Differences In DNA Content Research Articles

Genetic diversity and comparative study of genomic DNA extraction protocols in Tamarix L. species

The genus Tamarix consists of about 54 species that mainly grow in saline areas of deserts and semi-deserts. This genus is chemically characterized by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. Thus it is necessary to optimize extraction protocols to minimize the influence of these compounds to the lowest level. The present study compares the efficiency of five different approaches to extract total genomic DNA in Tamarix species, showing significant differences in the extracted DNA contents and quality,by using Kit (DNP TM Kit), CTAB DNA extraction method by Murray and Thompson, Sahu et al., Nalini et al. and Bi et al., for the extraction of DNA from Tamarix species. Our results showed significant differences in DNA contents between these five methods. The quantity and quality of extracted genomic DNA were checked by the spectrophotometer, Nano-Drop and and agarose gel electrophoresis analysis. Finally, a PCR-based method was also applied to verify the amplification efficiency for two molecular markers (ITS and ISSR).. In the present study, the genetic diversity of 96 Tamarix individuals species and 8 populations were studied using 10 ISSR markerswhile for nrDNA ITS 8 species samples were used. The method of Nalini et al., provided best results (207 ng/μL) in terms of quantity and quality ofDNA. Our results proposed that this method could be effective for plants with the same polysaccharides, proteins and polyphenols components. The advantage of this method is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, and also do not requires expensive chemicals such as proteinase K, liquid nitrogen. ,. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Tamarix species group and the reliability of this method has been discussed.

Whole genome sequences from non-invasively collected caribou faecal samples

Conservation genomics is an important tool to manage threatened species under current biodiversity loss. Recent advances in sequencing technology mean that we can now use whole genomes to investigate demographic history, local adaptation, inbreeding, and more in unprecedented detail. However, for many rare and elusive species only non-invasive samples such as faeces can be obtained, making it difficult to take advantage of whole genome data. We present a method to extract DNA from the mucosal layer of faecal samples to re-sequence high coverage whole genomes using standard laboratory techniques. We use wild collected faecal pellets collected from caribou (Rangifer tarandus), a species undergoing declines in many parts of its range in Canada and subject to comprehensive conservation and population monitoring measures. We compare four faecal genomes to two tissue genomes sequenced in the same run. Quality metrics were similar between faecal and tissue samples with the main difference being the alignment success of raw reads to the reference genome due to differences in low quality and endogenous DNA content, affecting overall coverage. One of our faecal genomes was only re-sequenced at low coverage (1.6 ×), however the other three obtained between 7 and 15 ×, compared to 19 and 25 × for the tissue samples. We successfully re-sequenced high-quality whole genomes from faecal DNA and are one of the first to obtain genome-wide data from wildlife faecal DNA in a non-primate species. Our work represents an important advancement for non-invasive conservation genomics.

Impact of Amplification Efficiency Approaches on Telomere Length Measurement via Quantitative-Polymerase Chain Reaction.

Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio.Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1–72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring.Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10–2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software.Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

Fecal microbiota transplantation does not alter bacterial translocation and visceral adipose tissue inflammation in individuals with obesity.

AimsVisceral adipose tissue inflammation is a fundamental mechanism of insulin resistance in obesity and type 2 diabetes. Translocation of intestinal bacteria has been suggested as a driving factor for the inflammation. However, although bacterial DNA was detected in visceral adipose tissue of humans with obesity, it is unclear to what extent this is contamination or whether the gut microbiota is causally involved. Effects of fecal microbiota transplantation (FMT) on bacterial translocation and visceral adipose tissue inflammation in individuals with obesity and insulin resistance were assessed.Material and MethodsEight individuals with clinically severe obesity (body mass index [BMI] >35 kg/m2) and metabolic syndrome received lean donor FMT 4 weeks prior to elective bariatric surgery. The participants were age‐, sex‐, and BMI‐matched to 16 controls that underwent no fecal transplantation. Visceral adipose tissue was collected during surgery. Bacterial translocation was assessed by 16S rRNA gene sequencing of adipose tissue and feces. Pro‐inflammatory cytokine expression and histopathological analyses of visceral adipose tissue were performed to assess inflammation.ResultsFecal microbiota transplantation significantly altered gut microbiota composition. Visceral adipose tissue contained a very low quantity of bacterial DNA in both groups. No difference in visceral bacterial DNA content between groups was observed. Also, visceral expression of pro‐inflammatory cytokines and macrophage infiltration did not differ between groups. No correlation between inflammatory tone and bacterial translocation was observed.ConclusionsVisceral bacterial DNA content and level of inflammation were not altered upon FMT. Thus, bacterial translocation may not be the main driver of visceral adipose tissue inflammation in obesity.

P–057 Preliminary evaluation of laser confocal Raman spectroscopy as a noninvasive method for detecting sperm chromosome aneuploidy

Abstract Study question To evaluate the efficiency and accuracy of Raman microspectra in detecting sperm chromosome balance state by DNA content difference. Summary answer Raman spectroscopy can identify the difference of X and Y sperm DNA content, but the accuracy still needed to be improved for clinical application. What is known already Aneuploid sperm fertilization affects embryo quality and leads to the waste of oocytes in Assisted Reproductive Technology (ART). Raman spectroscopy can identify substances and observe molecular changes through specific spectral patterns with high specificity and has become a new hot spot in ART. Previous research has used this technology to detect embryo culture medium to evaluate the aneuploidy of embryos. The DNA content of X and Y in sperm was different, which may serve as a marker for sperm aneuploidy detection by Raman spectroscopy. Study design, size, duration The significant difference in the morphology of the sex chromosomes of X and Y spermatozoa leads to a substantial difference in the DNA content. We perform Raman spectroscopy to identify the spectral differences of the sperms, especially the differences in sperm DNA content. We further verified the accuracy with fluorescence in situ hybridization (FISH). Participants/materials, setting, methods Spermatozoa were provided by healthy donors with normal aneuploidy, and analysis parameters met the current World Health Organization (WHO, 2010) standards. Sperm heads were detected by laser confocal Raman spectroscopy and obtained the corresponding spectra. The sperm chromosome information was classified by Standard principal component analysis (PCA) and identified by fluorescence in situ hybridization (FISH). Student’s t-test and Receiver operating characteristic (ROC) curve analysis was performed for further analysis. Main results and the role of chance Standard principal component analysis (PCA) after unqualified quality control divided spermatozoa into two groups according to the calculation and calibration results, 22 cases in group A and 31 cases in group B. Then, we conducted frequency distribution histogram statistics on the above data, and the results showed that there were differences in frequency distribution at I785 = 23,750 and Area714 –1162 = 3,250,000. The FISH analysis identified sex chromosomes of 59 spermatozoa, which was not exactly one-to-one correspondence with the results of PCA analysis. Then we further analyzed the sperm of 59 cases by statistical analysis. The results showed that there were significant differences between X sperm (n = 39) and Y sperm (n = 20) at 714–1162 cm–1 and 785 (P < 0.05). ROC curve analysis was used to evaluate the sensitivity of correlation between sperm DNA content and Raman spectra. The results showed that the corresponding thresholds of I785 = 24,986.5 and Area714–1162 cm–1 = 3,748,990 were the best for distinguishing the two kinds of sperm. When the sperm’s peak value of 785 or 714–1162cm–1 exceeds the above thresholds, X-sperm’s possibility greatly increased. The AUC of the ROC curve in both cases was 0.662 and 0.696, respectively. Limitations, reasons for caution Current Raman spectroscopy requires spermatozoa elution and fixation, which damage the sperms. Furthermore, current Raman spectral data are not obtained from the whole sperm head, limiting the accuracy of this technique. Wider implications of the findings: Our results indicated that Raman spectroscopy had potential application value for sperm aneuploidy detection and could be used as a noninvasive selector for normal haploid sperms in the ART. Trial registration number LL-SC–2018–038

Genome Size and Karyotype Studies in Five Species of Lantana (Verbenaceae)

Lantana species are an important component of the U.S. environmental horticulture industry. The most commonly produced and used species are L. camara and, on a smaller scale, L. montevidensis. Both were introduced to the United States from Central and/or South America. Lantana species native to the continental United States include L. canescens, L. depressa, L. involucrata, etc. and most of them have not been well exploited. This study was conducted to obtain information about somatic chromosome numbers, karyotypes, and genome size of these five species. Nuclear DNA content in these species ranged from 2.74 pg/2C (L. involucrata) to 6.29 pg/2C (L. depressa var. depressa). Four chromosome numbers were observed: 2n = 2x = 22 in L. camara ‘Lola’ and ‘Denholm White’, 2n = 4x = 44 in L. depressa var. depressa, 2n = 2x = 24 in L. canescens and L. involucrata, and 2n = 3x = 36 in L. montevidensis. Two basic chromosome numbers were observed: x = 11 in L. camara and L. depressa var. depressa, and x = 12 in L. canescens, L. involucrata, and L. montevidensis. Analysis of somatic metaphases resulted in formulas of 20m + 2sm for L. camara ‘Lola’ and ‘Denholm White’, 12m + 12sm for L. canescens, 44m for L. depressa var. depressa, 10m + 14sm for L. involucrata, and 32m + 4sm for L. montevidensis. Satellites were identified in all five species, but were associated with a different chromosome group in different species. L. depressa var. depressa had the longest total chromatin length (146.78 µm) with a range of 1.88 to 4.41 µm for individual chromosomes. The maximum arm ratio was observed in L. canescens, with a ratio of 2.5 in chromosome group 3. L. depressa var. depressa was the only species that had all of its centromeres located in the median region of the chromosome. The results show significant differences in nuclear DNA content, chromosome number, and karyotype among three native and two introduced lantana species and will help to identify, preserve, protect, and use native lantana species. The information will be helpful in assessing the ploidy levels in the genus by flow cytometry.

Hyperspectral microscopy combined with DAPI staining for the identification of hepatic carcinoma cells.

In this study, the DAPI staining is firstly reported for use in the identification of hepatic carcinoma cells based on hyperspectral microscopy. Nuclei in cancer cells usually contain more aneuploidies than that in normal cells, leading to the change of DNA content. Here, we stain hepatic carcinoma tissues and normal hepatic tissues with 4',6-diamidino-2-phenylindole (DAPI) which is sensitive to the DNA content as a fluorochrome binds to DNA. Consequently, the difference in DNA content between hepatic carcinoma cells and normal hepatic cells can be identified by the fluorescent spectral characteristics. Harnessing the hyperspectral microscopy, we find that the fluorescent properties of these two kinds of cells are different not only in the intensity but also in the spectral shape. These properties are exploited to train a support vector machine (SVM) model for classifying cells. The results show that the sensitivity and specificity for the identification of 1000 hepatic carcinoma samples are 99.3% and 99.1%, respectively.

Advances in gender preselection in swine

Gender preselection using isolated populations of X- and Y-chromosome bearing spermatozoa that have been separated on the basis of DNA content is currently possible in swine and other farm animals, as well as in humans. Semen from most livestock species can now be successfully separated into predominantly X or Y sperm populations before their use for intra-tubal insemination, deep-uterine insemination or for in vitro fertilization (IVF) to produce sexed offspring. Birth of progeny of the desired sex in cattle, sheep and swine under semi-practical conditions has successfully validated the sexing technology. Spermatozoa are separated on the basis of inherent differences in DNA content in the X- and Y-chromosome bearing sperm population using modified flow cytometry /cell sorting technology. Spermatozoa are stained with Hoechst 33342 which binds to the DNA in an amount proportional to the amount of DNA present in the individual spermatozoa. Over 300 animals from various species at several locations have been born using the USDA-Beltsville Sperm Sexing Technology for separating X and Y spermatozoa. Sex ratios are shifted from the normal 50:50 to 85 to 90% of one sex or the other. In swine, offspring have been born as the result of surgical intratubal insemination of separated spermatozoa and also from 11/F and embryo transfer. At the present time, standard swine artificial insemination techniques are not optimized for use with the small numbers of flow cytometrically separated X or Y sperm populations. Cattle, swine and rabbit offspring have been reproduced through the second generation with normal morphology and reproductive function. Numerous improvements have been made in the sexing technology since it was first reported in 1989. Increasing the speed of the sexing process to make the application of the technology available to a larger segment of the livestock industry is paramount, even with insemination technology designed for small numbers of spermatozoa.

Nuclear DNA content as an indicator of inflorescence colour stability of in vitro propagated solid and chimera mutants of chrysanthemum

In chrysanthemum, breeders seek for desirable characteristics of the inflorescence, which can first be established once the plant is mature. The present study aims to determine whether measurement of DNA content can be useful in the detection of somaclonal variants and/or separation of chimera components in chrysanthemum at the early in vitro multiplication stage. Eleven Chrysanthemum × morifolium (Ramat.) Hemsl. cultivars of the Lady group (a mother cultivar and ten of its radiomutants obtained by X-ray- or γ-irradiation; solid and periclinal chimeras) were propagated in vitro. Single-node explants were cultured in Murashige and Skoog (MS) medium, either without plant growth regulators (PGRs) or supplemented with 6-benzyladenine (BA) and indole-3-acetic acid (IAA). The nuclear DNA content was measured by flow cytometry (FCM) in the shoots produced in vitro. After acclimatization and growth of the plants in a glasshouse, inflorescence colour was recorded. The addition of PGRs to the medium almost doubled the mean number of shoots produced in vitro per explant, but caused a change in inflorescence colour of all (‘Lady Apricot’; periclinal chimera) or part of the plants (‘Lady Amber’; solid mutant and ‘Lady Salmon’; periclinal chimera). All radiomutants contained less DNA than the mother cultivar ‘Richmond’. There were significant differences in DNA content between plants of the same cultivar grown in media with or without PGRs for ‘Lady Apricot’ and ‘Lady Salmon’, but no phenotype alternation occurred in chrysanthemums produced in PGR-free medium compared to the original cultivars. Conversely, in medium with PGRs, chimeras produced flowers different from the original colour. In all except one cultivar (‘Lady Amber’; solid mutant) a lack of differences in genome size between plants grown in either medium coincided with a stable inflorescence colour. The occurrence of some plants of ‘Lady Amber’ with different inflorescence colour may be due to small DNA changes, undetectable by FCM. It can be concluded that FCM analysis of DNA content in young plantlets can be indicative of the stability of inflorescence colour in chrysanthemum, especially chimeric cultivars, and for mutant detection.

Effects Of Age And End-stage Osteoarthritis On Markers Of Skeletal Muscle Long INterspersed Element-1 Activity

PURPOSE: Long INterspersed Element-1 (L1) is the only active, autonomous transposable element (termed retrotransposon) in the mammalian genome. L1 retrotransposons can insert themselves into the genome and, consequently, have been associated with a number of diseases and aging. L1 transcripts that are not reverse transcribed into the genome can accumulate in the cytoplasm and activate an inflammatory response via the cGAS-STING pathway. The purpose of this study was to examine skeletal muscle L1 markers and STING protein levels in younger/healthy participants as well as older participants with end-stage osteoarthritis (OA) undergoing total hip or knee arthroplasty. METHODS: Skeletal muscle was obtained perioperatively from OA patients (62 ± 11 years old) undergoing total hip or total knee arthroplasty (THA n=4; TKA n=6; total n=10) who were enrolled in the TWEAK Trial (R01HD084124, NCT02628795) at UAB. Muscle samples were collected from both surgical (Sx) and contralateral (CTL) thighs. A third cohort of young, healthy individuals (Y; 22 ± 2 years old) (Auburn, AL, USA) served as a comparator group (n=10). RNA, DNA, and protein were isolated for analysis. L1 mRNA expression and DNA content were quantified using primer sets for L1.3, the most active element, as well as ORF1. DNA methylation status and chromatin state of the L1 promoter were also interrogated. Protein targets included ORF1p and STING. Dependent variables were analyzed using a one-way ANOVA with Fisher’s LSD post hoc where appropriate. RESULTS: Both L1.3 and ORF1 mRNA were higher in Sx vs Y (p=0.003 and p<0.001, respectively) as well as CTL vs Y (p<0.001). Protein expression was higher in Sx vs Y for both ORF1p (p=0.003) and STING (p=0.013). There were no between-group differences in DNA content for L1.3 or ORF1, or the methylation status and chromatin state of the L1 promoter. CONCLUSIONS: These data show higher L1-related mRNA expression in old vs. young adult muscle irrespective of OA status. Alternatively, indices of protein signaling were elevated in Sx muscle only, suggesting upregulation of the cGAS pathway may play a role in the inflammatory burden often unique to muscle surrounding an OA joint. Future work will enable us to better determine if increased muscle L1 activity contributes to localized muscle inflammation susceptibility with aging or OA.

New Biological Insights on X and Y Chromosome-Bearing Spermatozoa

A spermatozoon is a male germ cell capable of fertilizing an oocyte and carries genetic information for determining the sex of the offspring. It comprises autosomes and an X (X spermatozoa) or a Y chromosome (Y spermatozoa). The origin and maturation of both X and Y spermatozoa are the same, however, certain differences may exist. Previous studies proposed a substantial difference between X and Y spermatozoa, however, recent studies suggest negligible or no differences between these spermatozoa with respect to ratio, shape and size, motility and swimming pattern, strength, electric charge, pH, stress response, and aneuploidy. The only difference between X and Y spermatozoa lies in their DNA content. Moreover, recent proteomic and genomic studies have identified a set of proteins and genes that are differentially expressed between X and Y spermatozoa. Therefore, the difference in DNA content might be responsible for the differential expression of certain genes and proteins between these cells. In this review, we have compiled our present knowledge to compare X and Y spermatozoa with respect to their structural, functional, and molecular features. In addition, we have highlighted several areas that could be explored in future studies in this field.

TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry

In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 μM; 0.25 μM; 0.5 μM; 1.0 μM; 2.5 μM) for 8 wk followed by cultivation on hormone-free medium (AM0) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 μM, 15.0 μM, or 30.0 μM) followed by cultivation on AM0 for 8 wk. The highest number of axillary shoots per explant was achieved with 0.1-μM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-μM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-μM TDZ nor by those triggered by the 30.0-μM TDZ pulse treatment.

Diverse Stress-Inducing Treatments cause Distinct Aberrant Body Morphologies in the Chlamydia-Related Bacterium, Waddlia chondrophila.

Chlamydiae, such as Chlamydia trachomatis and Chlamydia pneumoniae, can cause chronic infections. It is believed that persistent forms called aberrant bodies (ABs) might be involved in this process. AB formation seems to be a common trait of all members of the Chlamydiales order and is caused by distinct stress stimuli, such as β-lactam antibiotics or nutrient starvation. While the diverse stimuli inducing ABs are well described, no comprehensive morphological characterization has been performed in Chlamydiales up to now. We thus infected mammalian cells with the Chlamydia-related bacterium Waddlia chondrophila and induced AB formation using different stimuli. Their morphology, differences in DNA content and in gene expression were assessed by immunofluorescence, quantitative PCR, and reverse transcription PCR, respectively. All stimuli induced AB formation. Interestingly, we show here for the first time that the DNA gyrase inhibitor novobiocin also caused appearance of ABs. Two distinct patterns of ABs could be defined, according to their morphology and number: (i) small and multiple ABs versus (ii) large and rare ABs. DNA replication of W. chondrophila was generally not affected by the different treatments. Finally, no correlation could be observed between specific types of ABs and expression patterns of mreB and rodZ genes.

Genome size evolution and chromosome numbers of species of the cryptanthoid complex (Bromelioideae, Bromeliaceae) in a phylogenetic framework

Abstract We describe the chromosome numbers and genome sizes of species of the cryptanthoid complex of Bromeliaceae in a phylogenetic framework and their relationship with habitat preferences. The 2C DNA contents varied 2.13-fold among species, ranging from 0.76 to 1.66 pg. A significant difference in DNA content was found among Cryptanthus, Hoplocryptanthus and Rokautskyia. Moreover, species from campos rupestres and the Atlantic Forest had lower and higher genome size values, respectively. The smaller genome sizes of Hoplocryptanthus spp. from campos rupestres may be related with the large genome constraint. The species show a highly conserved ploidy (with 2n = 32 and 34), although the genome sizes varied considerably. The observed variation in chromosome numbers seems to be influenced by dysploidy, but additional investigations are needed. Our study demonstrates that the genome size variation in the cryptanthoid complex species is not strictly related to the phylogenetic relationships and has probably been influenced by different evolutionary processes.

DNA content equivalence in haploid and diploid maize leaves

Main conclusionThe qPCR assay developed to differentiate haploid and diploid maize leaf samples was unsuccessful due to DNA content difference. Haploid cells are packed more closely together with less cellular expansion.Increased ploidy content (> 2 N) directly correlates with increased cell size in plants, but few studies have examined cell morphology in plants with reduced ploidy (i.e., haploids). To pioneer a scalable new ploidy test, we compared DNA content and cellular morphology of haploid and diploid maize leaves. The amount of genomic DNA recovered from standardized leaf-punch samples was equivalent between these two ploidy types, while both epidermal and mesophyll cell types were smaller in haploid plants. Pavement cells had a substantially smaller size than mesophyll cells, and this effect was more pronounced in the abaxial epidermis. Interveinal distance and guard cell size were significantly reduced in haploids, but the cell percentage comprising stomata did not change. These results confirm the direct correlation between ploidy content and cell size in plants, and suggest that reduced cell expansion predominantly explains DNA content equivalence between haploid and diploid samples, confounding efforts to develop a haploid detection method using DNA content.

Comparison of DNA extraction methods from Geranium (Geraniaceae)

The genus Geranium (Geraniaceae); with about 320 species throughout the temperate regions, is chemically characterised by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. It is necessary to optimise the extraction protocols to reduce the effects of the presence of these compounds to the lowest level. The present study compares the plant genomic DNA extraction Kit (DNP™ Kit), CTAB DNA extraction method by Murray and Thompson and Sahu et al., from the extracting DNA point of view Geranium species. The results showed significant differences in DNA contents between the three methods. Quantity and quality of extracted genomic DNAs were compared by employing the spectrophotometer, Nano-Drop, agarose gel electrophoresis, and polymerase chain reaction (PCR) methods and molecular marker such as (ITS and trnL-F) and ISSR. The method of Sahu et al., provided the best results (200 ng/µL) in terms of quantity and quality of DNA, therefore, this method was taken and optimised for DNA extraction. Our results proposed that this method could be effective for plants with same polysaccharides, proteins and polyphenols components. The advantage of this method is that it omits the use of liquid nitrogen and toxic phenols which are expensive. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Geranium species group and the reliability of this method has been discussed.

Haplotypes spanning centromeric regions reveal persistence of large blocks of archaic DNA.

Despite critical roles in chromosome segregation and disease, the repetitive structure and vast size of centromeres and their surrounding heterochromatic regions impede studies of genomic variation. Here we report the identification of large-scale haplotypes (cenhaps) in humans that span the centromere-proximal regions of all metacentric chromosomes, including the arrays of highly repeated α-satellites on which centromeres form. Cenhaps reveal deep diversity, including entire introgressed Neanderthal centromeres and equally ancient lineages among Africans. These centromere-spanning haplotypes contain variants, including large differences in α-satellite DNA content, which may influence the fidelity and bias of chromosome transmission. The discovery of cenhaps creates new opportunities to investigate their contribution to phenotypic variation, especially in meiosis and mitosis, as well as to more incisively model the unexpectedly rich evolution of these challenging genomic regions.

A new biological species in the Mercurialis annua polyploid complex: functional divergence in inflorescence morphology and hybrid sterility.

Background and AimsPolyploidy has played a major role in the origin of new plant species, probably because of the expansion of polyploid populations in the species’ ecological niche, and because reproductive isolation can be established between a new polyploid population and its diploid progenitor species. It is well established that most polyploid species are polyphyletic, with multiple independent origins, and that polyploid genomes may undergo rapid change after their duplication and hybridization associated with their origin. We considered whether multiple independent origins and rapid genomic change might lead to reproductive isolation between polyploid populations of the same ploidy but with potentially different evolutionary histories.MethodsWe tested our hypothesis by assessing differences in DNA content and morphology, the evolution of reproductive isolation, and the phylogenetic placement of two broadly sympatric hexaploid lineages of the wind-pollinated annual plant Mercurialis annua hitherto regarded as populations of the same species.Key ResultsThe two hexaploid lineages of M. annua have slightly divergent DNA content, and distinct inflorescence morphology. They also fall into largely different clades of a chloroplast phylogeny and are reproductively isolated from one another.ConclusionsThe distinct evolutionary histories of the two hexaploid lineages of M. annua have contributed to the remarkable reproductive diversity of the species complex. It seems likely that reproductive interference between them will eventually lead to the displacement of one lineage by the other via pollen swamping. Thus, whereas polyploidization can contribute to speciation, diversification might also be compromised by reproductive interference.

Advanced Parental Age at Conception and Sex Affects Mitochondrial DNA Copy Number in Human and Fruit Flies.

Aging is a multifactorial trait caused by early as well as late-life circumstances. A society trend that parents deliberately delay having children is of concern to health professionals, for example as advanced parental age at conception increases disease risk profiles in offspring. We here aim to study if advanced parental age at conception affects mitochondrial DNA content, a cross-species biomarker of general health, in adult human twin offspring and in a model organism. We find no deteriorated mitochondrial DNA content at advanced parental age at conception, but human mitochondrial DNA content was higher in females than males, and the difference was twofold higher at advanced maternal age at conception. Similar parental age effects and sex-specific differences in mitochondrial DNA content were found in Drosophila melanogaster. In addition, parental longevity in humans associates with both mitochondrial DNA content and parental age at conception; thus, we carefully propose that a poorer disease risk profile from advanced parental age at conception might be surpassed by superior effects of parental successful late-life reproduction that associate with parental longevity.

MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. &amp; Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry.