Tetraploid Clones Research Articles

Automated Cytogenetic Classifier (ACC) to Support Accurate Clinical Decision Making in Acute Myeloid Leukaemia

Introduction Cytogenetic analysis is a recommended test in newly diagnosed AML but requires accurate interpretation of the International System for Human Cytogenomic Nomenclature (ISCN). Keeping up to date across recent published classifications - WHO, the European Leukaemia Net (ELN) and International Consensus Classification (ICC) - is critical for accurate prognostic assessment, but can be challenging for clinicians and laboratory scientists. Furthermore, eligibility to access state-funded treatments in the UK is dictated by certain cytogenetic results. We previously developed a webapp that integrates cytogenetic, molecular and clinical features to aid accurate classification, prognostication and provide treatment recommendations in AML (Coats et al, BJH 2021). To further improve the webapp we have developed a cytogenetic classifier to automatically interpret reports written in ISCN format. In this study we aimed to validate the automated cytogenetic classifier (ACC) in identifying abnormalities that feature in the new WHO, ELN and ICC classifications. Methodology Software for the ACC was written in Python to: 1. count the number and type of karyotypic abnormalities (subdivided into number of monosomies, complete chromosome gains and structural changes) 2. identify presence of clinically relevant, named, abnormalities that feature in international classifications (categorised as an individual abnormalityg. t(9;11)(p21.3;q23.3), or a generic abnormality e.g. any translocation involving 11q23.3). Cytogenetic reports of 50 consecutive AML cases with abnormal karyotypes analysed by Bristol Haematological Oncology Diagnostic Service (BHODS) were selected (training cohort). Each report was uploaded to the ACC and its output independently checked for accuracy. Following review of the software's performance, adjustments to the ACC were made. The updated ACC (Figure 1) analysed 98 additional reports from two other laboratories, UCLH and OUH (validation cohort). Results The ACC analysed 49/50 cytogenetic reports from the BHODS laboratory. One report could not be read by the ACC. The correct quantity of abnormalities was identified in 49/49 cases analysed. Where relevant named abnormalities occurred, these were detected in 79/80 instances. One case of acute promyelocytic leukaemia containing an idic(17p) was not identified as containing an abnormal 17p. Additionally, in one report with a complex karyotype, a monosomy 13 was erroneously detected by the ACC. Adjustments were made to the ACC such that it could analyse all 50 cases with 100% accuracy. The updated ACC then analysed 98 cases from the validation cohort. The correct quantity of abnormalities was detected in 97/ 98 cases. In one case with a highly complex karyotype containing a tetraploid clone, the ACC incorrectly counted 48, as opposed to 45, abnormalities. Clinically relevant, named, abnormalities were correctly identified in 178/179 instances. In one case, containing a 4-way translocation, the ACC did not identify the t(9;11) abnormality. Discussion Here, we show that the updated ACC is accurate in identifying the number and type of karyotypic abnormalities relevant to AML from different laboratories. Reduced accuracy in counting the number of abnormalities in highly complex karyotypes (i.e. >40 abnormalities) is not likely to impact clinical interpretation. Further validation work is needed to analyse 3 and 4-way translocations. User warnings have been added to the ACC for karyotypes containing multiple-translocations and tetraploid/triploid clones to improve clinical safety. The ACC can support clinical decision-making by streamlining cytogenetic interpretation from across multiple clinical classifications. It can be used as a stand-alone programme or integrated into more sophisticated decision support tools. The ACC has the potential to be updated to interpret cytogenetic reports from other classifications or haematological malignancies. The ACC is available as a webapp (Streamlit (ts482-cytogenetics-calculator-extractorstreamlit-w7dq9p.streamlit.app). Figure 1. Flowchart of the methodology of the updated automated cytogenetic classifier (ACC).

Tetraploidization Increases the Motility and Invasiveness of Cancer Cells.

Polyploidy and metastasis are associated with a low probability of disease-free survival in cancer patients. Polyploid cells are known to facilitate tumorigenesis. However, few data associate polyploidization with metastasis. Here, by generating and using diploid (2n) and tetraploid (4n) clones from malignant fibrous histiocytoma (MFH) and colon carcinoma (RKO), we demonstrate the migration and invasion advantage of tetraploid cells in vitro using several assays, including the wound healing, the OrisTM two-dimensional cell migration, single-cell migration tracking by video microscopy, the Boyden chamber, and the xCELLigence RTCA real-time cell migration. Motility advantage was observed despite tetraploid cell proliferation weakness. We could also demonstrate preferential metastatic potential in vivo for the tetraploid clone using the tail vein injection in mice and tracking metastatic tumors in the lung. Using the Mitelman Database of Chromosome Aberrations in Cancer, we found an accumulation of polyploid karyotypes in metastatic tumors compared to primary ones. This work reveals the clinical relevance of the polyploid subpopulation and the strategic need to highlight polyploidy in preclinical studies as a therapeutic target for metastasis.

High-Frequency Dielectrophoresis Reveals That Distinct Bio-Electric Signatures of Colorectal Cancer Cells Depend on Ploidy and Nuclear Volume.

Aneuploidy, or an incorrect chromosome number, is ubiquitous among cancers. Whole-genome duplication, resulting in tetraploidy, often occurs during the evolution of aneuploid tumors. Cancers that evolve through a tetraploid intermediate tend to be highly aneuploid and are associated with poor patient prognosis. The identification and enrichment of tetraploid cells from mixed populations is necessary to understand the role these cells play in cancer progression. Dielectrophoresis (DEP), a label-free electrokinetic technique, can distinguish cells based on their intracellular properties when stimulated above 10 MHz, but DEP has not been shown to distinguish tetraploid and/or aneuploid cancer cells from mixed tumor cell populations. Here, we used high-frequency DEP to distinguish cell subpopulations that differ in ploidy and nuclear size under flow conditions. We used impedance analysis to quantify the level of voltage decay at high frequencies and its impact on the DEP force acting on the cell. High-frequency DEP distinguished diploid cells from tetraploid clones due to their size and intracellular composition at frequencies above 40 MHz. Our findings demonstrate that high-frequency DEP can be a useful tool for identifying and distinguishing subpopulations with nuclear differences to determine their roles in disease progression.

Morpho-genetic characterization of diploid and tetraploid taro (Colocasia esculenta L. Schott) cv. Kaliurang - An Indonesian local cultivar

Kaliurang is one of the local taro cultivars in Indonesia, potentially due to its high productivity and delicacy. This study analysed morpho-genetic variation between one diploid and three tetraploid Kaliurang taro clones from <i>in vitro</i> polyploid induction after plantlet acclimatization in the greenhouse for 6 weeks. Plants’ morphological characters were analysed using the unweighted pair group method with arithmetic mean (UPGMA) method based on 50 characters according to the taro descriptor from the International Plant Genetic Resources Institute (IPGRI). Furthermore, a morphometric characterization based on the Euclidean distance of 17 digitations from mature leaves, was analysed with the Analysis Phylogenic and Evolution package in R software. DNA band pattern was also performed with ISSR to analyse the clone’s genetic variance. The results showed morpho-genetic character diversity in diploid and tetraploid plants. A plant morphological study revealed that 22 out of 50 characters were distinct, with similarity coefficients ranging from 0.35 to 0.78. Leaf geometric morphometric analysis showed changes in five digitations and formed three clusters. Twelve ISSR primers out of twenty amplified the sequence of the Kaliurang taro genome to yield two clusters with a similarity coefficient of 0.71–0.83. Based on these three characterization approaches, tetraploid clones (K2 and K3) consistently differed morphologically from diploid clones (K0).

Morphological and Anatomical Comparison between Tetraploid Stevia rebaudiana (Bertoni) Bertoni and its Parental Diploid

Stevia rebaudiana (Bertoni) Bertoni is a perennial herbaceous plant that produces natural low glycemic-index sweeteners alternative to sugar cane. The genetic improvement of Stevia needs to be investigated to increase its productivity. Although this plant has a narrow genetic diversity, genetic improvement through polyploidization may succeed. Meanwhile, genotypic characters of tetraploid Stevia were previously reported. This study then observed the phenotypic characters of the tetraploid plants, which aimed to evaluate the morphological and anatomical characteristics of tetraploid Stevia clones compared to its wild-type parental diploid plant. Three tetraploids and one diploid clone were grown in the field for 12 weeks. The results of qualitative descriptive characters showed that the tetraploid clones B60.3H8, P1T22 and P3T5 had habitus type, shoot bud shape, leaf margin, leaf venation, shape, and type of stem, type of flower, and seed similar to the diploid parental clone. However, the quantitative characters showed that the tetraploid clones had leaf size, stem diameter, root size, length of the single flower, length of the flower stalk, and length of fruit which were more extensive than those of the parental diploid clone. The leaf color of tetraploids was a darker green, and the range of initial flowering times was earlier. This finding gives more understanding of the phenotypic and anatomical characteristics of Stevia tetraploids compared to their parental diploid plant.

Apple Autotetraploids—Phenotypic Characterisation and Response to Drought Stress

Polyploidization is an important source of variability for plant breeding. Polyploids are often characterised by increased resistance to biotic and abiotic stresses. Since drought and pathogen attack are the main threats to apple cultivation, obtaining new sources of resistance is an important issue for apple breeding. The newly obtained autotetraploid clones of apple cv. ‘Redchief’ showed superior resistance to fire blight. The aim of the presented research was the in-depth phenotypic characterisation of ‘Redchief’ tetraploids and assessment of their response to drought at the physiological and genetic level. The growth of own-rooted five-year-old trees of ‘Redchief’ tetraploids was poor compared with diploids; all growth parameters—the number and length of current season shoots, the total length of current season shoots per tree and the cross-section area of the trunk—were reduced in tetraploid clones. Grafting on M9 rootstock improved the growth characteristics of ‘Redchief’ tetraploids. Compared with diploid plants, the leaves of tetraploids were thicker, with altered shape, higher chlorophyll content, and larger stomata, but the stomatal density decreased. The leaf anatomical structure of tetraploids was changed, the adaxial and abaxial epidermis and both types of mesophyll were significantly thicker than in diploids. Moreover, the pollen grains of tetraploids were larger, but their viability and germination were reduced. Under conditions of limited water supply, the reduction in growth parameters was smaller and the physiological parameters were higher in the ‘Redchief’ tetraploid clone 4x-25 than in diploid plants. The expression of APX gene was higher in tetraploids than in diploids 15 days after drought stress induction. The results suggest the enhanced drought tolerance of the studied ‘Redchief’ autotetraploid clone compared with its diploid counterpart.

Potential use of polyploid eucalypt in forestry

Polyploidy induction has been used as a strategy to select new clones, potentially increasing the genetic progress obtained by eucalypt breeding in Brazil. Genetic variation occurs among polyploids produced by the chromosome duplication of a diploid clone, which can affect their performance. The present study aimed to evaluate the performance of polyploid eucalypt clones under field conditions to verify if they are more adapted and stable for forest exploration and if polyploid performance depends on its diploid. The evaluations were performed on 80 synthetic tetraploid clones obtained, in equal number, from two diploid clones of E. grandis x E. urophylla hybrids. The experiments were conducted in four environments in São Paulo and Mato Grosso do Sul States, Brazil. Measurements of DBH (diameter at breast height) and MAI (mean annual increment) of three-year-old trees were taken. Results showed that few polyploid clones showed similar performance to their diploid of origin. On average, the diploid clone A showed performance 41.1% higher than that presented by its polyploids and the diploid clone B presented performance 75.7% higher than that obtained by the polyploids originated from it, in MAI. The clones x environments interaction was relatively expressive, although predominantly simple in many cases, indicating that the ranking of clones was similar among the evaluated sites. More adapted and stable clones were identified, although in most cases their performances were lower than diploids. The performance of polyploid clones was not affected by the performance of their diploid. The results found were important and indicate that the selection of polyploids that outperform diploid clones should be pursued, although there are still many questions regarding the use of polyploidy in eucalypt breeding.

Prospects and methods of polyploid selection of new high-yielding miscanthus clones in the natural climatic conditions of Ukraine

Goal. Expanding the breeding trait collection of breeding genotypes of the genus Miscanthus (Anderson) and the genetic basis for new allotriploid clones by transferring components for hybridization of natural wild species Miscanthus sinensis and Miscanthus sacchariflorus to the tetraploid level.&#x0D; Methods. Cytological, biotechnological, fluorescent cytphotometry, field, laboratory.&#x0D; Results. The efficiency of polyploidisation for induction of new tetraploid forms of miscanthus in liquid nutrient media supplemented with colchicine (0.05% mass) and for stabilization of myxoploid shoots (0.005% mass) for 6 h of cultivation is examined. The period of exposure for Miscanthus sinensis (2x=2x= 38) and Miscanthus sacchariflorus (2x = 2x = 38) varied from 2 hours to 3 days depending on the genetic origin of the material, with transfer to a hormonal environment. The best indicators of tetraploid induction for Miscanthus sinensis were observed for the exposure period of 2 days with polyploidization efficiency (Db%) of 31.25% and 21.42%, and for Miscanthus sacchariflorus 2 and 6 hours with 35.0% and 27.3%, respectively. The technology of transfering rooted shoots into the soil on the Yaltushkiv Eperimental Breeding Farm was improved and the composition of the soil mixture that provided 99% rooting of culture seedlings at a humidity of 60−70% and air temperature of 35−55°С was found.&#x0D; Conclusions. New biotechnological tetraploid lines of Miscanthus sinensis (2xn=4x=76) and Miscanthus sacchariflorus (2xn=4x=76) were created in the conditions of liquid nutrient media supplemented with colchicine (0.05 % mass). It is investigated that flowering of new tetraploid clones in the conditions of Ukraine for the second year of vegetation occurs from late September to early October with formation of fertile pollen grains.

Growth response of diploid and tetraploid taro (Colocasia esculenta (L.) Schott) shoot culture to drought stress using polyethylene glycol

Taro genetic improvement through polyploidy induction is expected to be tolerant to abiotic stress. Several studies have shown that polyploidy plants have a higher adaptability to dry environments. In vitro selection technique for evaluation of plant tolerance to drought stress can be done by applying polyethylene glycol (PEG) as a selection agent. The aim of the research was to investigate the growth response of diploid and tetraploid taro shoot culture to drought stress using PEG. The experiment was conducted using a completely randomized design with two factors. The first factor was the concentration of PEG at 0, 5, 10, 15 and 20%. The second factor was Bentul taro clones which were 1 diploid clone and 2 tetraploid clones (clones 4.6.3 and 5.4.4). PEG was added to liquid MS medium containing 2 mg/l BAP. Observations of growth variables were carried out every week until the six weeks of culture. Fresh and dry weights, mortality percentage and proline content were determined at six weeks of culture. The results showed that the addition of PEG in liquid medium significantly affected the number of leaves, petiole length, number of roots, fresh and dry weights, as well as shoot mortality percentage. Differences in clones significantly affected the number of leaves, fresh and dry weights. The two factors tested (PEG and clones), gave an interaction on the number of leaves, fresh and dry weights. The proline content in all diploid and tetraploid clones showed an increase with increasing PEG concentration. The LC50 value in diploid clones was 9.82%, in tetraploid clones 4.6.3 and 5.4.4 were 14.14 and 15.45%, respectively. The results showed that PEG at 10% and 15% could be used for in vitro selection to drought stress both for diploid and tetraploid taro.

Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety.

Potato (Solanum tuberosum L.) is one of the most important crops with a worldwide production of 370 million metric tons. The objectives of this study were (1) to create a high-quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (2) to evaluate the new assembly’s usefulness for various genomic methods; and (3) to assess the performance of phasing in diploid and tetraploid clones, using linked-read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg1. Comparisons of the new dAg1_v1.0 sequence to other potato genome sequences point out the high divergence between the different potato varieties and illustrate the potential of using dAg1_v1.0 sequence in breeding applications.

Growth, stomata and trichome characteristics of diploid and tetraploid Artemisia annua L. plants

Tetraploid A. annua have been reported to have higher artemisinin level compared to diploid plants. This research aimed to evaluate growth, stomata, and trichome characteristics of diploids compared to tetraploid plants. Two accessions of diploids (K.I.B and K1.2.2) and two accessions of tetraploid plants (K2.2.9 and K3.2.1) V0M1 were grown in the field for 11 weeks. Growth of plants was investigated every week by measuring plant height, number of branches per plant, fresh and dry weights. Density and size of stomata were observed using light microscopy; the number of trichomes was observed by scanning electron microscopy. The artemisinin level was analyzed by HPLC. The results showed that the plant height of both diploid and tetraploid plants was comparable. From week-1 to week-11, all plants still increased in their plant height. Similarly, numbers of branches also increased except for diploid K.I.B plants. The highest growth rate of numbers of branches was found in tetraploid clone K3.2.1. However, the highest biomass production was found in diploid clone K1.2.2. The artemisinin level of tetraploid plants was higher than that of diploid plants. The level of ploidy was confirmed by flow cytometry analysis.

PECULIARITIES OF CREATION OF MISCANTHUS SINENSIS AND MISCANTHUS SACCHARIFLORUS TETRAPLOID LINES

The work aimed at creating tetraploid lines of Miscanthus sinensis and Miscanthus sacchariflorus species. To achieve this goal, we used methods of microclonal propagation, fluorescence cytophotometry, and genomic status differentiation using computer software of AP ‘Partec’ (Germany). It was found that the percentage of cultivated shoots of Miscanthus sacchariflorus for an exposure period of 1 day was 18.86 ± 5.37%. To compare, it was51.78 ± 6.51% in Miscanthus sinensis. The best indicators of tetraploid induction in Miscanthus sinensis were observed for the exposure to colchicine for 2 days with polyploidization efficiency of 31.25% and 21.42%, and in Miscanthus sacchariflorusf or 2 hours and 6 hours with rates of 35.0% and 27.3%, respectively. To stabilize the tetraploid level of genome ploidy, we used Murashige and Skoog liquid media (1962) supplemented with 0.005% colchicine and an exposure period of myxoploids for 6 hours. The flowering of new tetraploid clones in the conditions of Ukraine was observed on the second year of vegetation in late September and the beginning of October with the formation of fertile pollen grains. However, development of a microgametophyte depends on temperature conditions, both for Miscanthus sinensis (4x) and Miscanthus sacchariflorus (4x). Breeding schemes for the formation of anisoploid populations have also been developed: M sinensis (4x) x M sinensis (2x); M. sa?chariflorus (4x) x M sinensis (2x); M sinensis (4x) x M. sachariflorus (2x)

Genetic diversity and population structure of advanced clones selected over forty years by a potato breeding program in the USA

Knowledge regarding genetic diversity and population structure of breeding materials is essential for crop improvement. The Texas A&M University Potato Breeding Program has a collection of advanced clones selected and maintained in-vitro over a 40-year period. Little is known about its genetic makeup and usefulness for the current breeding program. In this study, 214 potato clones were genotyped with the Infinium Illumina 22 K V3 Potato Array. After filtering, a total of 10,106 single nucleotide polymorphic (SNP) markers were used for analysis. Heterozygosity varied by SNP, with an overall average of 0.59. Three groups of tetraploid clones primarily based on potato market classes, were detected using STRUCTURE software and confirmed by discriminant analysis of principal components. The highest coefficient of differentiation observed between the groups was 0.14. Signatures of selection were uncovered in genes controlling potato flesh and skin color, length of plant cycle and tuberization, and carbohydrate metabolism. A core set of 43 clones was obtained using Core Hunter 3 to develop a sub-collection that retains similar genetic diversity as the whole population, minimize redundancies, and facilitates long-term conservation of genetic resources. The comprehensive molecular characterization of our breeding clone bank collection contributes to understanding the genetic diversity of existing potato resources. This analysis could be applied to other breeding programs and assist in the selection of parents, fingerprinting, protection, and management of the breeding collections.

Tetraploid induction in savory (Satureja khuzistanica): cytological, morphological, phytochemical and physiological changes

Satureja khuzitanica, due to having phenolic compounds such as carvacrol in its essential oil and rosmarinic acid in its extract has strong antioxidant and antimicrobial properties and remarkable effects for treating several diseases. In this study, polyploidy induction was performed using 0.00, 0.05, 0.10, and 0.20% (w/v) colchicine and three treatment duration of 6, 12, and 24 h on one-week-old seedlings. Treated seedlings were transplanted to the medium culture and were regenerated. After three regenerations, tetraploid clones were identified by flow cytometry technique and chromosome counting. 2C DNA values in diploid (2n = 2x = 30) and induced tetraploid (2n = 4x = 60) were 1.43 ± 0.02 pg and 2.61 ± 03 pg, respectively. The highest tetraploid induction efficiency was 30%, obtained by colchicine concentration of 0.05% and the treatment duration of 6 h. The morphological, physiological, and phytochemical characteristics were measured in diploid and autotetraploid plants. Most of the studied traits in tetraploid plants including leaf length and width, stem diameter, stomata size, area, density, leaf guard cell size, flavonoid, phenol, soluble sugar, and essential oil yield increased significantly but stomata density decreased. In conclusion application of tetraploid induction in S. khuzistanica could be used to increase the quantity of secondary metabolites. It also can be promising to introduce a new genotype with higher phenolic compounds and drug effectiveness. The successful tetraploid induction of Khuzistan savory led to an increase in morphological parameters, biomass, essential oil yield, and phenolic compounds.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability.

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

6. Genetic characterization of tetraploid/near-tetraploid acute myeloid leukemia patients

Myelodysplastic syndromes and acute myeloid leukemias are clonal malignant neoplasms arising from myeloid progenitors that are characterized by dysplasia, ineffective hematopoiesis, and variable numbers of blasts. Cytogenetic analysis plays a fundamental role in defining the genetic basis of these diseases and can aid in the risk stratification of MDS and AML patients. Tetraploidy is a rare cytogenetic finding among AML and MDS patients and much of what is known about this abnormality is based on single-case reports. To identify additional tetraploid or near-tetraploid AML or MDS patients we queried The Ohio State University Clinical Cytogenetics database. Since 2002, the cytogenetics laboratory analyzed a total of 2,039 MDS or AML patients, 38 of which harbored tetraploid or near-tetraploid clones (38/2,039 = 1.86%). We performed a retrospective analysis on this subset of patients by conducting a thorough review of their electronic medical records. In addition to standard demographic data, we present time-to-event outcomes such that the prognostic significance of this abnormality may be better understood. Furthermore, we utilized CytoGPS, a bioinformatic tool that parses karyotypes and quantifies the cumulative gains and losses across datasets, to identify recurrent abnormalities among this group of patients. Preliminary analysis demonstrates enrichment for poor prognostic indicators such as deletion of 5q, deletion of 7q, and deletion of 17p. Finally, in 16 of our 38 patients we describe molecular results (single gene or multi-gene panel) obtained at or near the time the patients presented with a tetraploid clone. This analysis aims to validate the poor prognosis associated with tetraploid or near-tetraploid AML and to better understand the co-occurrence of cytogenetic and molecular genetic abnormalities.

Assessment of Lolium perenne tetraploid clones produced from a diverse diploid breeding population

Assessment of Lolium perenne tetraploid clones produced from a diverse diploid breeding population

Spindle Architectural Features Must Be Considered Along With Cell Size to Explain the Timing of Mitotic Checkpoint Silencing.

Mitosis proceeds through a defined series of events that is largely conserved, but the amount of time needed for their completion can vary in different cells and organisms. In many systems, mitotic duration depends on the time required to satisfy and silence the spindle assembly checkpoint (SAC), also known as the mitotic checkpoint. Because SAC silencing involves trafficking SAC molecules among kinetochores, spindle, and cytoplasm, the size and geometry of the spindle relative to cell volume are expected to affect mitotic duration by influencing the timing of SAC silencing. However, the relationship between SAC silencing, cell size, and spindle dimensions is unclear. To investigate this issue, we used four DLD-1 tetraploid (4N) clones characterized by small or large nuclear and cell size. We found that the small 4N clones had longer mitotic durations than the parental DLD-1 cells and that this delay was due to differences in their metaphase duration. Leveraging a previous mathematical model for spatiotemporal regulation of SAC silencing, we show that the difference in metaphase duration, i.e., SAC silencing time, can be explained by the distinct spindle microtubule densities and sizes of the cell, spindle, and spindle poles in the 4N clones. Lastly, we demonstrate that manipulating spindle geometry can alter mitotic and metaphase duration, consistent with a model prediction. Our results suggest that spindle size does not always scale with cell size in mammalian cells and cell size is not sufficient to explain the differences in metaphase duration. Only when a number of spindle architectural features are considered along with cell size can the kinetics of SAC silencing, and hence mitotic duration, in the different clones be explained.

Apple Autotetraploids with Enhanced Resistance to Apple Scab (Venturia inaequalis) Due to Genome Duplication-Phenotypic and Genetic Evaluation.

Among the fungal diseases of apple trees, serious yield losses are due to an apple scab caused by Venturia inaequalis. Protection against this disease is based mainly on chemical treatments, which are currently very limited. Therefore, it is extremely important to introduce cultivars with reduced susceptibility to this pathogen. One of the important sources of variability for breeding is the process of polyploidization. Newly obtained polyploids may acquire new features, including increased resistance to diseases. In our earlier studies, numerous tetraploids have been obtained for several apple cultivars with ‘Free Redstar’ tetraploids manifesting enhanced resistance to apple scab. In the present study, tetraploids of ‘Free Redstar’ were assessed in terms of phenotype and genotype with particular emphasis on the genetic background of their increased resistance to apple scab. Compared to diploid plants, tetraploids (own-rooted plants) were characterized with poor growth, especially during first growing season. They had considerably shorter shoots, fewer branches, smaller stem diameter, and reshaped leaves. In contrast to own-rooted plants, in M9-grafted three-year old trees, no significant differences between diplo- and tetraploids were observed, either in morphological or physiological parameters, with the exceptions of the increased leaf thickness and chlorophyll content recorded in tetraploids. Significant differences between sibling tetraploid clones were recorded, particularly in leaf shape and some physiological parameters. The amplified fragment length polymorphism (AFLP) analysis confirmed genetic polymorphism of tetraploid clones. Methylation-sensitive amplification polymorphism (MSAP) analysis showed that the level of DNA methylation was twice as high in young tetraploid plants as in a diploid donor tree, which may explain the weaker vigour of neotetraploids in the early period of their growth in the juvenile phase. Molecular analysis showed that ‘Free Redstar’ cultivar and their tetraploids bear six Rvi genes (Rvi5, Rvi6, Rvi8, Rvi11, Rvi14 and Rvi17). Transcriptome analysis confirmed enhanced resistance to apple scab of ‘Free Redstar’ tetraploids since the expression levels of genes related to resistance were strongly enhanced in tetraploids compared to their diploid counterparts.

Marker-assisted pyramiding of potato late blight resistance genes Rpi-rzc1 and Rpi-phu1 on di- and tetraploid levels

Late blight is a disease with the biggest economic impact on potato cultivation worldwide. Pyramiding of the resistance genes originating from potato wild relatives is a breeding strategy that has a potential to produce potato cultivars durably resistant to late blight. Growing such cultivars would allow limiting the intensive chemical control of the disease. The goal of this work was to transfer the late blight resistance gene Rpi-rzc1 from Solanum ruiz-ceballosii to the tetraploid level of cultivated potato and to pyramid it with the Rpi-phu1 gene. We obtained two diploid and, through 4x-2x cross, a tetraploid potato population segregating for the Rpi-rzc1 presence, as well as one diploid and one tetraploid population where both genes were introgressed. In total, 754 progeny clones were tested for resistance to late blight in detached leaflet assays. Pathogen isolates avirulent on plants with both genes and virulent on plants with the Rpi-phu1 were used. The selection was assisted by two PCR markers flanking the Rpi-rzc1 gene and a newly designed, highly specific intragenic marker indicating the Rpi-phu1 gene presence. We obtained 26 diploid and 49 tetraploid potato clones with pyramid of both genes that should enhance the durability and spectrum of their late blight resistance and that can be exploited in potato breeding. The specificity of the marker for the Rpi-phu1 gene and the precision of the Rpi-rzc1 mapping were improved in this work.