 and 5.12 pg.

Changing Ploidy as a Strategy: The Irish Potato Famine Pathogen Shifts Ploidy in Relation to Its Sexuality.

The oomycete Phytophthora infestans was the causal agent of the Irish Great Famine and is a recurring threat to global food security. The pathogen can reproduce both sexually and asexually, with high potential to adapt to various environments and great risk to break disease resistance genes in potato. As are other oomycetes, P. infestans is regarded to be diploid during the vegetative phase of its life cycle, although some studies reported trisomy and polyploidy. Using microsatellite fingerprinting, genome-wide assessment of single nucleotide polymorphisms, nuclear DNA quantification, and microscopic counting of chromosome numbers, we assessed the ploidy level of a comprehensive selection of isolates. All progenies from sexual populations of P. infestans in nature were found to be diploid, in contrast nearly all dominant asexual lineages, including the most important pandemic clonal lineages US-1 and 13_A2 were triploid. Such triploids possess significantly more allelic variation than diploids. We observed that triploid genotype can change to a diploid genome constitution when exposed to artificial stress conditions. This study reveals that fluctuations in the ploidy level may be a key factor in the adaptation process of this notorious plant destroyer and imposes an extra challenge to control this disease.

Prediction of autosomal STR typing success in ancient and Second World War bone samples

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.

Interspecific in vitro hybridization in genus Centaurium and identification of hybrids via flow cytometry, RAPD, and secondary metabolite profiles

Most of the species from the genus Centaurium Hill readily hybridize between themselves in natural habitats. Artificial interspecific hybrid plants were obtained under in vitro conditions using reciprocal crosses between 4 species: Centaurium erythraea, C. littorale, C. maritimum, and C. pulchellum. Among 76 crossing experiments performed, 3 types of crosses produced seeds and gave 20 interspecific hybrid plants. Nuclear DNA quantification, fingerprinting of molecular markers, and determination of variation in main secondary metabolite content were employed to confirm their hybrid nature. All 3 approaches were capable of detecting hybrid plants. RAPD molecular markers proved to be the most reliable, always placing the hybrids between their parents in dendrograms, while secoiridoid glycoside content also showed intermediate profiles in the hybrid plants in relation to the parents. Hence, each of the techniques employed can be recommended for elucidating the mechanisms of interspecific hybridization in natural populations of Centaurium species in further studies. This could help to comprehend their breeding system and recognize interspecific hybridization as one of the crucial steps in polyploid formation and speciation in this genus.

Genome size and karyotype diversity in Solanum sect. Acanthophora (Solanaceae)

In order to study the evolution of different cytogenetic characters in species of Solanum sect. Acanthophora in relationship to the known phylogeny for this group, the following techniques were used: CMA/DAPI chromosome banding; fluorescent in situ hybridization with probes for the 18-5.8-26S and the 5S rDNA genes in mitotic chromosomes; nuclear DNA quantification by flow cytometry. Depending on the species, 2–6 of the 12 basic chromosome pairs were identified. The heterochromatic banding patterns were shown to be species-specific. All species presented one chromosome pair bearing a 18-5.8-26S signal and one pair (rarely two) with a 5S signal, the two rDNA sites being non-syntenic. The techniques employed allowed us to establish two species groups within sect. Acanthophora: one with small, symmetric chromosomes, little heterochromatin and lower DNA content, and the other one with larger and more asymmetric chromosomes, more heterochromatin CMA+/DAPI− (associated with NOR or not) and a higher DNA content. An elevated karyotype asymmetry would be associated with a high amount of heterochromatin and a high DNA content. The trend within sect. Acanthophora would be towards a loss of heterochromatin, a reduction of chromosome size, and an increase in symmetry.

A nuclear DNA-based species determination and DNA quantification assay for common poultry species

DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

Quantitative real-time PCR (qPCR) assay for human–dog–cat species identification and nuclear DNA quantification

In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA.We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan® quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human–dog–cat origin with as little as 0.4pg of human and cat nuclear DNA, respectively, and 4.0pg of dog nuclear DNA.

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

BackgroundHigh throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.ResultsWe describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.ConclusionsExtracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells

Plant mitochondrial genomes are split into sub-genomes, i.e. genes are distributed across various sub-genomic molecules. To investigate whether copy numbers vary between individual mitochondrial genes, we used quantitative real-time PCR in combination with flow cytometric determination of nuclear DNA quantities to determine absolute per-cell-copy numbers of four mitochondrial genes in various Arabidopsis organs and the leaves of tobacco (Nicotiana tabacum) and barley (Hordeum vulgare). The copy numbers of the investigated mitochondrial genes (atp1, rps4, nad6 and cox1) not only differed from each other, but also varied between organs and changed during the development of cotyledons and leaves in Arabidopsis. We found no correlation between altered gene copy numbers, transcript levels and O(2) consumption. However, per cell, both the number of mitochondria and the number of gene copies increased with growing cell size. Gene copy numbers varied from approximately 40 (cox1 in young leaves) to approximately 280 (atp1 in mature leaves), and the mean number of mitochondria was approximately 300 in young leaves and 450 in mature leaves. Thus, cells are polyploid with respect to their mitochondrial genomes, but individual mitochondria may contain only part of the genome or even no DNA at all. Our data supports structural models of the mitochondrial genome in non-dividing cells of angiosperms that predict localization of the genes on sub-genomic molecules rather than master chromosomes. The data indicate control of the number of individual genes according to the genotype and developmental program(s) via amplification and/or degradation of sub-genomic molecules.

Change of DNA Content in Male and Female Gametes of Tobacco (<I>Nicotiana tabacum</I>)

The nuclear DNA content of male and female gametes of tobacco(Nicotiana tabacum) was measured using DAPI stain and microspectrofluorimetric measurement. Tobacco pollen is bicellular at anthesis,containing a vegetative cell and a generative cell,which divides to form two sperm cells in a pollen tube. The nuclear DNA content of generative cell in a pollen tube was at 2C level and that of two sperm cells in a pollen tube,which elongated in the style,at 1C level. Two sperm cells began to synthesize DNA after both were released in the degenerated synergid,and the quantity of nuclear DNA in both sperm cells approached 2C level before both fusing with egg and central cells. During this process,the nuclear DNA content of egg cell also began to increase and approached 2C level before fusing with sperm cell. After male and female gamete fused,the nuclear DNA content of zygote reached 4C level. Therefore,the fusion of male and female gametes of tobacco was at G2 of cell cycle,and this fusion of fertilization belonged to G2 type. This result displayed the fertilization multiformity in angiosperms.