Ploidy Level Research Articles

A Flow Cytometry-Based Assessment of the Genomic Size and Ploidy Level of Wild Musa Species in India.

In this paper, chromosome spreading was performed via protoplast isolation and a fast air-dry dropping method and flow cytometry were used with Raphanus sativus L. (Brassicaceae) as a standard for ploidy and genome size estimation. The results showed that genome size (2C) varied amongst Musa species, based on the ratio of G1 peak positions. The lowest genome size (2C) was found in M. balbisiana var. andamanica (1.051 ± 0.060 pg) and the highest genome size (2C) was recorded for Musa ABB.cv. Meitei-hei (1.812 ± 0.108 pg) for the section Eumusa. Among the species belonging to the section Rhodochlamys, M. rosae had the lowest 2C content of 1.194 ± 0.033 pg whereas the highest nuclear DNA content (2C) was observed in M. velutina (1.488 ± 0.203 pg). Cytogenetic analysis revealed that the chromosome number of 14 wild Musa species was 2n = 22, while 1 species-Ensete glaucum-showed a chromosome number of 2n = 18 (diploid), and for 3 species, the chromosome number was 2n = 33 (triploids). An association study based on the Pearson correlation coefficient showed 2C nuclear DNA content was significant and positively correlated with ploidy level (R = 0.9) and chromosome number (R = 0.84). The present study provides reliable information on the genome size and ploidy level of wild Musa species from the Indian region through flow cytometric analysis, which could be further utilized in taxonomic and crop improvement programs. For the first time, the nuclear DNA content of eight wild diploid and three triploid Indian species were estimated and reported. Genome size could be an effective indicator in identification of species and evolutionary studies in Musa with varying ploidy levels and morphological similarities.

E3 ligases MAC3A and MAC3B ubiquitinate UBIQUITIN-SPECIFIC PROTEASE14 to regulate organ size in Arabidopsis.

The molecular mechanisms controlling organ size during plant development ultimately influence crop yield. However, a deep understanding of these mechanisms is still lacking. UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by DA3, is an essential factor determining organ size in Arabidopsis (Arabidopsis thaliana). Here, we identified two suppressors of the da3-1 mutant phenotype, namely SUPPRESSOR OF da3-1 1 and 2 (SUD1 and SUD2), which encode the E3 ligases MOS4-ASSOCIATED COMPLEX 3A (MAC3A) and MAC3B, respectively. The mac3a-1 and mac3b-1 mutations partially suppressed the high ploidy level and organ size phenotypes observed in the da3-1 mutant. Biochemical analysis showed that MAC3A and MAC3B physically interacted with and ubiquitinated UBP14/DA3 to modulate its stability. We previously reported that UBP14/DA3 acts upstream of the B-type cyclin-dependent kinase CDKB1;1 and maintains its stability to inhibit endoreduplication and cell growth. In this work, MAC3A and MAC3B were found to promote the degradation of CDKB1;1 by ubiquitinating UBP14/DA3. Genetic analysis suggests that MAC3A and MAC3B act in a common pathway with UBP14/DA3 to control endoreduplication and organ size. Thus, our findings define a regulatory module, MAC3A/MAC3B-UBP14-CDKB1;1, that plays a critical role in determining organ size and endoreduplication in Arabidopsis.

Optimal conditions for pressure shock induction of triploidy in turbot and a comparison of induction efficiency with cold shock

The mass production of triploid turbot Scophthalmus maximus is expected to enhance the economic benefits of aquaculture due to its sterility, growth, and survival advantages. Among the methods for inducing triploidy in teleost, pressure shock has been considered a more consistent and reliable approach. In this study, the optimal parameters for inducing triploidy in turbot through hydrostatic pressure shock were investigated in a series of trials, including the intensity (55–75 MPa), timing (3.5–8.5 min after fertilization, maf) and duration (4–12 min). The ploidy level was determined by flow cytometry analysis. Under a water temperature of 14.5 ± 0.5 °C, treatment optima for pressure shock were determined to be 4.5–5.5 maf with 60 MPa for 6 min, resulting in 100% triploidy rate. A comparison of induction efficiency between pressure shock using this combination and cold shock initiated at 6.5 maf in −2 °C sea water for 25 min was carried out using eggs from three females. The higher hatching rates and triploidy rates, and lower abnormality rates were investigated with pressure shock treatment compared with cold shock treatment. The optimized parameters were successfully applied to three large batches of eggs (∼ 250, 300 and 330 mL) for mass production of triploid turbot. The total length and body weight of triploids were significantly lower than those of diploid counterparts at 2 months after hatching (mah), however, they exhibited a significant increase at 8 and 11 mah, respectively, and maintained this higher level thereafter. Additionally, the survival rates remained similar from 2 to 12 mah. The results of this preliminary study indicate that pressure shock rather than cold shock is more beneficial for the commercial production of triploid turbot under farming conditions.

High endoreduplication after drought-related conditions in haploid but not diploid mosses.

Endoreduplication, the duplication of the nuclear genome without mitosis, is a common process in plants, especially in angiosperms and mosses. Accumulating evidence supports the relationship between endoreduplication and plastic responses to stress factors. Here, we investigated the level of endoreduplication in Ceratodon (Bryophyta), which includes the model organism Ceratodon purpureus. We used flow cytometry to estimate the DNA content of 294 samples from 67 localities and found three well-defined cytotypes, two haploids and one diploid, the haploids corresponding to C. purpureus and Ceratodon amazonum, and the diploid to Ceratodon conicus, recombination occurring between the former two. The endoreduplication index (EI) was significantly different for each cytotype, being higher in the two haploids. In addition, the EI of the haploids was higher during the hot and dry periods typical of the Mediterranean summer than during spring, whereas the EI of the diploid cytotype did not differ between seasons. Endopolyploidy may be essential in haploid mosses to buffer periods of drought and to respond rapidly to desiccation events. Our results also suggest that the EI is closely related to the basic ploidy level, but less so to the nuclear DNA content as previously suggested.

Does the evolution of micromorphology accompany chromosomal changes on dysploid and polyploid levels in the Barnardia japonica complex (Hyacinthaceae)?

BackgroundChromosome number and genome size changes via dysploidy and polyploidy accompany plant diversification and speciation. Such changes often impact also morphological characters. An excellent system to address the questions of how extensive and structured chromosomal changes within one species complex affect the phenotype is the monocot species complex of Barnardia japonica. This taxon contains two well established and distinct diploid cytotypes differing in base chromosome numbers (AA: x = 8, BB: x = 9) and their allopolyploid derivatives on several ploidy levels (from 3x to 6x). This extensive and structured genomic variation, however, is not mirrored by gross morphological differentiation.ResultsThe current study aims to analyze the correlations between the changes of chromosome numbers and genome sizes with palynological and leaf micromorphological characters in diploids and selected allopolyploids of the B. japonica complex. The chromosome numbers varied from 2n = 16 and 18 (2n = 25 with the presence of supernumerary B chromosomes), and from 2n = 26 to 51 in polyploids on four different ploidy levels (3x, 4x, 5x, and 6x). Despite additive chromosome numbers compared to diploid parental cytotypes, all polyploid cytotypes have experienced genome downsizing. Analyses of leaf micromorphological characters did not reveal any diagnostic traits that could be specifically assigned to individual cytotypes. The variation of pollen grain sizes correlated positively with ploidy levels.ConclusionsThis study clearly demonstrates that karyotype and genome size differentiation does not have to be correlated with morphological differentiation of cytotypes.

Cell type specific polyploidization in the royal fat body of termite queens

Tissue-specific endopolyploidy is widespread among plants and animals and its role in organ development and function has long been investigated. In insects, the fat body cells of sexually mature females produce substantial amounts of egg yolk precursor proteins (vitellogenins) and exhibit high polyploid levels, which is considered crucial for boosting egg production. Termites are social insects with a reproductive division of labor, and the fat bodies of mature termite queens exhibit higher ploidy levels than those of other females. The fat bodies of mature termite queens are known to be histologically and cytologically specialized in protein synthesis. However, the relationship between such modifications and polyploidization remains unknown. In this study, we investigated the relationship among cell type, queen maturation, and ploidy levels in the fat body of the termite Reticulitermes speratus. We first confirmed that the termite fat body consists of two types of cells, that is, adipocytes, metabolically active cells, and urocytes, urate-storing cells. Our ploidy analysis using flow cytometry has shown that the fat bodies of actively reproducing queens had more polyploid cells than those of newly emerged and pre-reproductive queens, regardless of the queen phenotype (adult or neotenic type). Using image-based analysis, we found that not urocytes, but adipocytes became polyploid during queen differentiation and subsequent sexual maturation. These results suggest that polyploidization in the termite queen fat body is associated with sexual maturation and is regulated in a cell type-specific manner. Our study findings have provided novel insights into the development of insect fat bodies and provide a basis for future studies to understand the functional importance of polyploidy in the fat bodies of termite queens.

Effects of substrate, water flow, and turbulence in raceway culture of the high-value agarophyte Pterocladiella capillacea

One reason most commercial seaweed aquaculture is done extensively is that land-based intensive culture is expensive. Seaweed growth in land-based raceways can be bolstered by high water flows and turbulence, and a better understanding of this may mitigate production costs. We investigated flow conditions in raceway culture of Pterocladiella capillacea, a source of pharmaceutical grade agar. We tested the effects of ploidy level (carpospores, tetraspores), substrate (rope, glass slide), and flow type in raceways (turbulent, laminar) and the laboratory (no flow) on the survival and growth of sporelings. We also assessed the effect of flow rates (1.5, 4, 10 cm s−1) and flow type (turbulent, laminar) on the growth of adult P. capillacea. Sporeling survival 48 h post-settlement was unaffected by flow type, and carpospores survived better than tetraspores on ropes. After four weeks, sporelings in raceways survived better on ropes than on glass slides, and sporelings derived from carpospores survived better on ropes than those from tetraspores. Sporelings grown in raceways were 5.8 times larger than those in the laboratory, but flow types had little effect within raceways. Seaweeds grown from carpospores grew 1.2 times larger than tetraspore in the raceways. Flow rate and type had little effect on the growth of adults, and their growth was slow possibly because of smothery by epiphytes. This study suggests P. capillacea spores are amenable to raceway culture, but adults do not fare as well in similar conditions.

Best practices for instrument settings and raw data analysis in plant flow cytometry.

Flow cytometry (FCM) is now the most widely used method to determine ploidy levels and genome size of plants. To get reliable estimates and allow reproducibility of measurements, the methodology should be standardized and follow the best practices in the field. In this article, we discuss instrument calibration and quality control and various instrument and acquisition settings (parameters, flow rate, number of events, scales, use of discriminators, peak positions). These settings must be decided before measurements because they determine the amount and quality of the data and thus influence all downstream analyses. We describe the two main approaches to raw data analysis (gating and histogram modeling), and we discuss their advantages and disadvantages. Finally, we provide a summary of best practice recommendations for data acquisition and raw data analysis in plant FCM.

Contribution of Spontaneous Mutations to Quantitative and Molecular Variation at the Highly Repetitive rDNA Locus in Yeast.

The ribosomal DNA array in Saccharomyces cerevisiae consists of many tandem repeats whose copy number is believed to be functionally important but highly labile. Regulatory mechanisms have evolved to maintain copy number by directed mutation, but how spontaneous variation at this locus is generated and selected has not been well characterized. We applied a mutation accumulation approach to quantify the impacts of mutation and selection on this unique genomic feature across hundreds of mutant strains. We find that mutational variance for this trait is relatively high, and that unselected mutations elsewhere in the genome can disrupt copy number maintenance. In consequence, copy number generally declines gradually, consistent with a previously proposed model of rDNA maintenance where a downward mutational bias is normally compensated by mechanisms that increase copy number when it is low. This pattern holds across ploidy levels and strains in the standard lab environment but differs under some stressful conditions. We identify several alleles, gene categories, and genomic features that likely affect copy number, including aneuploidy for chromosome XII. Copy number change is associated with reduced growth in diploids, consistent with stabilizing selection. Levels of standing variation in copy number are well predicted by a balance between mutation and stabilizing selection, suggesting this trait is not subject to strong diversifying selection in the wild. The rate and spectrum of point mutations within the rDNA locus itself are distinct from the rest of the genome and predictive of polymorphism locations. Our findings help differentiate the roles of mutation and selection and indicate that spontaneous mutation patterns shape several aspects of ribosomal DNA evolution.

Climate-driven past and present interspecies gene flow may have contributed to shape microscale adaptation capacity in Tillandsia lomas in hyperarid south American desert systems

Epiarenic (sand-growing) Tillandsia vegetation in the hyperarid and arid region of the Chilean-Peruvian Atacama Desert represents an extreme case of adaptation in plant species-poor ecosystems. The involved species exist at the limit of terrestrial life and form mono/oligo-specific and very characteristic structures within the landscape. Covering thousands of square kilometers they represent the major carbon sink in the hyperarid Atacama core. The various Tillandsia species and respective vegetation may have evolved and adapted independently to this extreme environment. The most abundant vicariant diploid species are T. landbeckii in Chile and T. purpurea in Peru. Spatio-temporally varying distribution range overlaps may have caused potentially adaptive gene flow between different species leading to present day gene pools. Using species distribution modelling we explored the idea that from Last Glacial Maximum (LGM) onwards both species shifted their distribution ranges, which resulted in the formation of varying suture zones from Peru towards northern Chile. We further explored genetic data from a Tillandsia loma vegetation in Southern Peru with three sympatrically growing species exemplifying inter-species gene flow crossing even ploidy levels. This mechanism highlights a strategy to evolve and adapt more rapidly to environmental changes in extreme arid and hyperarid habitats and provides an opportunity for Tillandsia populations to efficiently conserve new genotypes via subsequent clonal propagation.

Implementation of different relationship estimate methodologies in breeding value prediction in kiwiberry (Actinidia arguta)

In dioecious crops such as Actinidia arguta (kiwiberries), some of the main challenges when breeding for fruit characteristics are the selection of potential male parents and the long juvenile period. Currently, breeding values of male parents are estimated through progeny tests, which makes the breeding of new kiwiberry cultivars time-consuming and costly. The application of best linear unbiased prediction (BLUP) would allow direct estimation of sex-related traits and speed up kiwiberry breeding. In this study, we used a linear mixed model approach to estimate narrow sense heritability for one vine-related trait and five fruit-related traits for two incomplete factorial crossing designs. We obtained BLUPs for all genotypes, taking into consideration whether the relationship was pedigree-based or marker-based. Owing to the high cost of genome sequencing, it is important to understand the effects of different sources of relationship matrices on estimating breeding values across a breeding population. Because of the increasing implementation of genomic selection in crop breeding, we compared the effects of incorporating different sources of information in building relationship matrices and ploidy levels on the accuracy of BLUPs’ heritability and predictive ability. As kiwiberries are autotetraploids, multivalent chromosome formation and occasionally double reduction can occur during meiosis, and this can affect the accuracy of prediction. This study innovates the breeding programme of autotetraploid kiwiberries. We demonstrate that the accuracy of BLUPs of male siblings, without phenotypic observations, strongly improved when a tetraploid marker-based relationship matrix was used rather than parental BLUPs and female siblings with phenotypic observations.

Genetic variation in Loudetia simplex supports the presence of ancient grasslands in Madagascar

Societal Impact StatementRecognizing Loudetia‐dominated grasslands were widespread prior to human colonization highlights that open ecosystems were and continue to be an important component of Madagascar's biodiversity. A better understanding of the plant species that form grassland ecosystems is necessary for effective land management strategies that support livelihoods, but substantial financial and logistical barriers exist to implementing conservation genetic studies using contemporary genomic tools. Some challenges for population genetic analyses of non‐model polyploids lacking reference genomes can be ameliorated by developing computational resources that leverage a cost‐effective data generation strategy that requires no prior genetic knowledge of the target species. This may benefit conservation programs with small operating budgets while reducing uncertainty compared to status quo microsatellite assays.Summary The extent of Madagascar's grasslands prior to human colonization is unresolved. We used population genetic analyses of a broadly dominant C4 fire‐adapted grass, Loudetia simplex, as a proxy for estimating grassland change through time. We carefully examined the utility of target‐enrichment data for population genetics to make recommendations for conservation genetics. We explored the potential of estimating individual ploidy levels from target‐enrichment data and how assumptions about ploidy could affect analyses. We developed a novel bioinformatic pipeline to estimate ploidy and genotypes from target‐enrichment data. We estimated standard population genetic summary statistics in addition to species trees and population structure. Extended Bayesian skyline plots provided estimates of population size through time for empirical and simulated data. All Malagasy L. simplex individuals sampled in this study formed a clade and possibly indicated an ancestral Central Highland distribution of 800 m in altitude and above. Demographic models suggested grassland expansions occurred prior to the Last Interglacial Period and supported extensive grasslands prior to human colonization. Though there are limitations to target‐enrichment data for population genetic studies, we find that analyses of population structure are reliable. Genetic variation in L. simplex supports widespread grasslands in Madagascar prior to the more recent periods of notable paleoclimatic change. However, the methods explored here could not differentiate between paleoclimatic change near the Last Glacial Maximum and anthropogenic effects. Target‐enrichment data can be a valuable tool for analyses of population structure in the absence a reference genome.

Ploidy Determination in Banana Hybrids through Stomatal Studies and Chloroplast Count

Stomata are probably a viable taxonomy distinguishing feature. The study of stomata characteristics of synthetic banana hybrids and their parentage has been carried out. Ploidy determination is essential in banana breeding programs to understand the genetic makeup of hybrid plants. The purpose of this study was to determine the ploidy level of hybrids in contrast with the properties of plant stomata in synthetic banana hybrids with those of their parent plants. Therefore, by examining components such as stomata location, number of epidermal cells, number of stomata cells, stomata length, width, area, intensity and density, as well as chloroplast count, it is possible to compare and contrast the traits of synthetic banana hybrids and their parentage which have different genomes. The entire mount approach was used to make an incision for the purpose of observing stomata. By characterising the stomata traits of the synthetic hybrids and their parentage, the results were then descriptively and qualitatively assessed. Three different locations on the abaxial of the leaf were taken for the determination. The samples were collected from close to the petiole (R1), middle (R2), and distal end (R3), stomata were observed using the replica method. Results showed that more number of stomata was observed in R2of leaf abaxial. The chloroplast count in pairs of stomatal guard cells from the accessions was also measured. The chloroplast density was determined in pairs of stomatal guard cells from the accessions. Based on morphological descriptions, it was observed that 16.66% of the accessions were diploid, 50% were triploid and 33.33% were tetraploid. When considering the chloroplast count, the accessions were categorized into groups, certain accessions classified as tetraploids, others as triploids, and diploids.

Diverse eastern gamagrass ecotypes: General characteristics, ploidy levels, and biogeography

AbstractEastern gamagrass (EG), Tripsacum dactyloides, is a diverse warm‐season, perennial bunchgrass that is widespread in the eastern half of the United States. The objectives of this study were to obtain a large assortment of ecotypes, measure and quantify some of their morphological aspects, and determine ploidy level for many of these ecotypes. Nearly 300 ecotypes were collected from 10 states and planted in a common garden in Temple, TX. Plants were measured throughout the growing season and ploidy was determined using flow cytometry. Plant morphology measurements were variable, though none were statistically significantly correlated when compared pairwise to ploidy levels, origin, landform, precipitation, or temperature. However, multivariate analyses showed that ploidy was significantly correlated with plant volume, plant height, and leaf width. Diploids were more narrowly restricted to drier habitats, while the tetraploids were widespread. Tetraploids have also a higher volume than diploids. Likewise, mean annual precipitation was correlated with plant height and leaf width. Landform (wet vs. dry) for collection sites was significantly correlated with plant volume in the multivariate analysis. Though not statistically significant, the southernmost populations start growing earlier than plants collected further north. This new insight into EG ecology via the collected data is useful to better answer producer questions about climate and management operations as they differ among ecotypes.

Characterization of traditional small potato (Solanum tuberosum L.) cultivars for nutritional, quality traits and ploidy level

Traditional small potato cultivars of North Eastern region of India are an important food source, but no documentation is availableregarding their ploidy level and nutritional worth. The study was undertaken to explore these cultivars to determine their ploidy levelby flow cytometry method. Fluorescence-activated cell sorter (FACS) based method was used for the analysis of ploidy level and theknown diploid potato clone Solanum chacoense was used as the reference clone. The ploidy level of these cultivars was determined bycomparing the value of mean peak position of G1 of reference diploid clone with mean peak position of G1 of small potato cultivars.Results indicated that all the 11 small potato cultivars collected from the Northeastern region were tetraploid with an average genomesize of 837.38 Mb although they were suspected to be diploid based on the tuber characters and overall plant morphology. Thesecultivars were characterized by having anthocyanin pigmentation of newly emerged sprouts and eye on tuber, small and narrow leaf,long stolon length (average 7.38 cm) and high number (average 33.90/plant) of very small (average 7.41 g) tubers compared to thecommercial potato varieties. However, these cultivars surpassed India’s commercial tetraploid potato varieties for dry matter, starch,ascorbic acid, β-carotene and total phenol contents. Appreciably rich nutritional profile coupled with matching levels of ploidy not onlyenhanced the biodiversity of cultivated potato but also suggested the possibility of utilizing these cultivars in the breeding programmeto enhance nutrient and antioxidant contents.

The Abundant and Unique Transcripts and Alternative Splicing of the Artificially Autododecaploid London Plane (Platanus × acerifolia).

Transcription and alternative splicing (AS) are now appreciated in plants, but few studies have examined the effects of changing ploidy on transcription and AS. In this study, we showed that artificially autododecaploid plants of London plane (Platanus × acerifolia (Aiton) Willd) had few flowers relative to their hexaploid progenitors. Transcriptome analysis based on full-length Oxford Nanopore Technologies (ONTs) and next-generation sequencing (NGS) revealed that the increased ploidy level in P. × acerifolia led to more transcribed isoforms, accompanied by an increase in the number of isoforms per gene. The functional enrichment of genes indicated that novel genes transcribed specifically in the dodecaploids may have been highly correlated with the ability to maintain genome stability. The dodecaploids showed a higher number of genes with upregulated differentially expressed genes (DEGs) compared with the hexaploid counterpart. The genome duplication of P. × acerifolia resulted mainly in the DEGs involved in basic biological pathways. It was noted that there was a greater abundance of alternative splicing (AS) events and AS genes in the dodecaploids compared with the hexaploids in P. × acerifolia. In addition, a significant difference between the structure and expression of AS events between the hexaploids and dodecaploids of Platanus was found. Of note, some DEGs and differentially spliced genes (DSGs) related to floral transition and flower development were consistent with the few flower traits in the dodecaploids of P. × acerifolia. Collectively, our findings explored the difference in transcription and AS regulation between the hexaploids and dodecaploids of P. × acerifolia and gained new insight into the molecular mechanisms underlying the few-flower phenotype of P. × acerifolia. These results contribute to uncovering the regulatory role of transcription and AS in polyploids and breeding few-flower germplasms.

Cytogeography of Gagea bohemica (Liliaceae) outside the Mediterranean: two ploidy levels, spatial differentiation of cytotypes, and occurrence of mixed-ploidy populations

Gagea bohemica s.l. is a morphologically and karyologically highly variable group with many morphologically similar “narrow” taxa currently considered as a single variable species. It is predominantly distributed in Mediterranean and warmer parts of temperate belt of Europe. The large-scale data on its cytogeography and population cytotype structure which could provide a basis for taxonomy are lacking, only scattered data on ploidy have been published from various parts of its range. In this study, we sampled 106 populations in broader Central Europe, the northeastern Balkan Peninsula and the northwestern Black Sea coast in order to analyze their ploidy level, genome size and pollen stainability. Two cytotypes, i.e., tetraploid (2n = 48) and pentaploid (2n = 60), were found in the study area using chromosome counting and flow cytometry, both in pure and mixed-ploidy populations. Pure pentaploid populations are mainly distributed in Austria, Czechia, northwestern Hungary and Slovakia while tetraploid cytotype in pure and mixed-ploidy populations forming two lineages which are concentrated into two disjunct geographical areas: a western lineage in Germany and Switzerland and an eastern one in Bulgaria, southeastern Hungary, northern Greece, Romania, Serbia and Ukraine. The two lineages differ in their genome size regardless of their ploidy, indicating their independent origin. Analysis of pollen stainability using a modified Alexander stain revealed an unusual pattern with tetraploids having a lower pollen stainability (mean 44.29%) than pentaploids (mean 70.70%), but the western and eastern populations differed again from each other.

Karyotype analysis of Chinese chive germplasms with different ploidy levels and their evolutionary relationships

Karyotype analysis of Chinese chive germplasms with different ploidy levels and their evolutionary relationships

Chromosome constitution and genetic relationships of Morus spp. revealed by genomic in situ hybridization

BackgroundMulberry (Morus spp.) is an economically important woody plant, which has been used for sericulture (silk farming) for thousands of years. The genetic background of mulberry is complex due to polyploidy and frequent hybridization events.ResultsComparative genomic in situ hybridization (cGISH) and self-GISH were performed to illustrate the chromosome constitution and genetic relationships of 40 mulberry accessions belonging to 12 species and three varietas in the Morus genus and containing eight different ploidy levels. We identified six homozygous cGISH signal patterns and one heterozygous cGISH signal pattern using four genomic DNA probes. Using cGISH and self-GISH data, we defined five mulberry sections (Notabilis, Nigra, Wittiorum, and Cathayana, all contained only one species; and Alba, which contained seven closely related species and three varietas, was further divided into two subsections) and proposed the genetic relationships among them. Differential cGISH signal patterns detected in section Alba allowed us to refine the genetic relationships among the closely related members of this section.ConclusionsWe propose that GISH is an efficient tool to investigate the chromosome constitution and genetic relationships in mulberry. The results obtained here can be used to guide outbreeding of heterozygous perennial crops like mulberry.

The effect of induced polyploidy on phytochemistry, cellular organelles and the expression of genes involved in thymol and carvacrol biosynthetic pathway in thyme (Thymus vulgaris).

Induced polyploidy usually results in larger vegetative and reproductive plant organs. In order to study the effect of chromosome doubling on Thymus vulgaris, three levels of colchicine concentration including 0.1, 0.3 and 0.5% (w/v) were applied for 6, 12 and 24 hours on apical meristem of 2- and 4-leaf seedlings. Ploidy level was evaluated by flow cytometry and microscopic chromosome counting. Chemical composition of essential oils extracted by hydro-distillation was analyzed by gas Chromatography/mass spectrometry (GC/MS) and gas Chromatography (GC). The application of 0.3% colchicine at 4-leaf seedling for 6 hours resulted in the highest survival rate and the highest number of tetraploid plants. Cytogenetic and flow cytometry analyses confirmed the increase of chromosome number from 2n=2x=30 in diploids to 2n=4x=60 in induced tetraploids. Tetraploid plants had larger leaves, taller and thicker stems, dense branching, longer trichome, larger stomata, larger guard cells, and decreased number of stomata. The number of chloroplasts and mitochondria increased significantly in tetraploid plants by 1.66 and 1.63 times, respectively. The expression of CYP71D178, CYP71D180 and CYP71D181 increased in tetraploids by 3.27, 7.39 and 2.15 times, respectively, probably resulting in higher essential oil compounds, as tetraploids outyielded the diploid plants by 64.7% in essential oil, 40.9% in thymol and 18.6% in carvacrol content.