2C DNA Content Research Articles

Genome size and chromosome number variation in sugarcane (Saccharum spp.) families and phenotypically contrasting Saccharum genotypes

AbstractModern sugarcane (Saccharum spp.) cultivars originated from an intricate hybridization process, resulting in complex polyploid and aneuploid genomes. Flow cytometry (FCM), which has emerged as a precise and fast method for research on genome size in plants, has potential to unveil key aspects of sugarcane cytogenetics. In this study, FCM probes were conducted to estimate 2C DNA content in 175 Saccharum genotypes embracing phenotypically contrasting canes (commercial cultivars, energy‐cane hybrids, and a Saccharum spontaneum accession) and full‐sib sugarcane families. FCM‐mediated DNA content estimates exhibited high accuracy in predicting chromosome numbers, offering a time‐efficient alternative to classical cytogenetic procedure. Low‐magnitude correlations detected between DNA content and phenotypic traits suggest that indirect selection of agronomic traits using FCM‐mediated DNA content estimates may not be efficient. DNA content variation patterns in sugarcane families varied according to the crossing, and revealed transgressive variations associated with rampant aneuploidy. Altogether, the findings uncover that loss or gain of genetic material constitutes a source of high genetic variability that may substantially impact on crop breeding.

Variability in genome size of Trigonella foenum-graecum, Trigonella corniculata and Trigonella caerulea as estimated by flow cytometry indicates complex evolutionary history of fenugreek.

The determination of genome size is a fundamental step which provides a basis to initiate studies aimed at deciphering the genetic similarity of a species and to carry out other genomics based investigations. Fenugreek (Trigonella spp.) is an important spice crop which has numerous health promoting phytochemicals. Many species within this genus are known for their various health benefits owing to the presence of a wide diversity of important phytochemicals like diosgenin, trigonelline, fenugreekine, galactomannan, 4-hydroxy isoleucine, etc. It is a multipurpose crop being cultivated for food, animal feed and industrial purposes. Despite its importance, research on the genomics aspect of fenugreek remains scant. In the absence of sufficient genomic information, crop improvement in fenugreek is severely lagging. Estimation of genome size of a species is the preliminary step for initiation of any genomic studies and therefore in the present study we have estimated the genome size for fenugreek. Here, we have determined the genome sizes of three different Trigonella spp. namely T. foenum-graecum, T. corniculata and T. caerulea through flow cytometry (FC). The 2C DNA content values were found to be 6.05 pg (T. foenum-graecum), 1.83 pg (T. corniculata) and 1.96 pg (T. caerulea). The genome size of T. foenum-graecum is approximately three times the genome size of T. corniculata and T. caerulea. This variation in genome size of more than three-fold indicates the level of genetic divergence among the three species, though within the same genus. The differences observed in the genome sizes of the three species provide conclusive evidence of their genetic divergence. Additionally, the information about the genome size would provide an impetus to the structural and functional genomics-based research in this crop.

Genome size variation and evolution during invasive range expansion in an introduced plant.

Plants demonstrate exceptional variation in genome size across species, and their genome sizes can also vary dramatically across individuals and populations within species. This aspect of genetic variation can have consequences for traits and fitness, but few studies attributed genome size differentiation to ecological and evolutionary processes. Biological invasions present particularly useful natural laboratories to infer selective agents that might drive genome size shifts across environments and population histories. Here, we test hypotheses for the evolutionary causes of genome size variation across 14 invading populations of yellow starthistle, Centaurea solstitialis, in California, United States. We use a survey of genome sizes and trait variation to ask: (1) Is variation in genome size associated with developmental trait variation? (2) Are genome sizes smaller toward the leading edge of the expansion, consistent with selection for "colonizer" traits? Or alternatively, does genome size increase toward the leading edge of the expansion, consistent with predicted consequences of founder effects and drift? (3) Finally, are genome sizes smaller at higher elevations, consistent with selection for shorter development times? We found that 2C DNA content varied 1.21-fold among all samples, and was associated with flowering time variation, such that plants with larger genomes reproduced later, with lower lifetime capitula production. Genome sizes increased toward the leading edge of the invasion, but tended to decrease at higher elevations, consistent with genetic drift during range expansion but potentially strong selection for smaller genomes and faster development time at higher elevations. These results demonstrate how genome size variation can contribute to traits directly tied to reproductive success, and how selection and drift can shape that variation. We highlight the influence of genome size on dynamics underlying a rapid range expansion in a highly problematic invasive plant.

Genome size estimation of false daisy, cheek weed, pot marigold and marigold

We report the genome size estimated using flow cytometry for four closely related species, including false daisy (Eclipta prostrate), cheek weed (Ageratum conyzoides), pot marigold (Calendula officinalis), and marigold (Tagetes erecta) belonging to Asteraceae family. The detected genome size for false daisy, cheek weed, pot marigold, and marigold was, 2.435, 3.266, 3.413, and 1.897, Gbp, respectively, while their respective 2C DNA content was 2.5, 3.3, 3.5, and 1.9, pg. The information on genome size presented here will be useful for understanding genomic evolution and will also clear the way for additional genomic research in these species.

Ecotype variations in karyotypes and 2C DNA content in Drimia indica (Roxb.) Jossop: An important medicinal plant

Indian squill (Drimia indica) belonging to the family Asparagaceae is a highly medicinal herb with several therapeutic properties. Detailed karyotype analysis and 2C DNA contents were reported in twelve ecotypes of D. indica from different geographical locations in the state of Odisha, India. The diploid somatic chromosome 2n = 20 was recorded in four ecotypes ODi-16 (Kendrapada), ODi-23 (Tikarpada), ODi-24 (Daspalla), and ODi-26 (Odagaon) while tetraploid having 2n = 40 was newly reported in one ecotype from Nuagaon (Eco-ODi-06). Aneuploid chromosome number 2n = 26 was obtained in ecotypes from Similipal and Satkosia besides 2n = 32 was newly reported in ecotypes from Bitarkanika and Narasinghpur. The ecotypes ODi-22 (Nayagarh) and ODi-11 (Nilagiri) were found to be with 2n = 16 and 2n = 22 respectively. The 2C DNA content analysis showed a 2.19-fold increase which is confirmed by an increase in the number and chromosome length of ecotypes. The 2C DNA values were found highest at 36.26 p.g. (2n = 40) in Eco-ODi-06 (Nuagaon) and lowest 16.54 p.g. (2n = 16) in Eco-ODi-22 of Nayagarh that corresponds with ploidy and genome size. The asymmetry in the karyotypes showed non-significant clusters among the ecotypes except ecotypes ODi-26 (Odagaon) and ODi-2a (Bhitarkanika) which are most asymmetric having advanced karyotypic features as compared to the ecotypes ODi-01, ODi-08 and ODi-22 having symmetric karyotypes. The chromosome characteristics along with genome size analysis of D. indica can be very useful in establishing genome-specific cytotypes which can be used for breeding programs for crop improvement. Furthermore, enhancement of therapeutic phytoconstituents and cyto-taxonomic identification from adulterants and confirmation of allopolyploidy or autopolyploidy through genomic in situ hybridization could unravel the potential of this plant in relation to secondary metabolite production.

Genome size, chromosome number variation and its correlation with stomatal characters for assessment of ploidy levels in a core subset of mulberry (Morus spp.) germplasm

The large size of the germplasm collection along with scanty information on their cytological and genome constitution have hindered well-planned breeding schemes in mulberry. To address the issue, a study was undertaken to investigate the variability in DNA content and genome size, chromosome number, ploidy and its relation with important stomatal characteristics among 162 mulberry germplasm collection. These germplasm comprise a core subset of 150 collections along with a representative collection of different mulberry species including the wild. Among the germplasm belonging to 16 species, we identified 122 diploids (2n = 28), 4 aneuploids (2n = 30), 13 triploids (2n = 42), 15 tetraploids (2n = 56), 7 hexaploids (2n = 84) and 1 dodecosaploid (2n = 308) based on the chromosome count. Most of the cultivated mulberries are found to be diploids. The mean nuclear 2C DNA content estimated by Flow cytometry, varied from 0.723 ± 0.006 pg (M. australis, 2n = 2x) to 7.732 pg (M. nigra, 2n = 22x). The 2C DNA content positively correlated with the ploidy status and stomatal length (r = 0.814, p < 0.001). Based on the 1Cx value, the study also suggests that the majority of the polyploid species have experienced genome downsizing in relation to their diploid progenitors. This study provides the most essential information on chromosome number, ploidy and DNA content to facilitate the utilization of a core subset of germplasm in the mulberry breeding program.

Effect of Green Synthesized ZnO-NPs on Growth, Antioxidant System Response and Bioactive Compound Accumulation in Echinops macrochaetus, a Potential Medicinal Plant, and Assessment of Genome Size (2C DNA Content).

Echinops macrochaetus is a medicinal plant that can be used to cure various diseases. In the present study, plant-mediated zinc oxide nanoparticles (ZnO-NPs) were synthesized using an aqueous leaf extract of the medicinal plant Heliotropium bacciferum and characterized using various techniques. E. macrochaetus was collected from the wild and identified using the internal transcribed spacer sequence of nrDNA (ITS-nrDNA), which showed the closeness to its related genus in a phylogenetic tree. The effect of synthesized biogenic ZnO-NPs was studied on E. macrochaetus in a growth chamber for growth, bioactive compound enhancement and antioxidant system response. The irrigation of plants at a low concentration of ZnO-NPs (T1 = 10 mg/L) induced more growth in terms of biomass, chlorophyll content (273.11 µg/g FW) and carotenoid content (135.61 µg/g FW) than the control and other treatments (T2-20 mg/L and T3-40 mg/L). However, the application of a high concentration of ZnO-NPs (20 and 40 mg/L) increased the level of antioxidant enzymes (SOD, APX and GR), total crude and soluble protein, proline and TBARS contents. The accumulations of the compounds quercetin-3-β-D-glucoside, luteolin 7-rutinoside and p-coumaric acid were greater in the leaf compared to the shoot and root. A minor variation was observed in genome size in treated plants as compared to the control group. Overall, this study revealed the stimulatory effect of phytomediated ZnO-NPs, which act as bio-stimulants/nano-fertilizers as revealed by more biomass and the higher production of phytochemical compounds in different parts of the E. macrochaetus.

Mixed-Ploidy and Dysploidy in Hypericum perforatum: A Karyomorphological and Genome Size Study.

Karyomorphology and genome size of 15 St John's wort (Hypericum perforatum L.) populations are reported for the first time. Root tips and fresh young leaves were used for karyological studies and flow cytometric (FCM) measurements, respectively. The chromosome length varied from 0.81 µm to 1.16 µm, and chromosome types were determined as "m". Eight different somatic chromosome numbers were found (2n = 16, 22, 24, 26, 28, 30, 32, 38). Based on the observed basic (x) chromosome numbers of x = 8, 11, 13, 14, 15, 19, this may correspond to diploid (2x), triploid (3x), tetraploid (4x), respectively. Interestingly, we found mixoploidy (3x - 4x) in the root tips of one of the populations. Hybridization, polyploidy and dysploid variation may be the main factors associated with the chromosome number evolution of this species. FCM showed that 2C DNA contents vary from 0.87 to 2.02 pg, showing more than a 2-fold variation. The mean amount of 2C DNA/chromosome and the mean of monoploid genome size were not proportional to ploidy.

Comparative Study on Genome Size and Phytochemical Profile of Three Potential Species of Acacia: Threatened and Endemic to Saudi Arabia

Acacias are widely distributed in tropical and subtropical regions of the world and have both economic as well as medicinal value. The estimation of genome size is very important as it changes due to the change in noncoding DNA sequence as well as genome duplication among organisms for their evolutionary aspects. Three potential species of the genus Acacia including Acacia etbaica, Acacia johnwoodii and Acacia origena, which are threatened and nearly endemic to Saudi Arabia, were collected. The present study was carried out to determine the genome size (2C DNA contents), total phenolic content (TPC), total flavonoid (TFC) and some bioactive compounds in these species for their comparison. The genome size ranged from 1.91 pg (A. etbaica) to 2.45 pg/2C (A. origena) among the Acacia species, which correspond to genome sizes 1843.15–2364.25 Mbp, respectively. The variation was observed in genome size within Acacia species as nuclei were extracted using different extraction buffers except for GB and MB01 buffers. The FTIR analysis revealed the presence of various functional groups in compounds that might be responsible for different types of phytochemicals in these Acacia species. Total flavonoid content (TFC) ranged from 0.647 (A. origena) to 1.084 mg QE/g DW (A. etbaica), whereas the total phenolic f content (TPC) ranged between 15.322 (A. origena) to 28.849 (A. johnwoodii) mg/g DW of GAE. HPLC analysis revealed the presence of quercetin 3-β-D-glucoside and luteolin 7-rutinoside in the leaves of all three Acacia species in considerable amounts, and these might have good health-promoting effects. This is our first study on genome size (2C DNA content) using flow cytometry and phytochemical profiling on these Acacias. Thus, estimated genome size and phytochemical study of these species could help to understand the biosynthesis of secondary metabolites under various genes and the evolutionary relationships among them.

Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature “nurturing” state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.

Genetic and nuclear DNA content variation of stevia (Stevia rebaudiana Bertoni) accessions grown in Turkey

The objectives of this study were to make morphological and molecular characterization of Turkish stevia accessions. The field data showed significant variations (P &lt; 0.05) for all morphological parameters (plant height, number of main stems per plant, number of secondary stems, fresh and herb yields) exception with number of secondary stems. The primers of MtP5CS, AtNHX1, MtProDH, Mt-Actin MtSOS1and MtSOS2 genes were used as loci specific DNA markers. All markers were amplified in Stevia genome and provided a mean of 74.16% polymorphism rate. Flow cytometer analysis showed no statistically significant differences (P &lt; 0.16) for 2C DNA content with a mean of 1.62 pg. The UPGMA analysis revealed that Samsun accession was distinctly separated from the others and had the lowest 2C DNA content (1.52 pg). This study first time showed that the stevia accessions grown in Turkey have limited genetic and no nuclear DNA content variations. The results also revealed that gene targeted functional markers associated with salt and drought tolerance have a great potential to be used as DNA markers to determine the genetic variation in plants

The genetic diversity of Asplenium viride (Aspleniaceae) fern colonizing heavy metal-polluted sites

Asplenium viride is a diploid species, belonging to the largest genus of the cosmopolitan fern family Aspleniaceae and occurring on various types of alkaline rocks. It is known to colonize sites with high concentrations of heavy metals, exhibiting changes in frond morphology. A. viride can sometimes form new substrate-dependent ecotypes that can be morphologically and genetically different from parental populations. This study aimed to evaluate the morphological and genetic diversity of A. viride, and test for a potential correlation between variability and heavy metal concentration. Analysis of A. viride specimens from one metalliferous and five non-metalliferous sites showed elevated concentrations of heavy metals in roots of metalliferous plants. The concentrations were higher in roots than in aboveground organs, especially in the case of Cd and Pb, suggesting an excluder strategy for these metals. Both metalliferous and non-metaliferous sites were populated by plants with similar ploidy levels. The overall genetic diversity was low (HT = 0.25) and concentrated between populations (GST = 0.62). The obtained 2C DNA content ranged from 8.67 pg/2C to 8.69 pg/2C. STRUCTURE analysis revealed two groups among the studied populations which did not correlate with heavy metal concentrations and were not significantly supported by AMOVA. This suggests that factors influencing genetic diversity of A. viride are a consequence of intragametophytic selfing caused by patchy habitats and subsequent founder effects, resulting from long-distance colonization by single spores. The species has a potential to colonize heavy metal polluted sites; however, it does not seem to form genetically distinct ecotypes at those sites.

Abstract 5379: Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC)

Abstract Inhibition of substrate binding to Cyclin A has been postulated to be synthetically lethal in retinoblastoma (Rb) mutated cancers (Chen et al 1999). While compounds that target the cyclin-associated kinases are approved for clinical use, attempts to target the protein-protein interactions between cyclins and their substrates, such as E2F, have so far been unsuccessful. Using structure-guided design we have developed cell-permeable macrocycle compounds that selectively inhibit the RxL-mediated binding of substrates to Cyclin A. We used NCI-H1048, an Rb-/- SCLC cell line, to study the effects of these Cyclin A targeting macrocyclic compounds on cell proliferation, apoptosis, and cell cycle regulation. Proliferation of H1048 cells was inhibited ~90% at 100nM of compound A whereas no inhibition was observed at up to 10 uM in WI-38 cells, a human non tumor lung fibroblast cell line, or with an inactive isomer of compound A in NCI-H1048 cells. Cyclin A inhibition in H1048 cells resulted in accumulation of cells at G2/M, an increased proportion of cells with 4C DNA content by FACS, hyperphosphorylation of RPA and upregulation of both phosphorylated histone H3 (pHH3) and phosphorylated FOXM1 (pFOXM1). Substantial apoptosis (10 to 20-fold) measured using caspase 3/7 activity was induced after just 4 hours incubation of H1048 cells with Cyclin A inhibitors. In vivo efficacy studies in a murine H1048 xenograft model showed reduction in tumor growth. We analyzed sensitivity to these compounds in tumor cell lines representing multiple indications and observed inhibition of cell proliferation at 1 uM or below in 6 out of 10 SCLC cell lines, where all of the responding cell lines were Rb dysfunctional. Sensitivity to these compounds in a larger panel of SCLC and other cancer cell lines is currently under investigation to further evaluate the broader utility of cyclin inhibition. Macrocycle inhibitors that inhibit Cyclin A RxL-mediated substrate binding have demonstrated G2/M accumulation and apoptosis in Rb -/- SCLC in vitro consistent with synthetic lethality, and anti-proliferative effects in vitro and in vivo. Inhibiting cyclins is mechanistically distinct from strategies that inhibit cell cycle kinases, and is expected to be clinically useful, hence the compound series is being optimized for clinical development. Citation Format: Pablo D. Garcia, Catherine E. Gleason, Miles Membreno, Frances Hamkins-Indik, Gavin Situ, Bernard Levin, Evelyn W. Wang, Siegfried Leung, Breena Fraga, Andrew Bockus, James Aggen, David Spellmeyer, David J. Earp, Rajinder Singh. Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5379.

The variability of nuclear DNA content of different Pelargonium species estimated by flow cytometry.

Pelargonium is a versatile genus mainly from the Cape Region, South Africa. The genus is divided into four subgenera and 16 sections characterized by several groups of chromosomes sizes and numbers. The DNA content of species from all subgenera and sections of Pelargonium, except for the sections Subsucculentia and Campylia was estimated using flow cytometry. Nuclei of Pelargonium samples (leaf or petal tissue) and an internal plant standard (leaf tissue) were isolated together and stained with propidium iodide. The DNA content was estimated providing that the 2C peaks of sample and standard be in linearity in the flow cytometer histograms. In total, 96 Pelargonium accessions of 60 species (22 Pelargonium species for the first time) were analyzed. The 2C DNA content ranged from 0.84 pg (P. longifolium, section Hoarea) to 6.69 pg (P. schizopetalum, section Magnistipulacea) and the corresponding 1Cx DNA content from 0.42 pg (P. longifolium) to 1.72 pg (P. transvaalense. This demonstrates the high plasticity within the genus Pelargonium. Some species, such as P. peltatum accessions revealed a pronounced endopolyploidization in leaves but not in petals underlining the importance to choose the right tissue as sample for the flow cytometry analysis. The reported genome sizes are a step forward towards the characterization of the Pelargonium collection within the German Gene Bank for Ornamental Plants and a valuable base for future sequencing programs of the Pelargonium genomes.

Karyological and nuclear DNA content variation of the genus Asparagus.

Asparagus wild relatives could be a promising possibility to extent the genetic variability of garden asparagus and for new cultivars with favorable traits such as high yield stability, disease resistance and stress tolerance. In order to achieve an efficient use in breeding, a detailed cytogenetic characterization of the accessions is necessary. This study worked on 35 Asparagus accessions, including A. officinalis cultivars (‘Darlise’, ‘Ravel’ and ‘Steiners Violetta’) and Asparagus wild relatives, for which the number of chromosomes, their size, the nuclear DNA content, and the genomic distribution of 5S and 45S rDNA were analyzed. Different ploidy levels (diploid, triploid, tetraploid, pentaploid and hexaploid) were found. Furthermore, the size of the chromosomes of all diploid Asparagus accessions was determined which led to differences in the karyotypic formula. A. plocamoides harbors the smallest chromosome with 1.21 μm, whereas the largest chromosome with 5.43 μm was found in A. officinalis. In all accessions one 5S rDNA locus per genome was observed, while the number of 45S rDNA loci varied between one (A. albus, A. plumosus, A. stipularis) to four (A. setaceus). In most Asparagus accessions, the 5S and 45S rDNA signals were located on different chromosomes. In contrast, the genomes of A. africanus, A. plocamoides, A. sp. (a taxonomically unclassified Asparagus species from Asia) and A. verticillatus (diploid accessions) have one 5S and one 45S rDNA signal on the same chromosome. The measured 2C DNA content ranges from 1.43 pg (A. plocamoides, diploid) to 8.24 pg (A. amarus, hexaploid). Intraspecific variations for chromosome number, karyotypic formula, signal pattern with 5S and 45s rDNA probes and DNA content were observed. Interspecific variations were also recognized in the genus Asparagus.

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.

Genome Estimation and Phytochemical Compound Identification in the Leaves and Callus of Abrus precatorius: A Locally Endangered Plant from the Flora of Saudi Arabia.

Abrus precatorius is considered to be a valuable source of natural products for the development of drugs against various diseases. Herein, the genome size and phytochemical compounds in the leaves and callus of A. precatorius were evaluated. The endangered A. precatorius was collected from the Al-Baha mountains, Saudi Arabia and identified based on the phylogenetic analysis of a DNA sequence amplified by ITS1 and ITS4 primers. The callus was induced by the culture of stem explants onto Murashige and Skoog medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4D) and 6-Benzylaminopurine (BAP). The callus with the highest fresh weight (2.03 g) was obtained in the medium containing 0.5µM BA and 5 µM 2,4-D after 8 weeks of culture; thus, the callus of this combination was selected for the genome estimation and phytochemical compound extraction. The genetic stability of the leaves from the donor as well as in the regenerated callus was analyzed by flow cytometry with optimized tomato (2C = 1.96 pg) as an external reference standard. The 2C DNA content was estimated to 1.810 pg ± 0.008 and 1.813 pg ± 0.004 for the leaves and callus, respectively. Then, the total phenol and total flavonoid contents in the methanol extract of the callus and leaves were measured using a spectrophotometer and the High-performance liquid chromatography (HPLC ) methods. The results showed that the methanolic extract of the leaves was higher in total phenols and total flavonoids than the callus extract. Finally, the extracts of callus and leaves were analyzed for phytochemical compound through the Gas chromatography and Mass spectroscopy (GC-MS). A total of 22 and 28 compounds were detected in the callus and leaves, respectively. The comparative analysis showed that 12 compounds of the secondary metabolites were present in both extracts.

Morphological, molecular and phytochemical variations induced by colchicine and EMS chemical mutagens in Crocus sativus L.

Crocus sativus L., also known as saffron, is one of the most important medicinal and spice plants throughout the world. The plant is a rich source of apocarotenoids such as crocetin esters, picrocrocin, and safranal. The purpose of this study was to investigate the effect of colchicine and ethyl methanesulfonate (EMS) mutagens on possible inducing new variation in C. sativus. Accordingly, corms were exposed to EMS (0.1% and 0.2%) and colchicine (0.05% and 0.025%) for three incubation times. The lowest survival rate of corms was related to EMS treatments. The relative expression of ALDH, BGL, and CCD2 genes under 0.025% colchicine treatment for 12 h revealed a 2 fold increase compared with the control. The flow cytometric measurements (FCM) of the nuclear DNA content of the colchicine-treated plants did not reveal any significant changes in 2C DNA content. The results, manifest the potential of mutagens to create new variations in the plant.

Nuclear DNA contents and ploidy levels of North American Vaccinium species and interspecific hybrids

Breeding strategies for improving blueberry (Vaccinium corymbosum and V. virgatum) cultivars often include introgressing regionally adapted species into the cultivated gene pools through interspecific hybridization. However, these approaches are complicated by variation in ploidy, triploid blocks and infertility, production of unreduced gametes, and aneuploidy. The objective of this study was to use flow cytometry, k-mer distribution analysis, and known pedigree information to evaluate genome sizes (2C nuclear and 1Cx monoploid), and ploidy of diverse accessions from Vaccinium sections and species. A total of 369 accessions, including a diversity panel (DP) of 251 inter- and intra-specific hybrid Vaccinium accessions, as well as 118 non-hybrid Vaccinium species across multiple sections, were sampled from the North Carolina State University blueberry breeding program and the National Clonal Germplasm Repository. The nuclear DNA content was analyzed via flow cytometry. The mean (range) DNA content of diploid, tetraploid, and hexaploid reference species were 1.20 pg (0.99 pg in V. crassifolium ‘Well's Delight’ to 1.41 pg in V. caesariense NC79–24), 2.37 pg (2.11 pg in V. corymbosum ‘Concord’ to 3.01 pg in V. corymbosum DE599), and 3.64 pg (3.24 in V. constablaei NC83–21–2 to 3.80 in V. virgatum ‘Premier’ and NC4790), respectively. Of the 369 unique accessions analyzed for ploidy, 259 were tetraploid, 46 were diploid, one was triploid, 51 were pentaploid or aneuploid with 2C values between tetraploid and hexaploid values, and 12 were hexaploid. Tetraploid hybrid pedigrees, which involved hexaploid crosses within three prior generations, had a 2C value range between 2.22 pg and 2.59 pg. Interspecific pentaploid and aneuploid progeny 2C DNA content ranged from 2.61 pg to 3.15 pg. We speculate some of these progeny to be near tetraploids with extra chromosomes from hexaploid progenitors. Further karyotyping of these individuals is necessary to ascertain aneuploidy anomalies. This research provides an expanded knowledge base of genome sizes, ploidy, and reproductive pathways for diverse species and hybrids to enhance future breeding, improvement, and the genomic study of blueberry.

Analysis of Hypericum accessions by DNA fingerprinting and flow cytometry

Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies.