Diploid Controls Research Articles

Chromosomal aberrations and early mortality in a non-mammalian vertebrate: example from pressure-induced triploid Atlantic salmon.

In commercial aquaculture, the production of triploid fish is currently the most practical approach to prevent maturation and farm-to-wild introgression following escapes. However, triploids often exhibit poor welfare, and the underlying mechanisms remain unclear. Inheritance issues associated with sub-optimal hydrostatic pressure treatments used to induce triploidy, or the genetic background of parental fish, have been speculated to contribute. We tested this by quantifying the frequency and type of chromosomal aberrations in Atlantic salmon subjected to a gradient of sub-optimal pressure treatments (Experiment 1) and from multiple mothers (Experiment 2). From these experiments, we genotyped a subsample of ~900 eyed eggs and all ~3300 surviving parr across ~20 microsatellites. In contrast to the low frequency of chromosomal aberrations in the diploid (no hydrostatic pressure) and triploid (full 9500 PSI treatment) controls, eyed eggs subjected to sub-optimal pressure treatments (6500-8500 PSI) had a higher incidence of chromosomal aberrations such as aneuploidy and uniparental disomy, corresponding to lower triploidization success and higher egg mortality rates. We also observed maternal effects on triploidization success and incidence of chromosomal aberrations, with certain half-sibling families exhibiting more aberrations than others. Chromosomal aberrations were rare among surviving parr, suggesting a purge of maladapted individuals during early development. This study demonstrates that sub-optimal hydrostatic pressure treatments and maternal effects not only influence the success of triploidization treatments, but may also affect the incidence of chromosomal aberrations and early mortality. The results have important implications for aquaculture breeding programs and their efforts to prevent farm-to-wild introgression.

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Abstract A012: Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders

Abstract Monosomy 7 is among the most frequent cytogenetic abnormalities in hematological disorders and is associated with short survival and drug resistance. Despite its high prevalence and detrimental impact, the therapeutic vulnerabilities underlying monosomy 7-associated blood disorders remain largely elusive, impeding progress toward improved patient care. The homeostatic cellular requirement for a normal dosage of essential genes creates an opportunity to target vulnerabilities that arise due to reduced levels of proteins encoded by a haploinsufficient essential gene. Briefly, the loss of one copy of a dosage-sensitive essential gene (gene X), in combination with the inhibition of itself or a related gene (gene Y), or an associated pathway results in lethal consequences for cells. The remarkable and selective clinical efficacy of lenalidomide for the treatment of del(5q) MDS has demonstrated how allelic haploinsufficiency underlies the sensitivity to this drug by synthetic lethality. Differential expression analysis of gene and protein expression in primary AML samples with monosomy 7 revealed significant downregulation of multiple proteasome pathway members at the protein level, but not at the RNA level. Primary AML samples with -7/del(7q) exhibited increased sensitivity (low IC50) to the proteasome inhibitor bortezomib, as evidenced by two independent ex vivo drug screening cohorts (the Beat AML and the FIMM study). Chromosome 7 harbors four proteasome subunits, PSMA2, PSMC2, PSMG3, and SEM1. We performed gene expression, protein expression, copy number analysis, and individual gene knockout experiments. The results have revealed PSMA2 to be a haploinsufficient essential gene on chromosome 7. PSMA2 knockout confers leukemia a growth disadvantage for multiple AML cell clines in both TP53 wild-type and knockout backgrounds. We generated PSMA2 isogenic hemizygous deletion and diploid single-cell clones. PSMA2 hemizygous deletion cells exhibited approximately half the protein expression compared to diploid controls, confirming that PSMA2 is a haploinsufficient gene. PSMA2hemizygous deletion single-cell clones showed significantly enhanced sensitivity to all three evaluated proteasome inhibitors (bortezomib, ixazomib, and carfilzomib), aligning with the sensitivity observed in primary -7/del(7q) leukemia samples. PSMA2 hemizygous deletion cell clones displayed increased p38 and decreased pERK levels upon treatment with proteasome inhibitors, potentially contributing to their increased sensitivity to proteasome inhibitors. Proteomics analysis and in vivo validation is ongoing. As such, we have identified haploinsufficient essential gene PSMA2 mediated proteasome pathway vulnerability in monosomy 7 associated leukemia and further showed that proteasome inhibitors as promising therapeutic approaches for treating hematological disorders associated with monosomy 7. Citation Format: Haijiao Zhang, Basil Allen, Daniel Bottomly, Peter Ryabinin, Schannon K. Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A012.

Mitovesicles secreted into the extracellular space of brains with mitochondrial dysfunction impair synaptic plasticity

BackgroundHypometabolism tied to mitochondrial dysfunction occurs in the aging brain and in neurodegenerative disorders, including in Alzheimer’s disease, in Down syndrome, and in mouse models of these conditions. We have previously shown that mitovesicles, small extracellular vesicles (EVs) of mitochondrial origin, are altered in content and abundance in multiple brain conditions characterized by mitochondrial dysfunction. However, given their recent discovery, it is yet to be explored what mitovesicles regulate and modify, both under physiological conditions and in the diseased brain. In this study, we investigated the effects of mitovesicles on synaptic function, and the molecular players involved.MethodsHippocampal slices from wild-type mice were perfused with the three known types of EVs, mitovesicles, microvesicles, or exosomes, isolated from the brain of a mouse model of Down syndrome or of a diploid control and long-term potentiation (LTP) recorded. The role of the monoamine oxidases type B (MAO-B) and type A (MAO-A) in mitovesicle-driven LTP impairments was addressed by treatment of mitovesicles with the irreversible MAO inhibitors pargyline and clorgiline prior to perfusion of the hippocampal slices.ResultsMitovesicles from the brain of the Down syndrome model reduced LTP within minutes of mitovesicle addition. Mitovesicles isolated from control brains did not trigger electrophysiological effects, nor did other types of brain EVs (microvesicles and exosomes) from any genotype tested. Depleting mitovesicles of their MAO-B, but not MAO-A, activity eliminated their ability to alter LTP.ConclusionsMitovesicle impairment of LTP is a previously undescribed paracrine-like mechanism by which EVs modulate synaptic activity, demonstrating that mitovesicles are active participants in the propagation of cellular and functional homeostatic changes in the context of neurodegenerative disorders.

Triploid brown trout, Salmo trutta, develop functional gonads with age and are able to interbreed with diploid counterparts.

The study investigated if gonad maturation in triploid brown trout, Salmo trutta, was entirely suppressed or only delayed, and if triploids could interbreed with diploid counterparts. Ten percent of the total number of 3-year-old triploid S. trutta, 15% of 4-year-old fish, and 17% of 5-year-old fish produced semen. Three and 4 years old triploid fish did not produce eggs, but 15% of the 5-year-old fish did so. The quantity and sperm motility of triploid semen did not differ from diploids, but the sperm concentration was significantly lower. When diploid eggs were fertilized with triploid semen (3n × 2n crosses), the percentage of eyed stage embryos, of hatched larvae, and of normal-shaped larvae did not differ from the diploid controls. Circa 90% of 3n × 2n crosses had a ploidy level of 2.4n. In the remaining percentage of 3n × 2n crosses, the ploidy level was ≥2n and <2.4n. In sperm competition experiments where diploid eggs were fertilized with a mixture of diploid and triploid semen, 52% of the originating larvae had a ploidy level of 2n, 43% of 2.4n, and 5% of the fish were not exactly classified. From the start of feeding to an age of 248 days, the mortality rate of 3n × 2n interploid crosses and of 2n × 2n controls was similar. The growth of interploid crosses was significantly higher than that of controls. In triploid mature females, the egg mass per kilogram of body weight was significantly lower than in diploids. The mass of the non-hardened eggs and the percentile weight increase during hardening did not differ from diploid eggs. When triploid eggs were fertilized with diploid semen (2n × 3n crosses), the development rate to normal hatched larvae was less than 10%. All originating larvae had a ploidy level of 3n. From the start of feeding to an age of 248 days, 2n × 3n crosses had a higher mortality rate (15%) than diploid controls (<5%). Growth of this type of interploid crosses was reduced in comparison to controls. Therefore, triploids introduced into natural waters for recreational fisheries or escaping from farms may interbreed with diploid counterparts. This not only alters the genotypes of local populations but also changes the ploidy levels.

Generation and genetic analysis of a rainbow trout (Oncorhynchus mykiss) clonal line produced by gynogenesis

The efficiency of heat-shock treatments for producing gynogenetic double haploids was evaluated using a rainbow trout (Oncorhynchus mykiss) strain locally adapted in Brazil (2002 broodstock). Eleven and 15 females produced by conventional reproduction (FC) or meiotic gynogenesis (FG) were used as egg donors, respectively. Oocytes from each female were randomly divided into five uneven lots which were submitted to different treatments: (C2n) Normal diploid control, (H) Haploid control, (G-Me) Meiotic gynogenetic, (G-Mi) Mitotic gynogenetic, and (4 n) Control of suppression of the 1st cell cleavage. Homozygous yellow-colored (Dominant albino - DA) males were used as semen donors. Gynogenesis was induced by insemination of eggs with DA semen genetically inactivated by ultraviolet irradiation. Diploidization of gynogenic individuals was induced by an early heat shock to block 2nd polar body extrusion (G-Me), or a late heat shock to suppress first mitosis (G-Mi). Overall mean survival rates to eyed-egg and first feeding stages of treated groups, relative to respective C2n control group were: 19.21 and 14.90%, 13.02 and 8.09%, and 29.94 and 16.48%, for treatments G-Me, G-Mi and 4 n, respectively. When compared with C2n, all treatments showed lower survival rates at both stages. Survival of G-Me and G-Mi progeny from FG females were significantly higher when compared to progeny from FC females for both stages. Progenies from two different females observed to be 100% homozygous in analyzed informative microsatellite markers were used in subsequent cycles of gynogenic reproduction. One line originally derived from a single gynogenic female was reproduced for nine successive generations of meiotic gynogenetic reproduction. Forty animals from the ninth successive generation of gynogenetic reproduction were analyzed with a low-density SNP (Single Nucleotide Polymorphism) panel. All individuals were observed to be homozygous for identical alleles at all SNPs, therefore indicating that the established mitotic gynogenetic line is composed of 100% clonal individuals.

Initial investigations into the production of triploid sunshine bass using temperature shock

AbstractObjectiveThe sunshine bass (female White Bass Morone chrysops × male Striped Bass M. saxatilis) is an important food fish raised in U.S. aquaculture. Spawning is performed by manually stripping and fertilizing White Bass eggs with Striped Bass sperm. Embryos hatch in approximately 48 h. Mature sunshine bass females can become fertile and produce viable eggs, which is problematic in commercial food‐fish production; therefore, triploid fish are desired.MethodsThe present nonreplicated study incorporated three warm and three cold temperature shocks, along with three postfertilization initiation times and three exposure times for each temperature, during initial stages of incubation to induce triploidy. After exposure to temperature shocks, fertilized eggs were incubated in experimental hatching systems. The best performing cold and warm temperatures and times (and an ambient diploid control) were then used to incubate fertilized eggs in a commercial‐scale trial using standard industry practices with a single batch of eggs. Larvae were stocked at about 185,000 larvae/ha (75,000 larvae/acre) in newly filled and fertilized ponds. Fry were sampled weekly and harvested at 30 days.ResultThere were 15 survivors from the pond containing fish derived from cold‐shocked fertilized eggs; these individuals were tested via Coulter Counter, and no triploids were detected. Testing of 50 survivors from the pond containing fish derived from warm‐shocked fertilized eggs indicated that 14% were triploid.ConclusionTherefore, initial trials indicate that warm temperature shock is preferable to cold shock for inducing a greater percentage of triploid sunshine bass.

Electrophysiological effects of mitochondria‐derived extracellular vesicles: implications for memory dysfunction in neurodegenerative disorders

AbstractBackgroundMitovesicles are a newly identified subtype of small extracellular vesicles (EVs) of mitochondrial origin. We recently developed a method to separate mitovesicles from other EVs after their isolation from the extracellular space of murine and human brain tissues. This method permits the study of the roles of mitovesicles in physiology and disease.Given their origin, we hypothesized that mitochondrial dysfunction, a hallmark of both Alzheimer’s disease (AD) and Down syndrome (DS), could induce alterations in mitovesicles, thus perturbing brain homeostasis and contributing to propagation of pathology. Therefore, we studied mitovesicle components and functionality after their isolation from the brain of a mouse model of DS (hereafter Ts2) as compared to diploid (2N) controls, as well as from frontal cortices of individuals with DS as compared to their age‐matched 2N controls.MethodBrain mitovesicles were isolated and analyzed by Western blotting, electron microscopy, nanotrack analysis, and monoamine neurotransmitter degradation assay as recently described (D’Acunzo, et al., 2022, PMID: 35962195). Coronal hippocampal slices from wild‐type mice were perfused with different subtypes of brain EVs, pretreated or not with clorgiline and pargyline (1 μM each), irreversible inhibitors of the monoamine oxidases type A and B (MAO‐A and MAO‐B), respectively. Long‐term potentiation (LTP) was induced at the level of the Schaeffer collateral fibers through a theta‐burst stimulation (Fà, et al., 2014, PMID: 24643165).ResultMitovesicle number is higher in brains of DS individuals and of Ts2 mice and contain a higher MAO‐B protein amount and enzymatic activity as compared to diploid controls, suggesting a potential mitovesicle‐dependent impairment of neuroamine homeostasis in DS. In agreement with that, Ts2 (but not 2N) mitovesicles stimulated a decreased LTP in recipient hippocampal slices as compared to vehicle control (Fig. 1; mtVs: mitovesicles). This biological activity was abolished when MAO‐B (but not MAO‐A) was blocked. The other EV subtypes of both genotypes, microvesicles and exosomes, did not trigger an effect on LTP. DS changes were found to be sex independent.ConclusionPathogenic mitovesicles are previously undescribed active players of the brain extracellular milieu by regulating LTP via MAO‐B, and potentially contribute to memory formation deficiencies typically found in AD and DS.

Triploidy verification of Schizothorax richardsonii using erythrocyte cell and nucleus measurement

The individuals of diploid and triploid Schizothorax richardsonii (Gray, 1832) were identified using erythrocyte indices. The Erythrocyte cell's major axis of the triploid was 17% higher than the diploid. The increase in the nucleus major axis size of the erythrocyte of the triploid was also greater by 8% concerning the diploid fishes. The erythrocyte measurement ranged between 13.29±1.06 to 14.45±0.97 for diploid 15.91±1.32 to 16.49±1.23 for triploid erythrocyte nucleus 6.16±0.67 to 6.40±0.63 and 6.70±0.71 to 6.94±0.56 for diploid and triploid respectively. The data were analyzed by One Way of Analysis of Variance (ANOVA) for both diploid (control) and triploid (treated) erythrocyte and nucleus measurement which showed significant differences with p&lt;0.05. These parameters, thus, can successfully be used in discriminating diploid and triploid S. richadrsonii.

Induced Tetraploidy in the African Catfish, &lt;i&gt;Clarias anguillaris&lt;/i&gt;

Tetraploidy was successfully induced in Clarias anguillaris. Hatchability in tetraploids ranged between 50% and 70%, and above 95% in diploid control. About 80% and 100% of the tetraploid and diploid fry survived in the first seven days, respectively. Tetraploids showed better growth performance (12.61 g) but not significant compared to 11.2 g for the diploid. Metaphase chromosome complement of tetraploid was 108 and diploid was 54. About 20% of cells in tetraploid preparation showed 3n and 2n chromosome complements. About 1% phenotypic abnormalities were recorded after hatching in tetraploid but absent in the control.

Zebrafish (Danio rerio) Gynogenetic Production by Heat Shock: Comparison Between Mitotic and Meiotic Treatment.

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm2) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm2 was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

A comparative study of morphological characteristics in diploid and tetraploid (auto and allotetraploids) Citrullus genotypes

ABSTRACT In this study, ploidy levels were determined by stomatal observations and flow cytometry analysis of plants polyploidised by the application of 0.05% colchicine to seedlings at the first true leaf stage. In the study of developing polyploid watermelon rootstocks, the survival rate of the plants was 77%, and the polyploidisation rates were 11% and 3% according to stomatal observations and flow cytometry analysis, respectively. According to the results of flow cytometry, 22 polyploid genotypes were determined. Auto- (12) and allotetraploids (10) of Citrullus genotypes were developed, and their plant growth performance was determined in hydroponic culture in comparison with diploids, commercial rootstocks (RS841, ‘Argentario’) and watermelon cultivar (‘Crimson Tide’). Putative tetraploids and their diploid controls were grown in hydroponic culture for 21 days, and their vegetative growth performances were determined. The results showed that the increases in plant biomass depending on polyploidisation were 100% in autotetraploids and 156% in allotetraploids as compared to diploid controls.

Production of double haploid watermelon via maternal haploid induction.

Production of double haploid watermelon via maternal haploid induction.

De Novo Transcriptome Analysis of the Early Hybrid Triploid Loach (Misgurnus anguillicaudatus) Provides Novel Insights into Fertility Mechanism

As a new freshwater aquaculture product, triploid loaches (Misgurnus anguillicaudatus) are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility. In this study, a natural tetraploid loach (4n = 100) (♀) was crossed with a diploid loach (2n = 50) (♂), thus creating the hybrid triploid loach (3n = 75). The histological observations of triploid offspring and diploid controls at 4 days post-hatching (dph), 15dph, 22dph, and 50dph showed that most of the hybrid triploid loaches were abortive in the early gonad differentiation process. To explore its fertility mechanism, through transcriptome analyses of triploid offspring and diploid controls at four periods, 10 differentially expressed genes related to the early fertility mechanism were identified: amh, hormad1, rec8, h2b, plvap, zp3, h2a, nrb0b1, ddx4, and esr2. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, two pathways were identified that are closely related to the early fertility mechanism at 50dph: the estrogen signaling pathway and steroid biosynthesis. The findings laid a foundation for further exploration of their molecular inhibition mechanism in hybrid triploid loaches.

Production and Biological Assessment of VNIISPK Cultivars of Various Ploidy for the Zone of Temperate Continental Climate.

Increasing the reliability of fruit crops in unstable weather conditions of a temperate continental climate has become particularly relevant. This research was carried out based on a bioresource collection from the Russian Research Institute of Fruit Crop Breeding (VNIISPK BRC). Apple cultivars (diploids and triploids) of different maturation periods were studied. Triploid cultivars (3×) of summer maturation were developed using diploid gametes of the ‘Papirovka tetraploid’ cultivar as a donor (2-4-4-4×), triploids of winter maturation were developed using 13-6-106 (Suvorovetz: open pollination), and ‘Wealthy tetraploid’ (2-4-4-4×) or ‘Giant Spy’ (2-4-4-4×) were used as donors of diploid gametes. ‘Antonovka’ and ‘Melba’ were taken as control cultivars. The aim of this work was to evaluate the apple breeding cultivars according to the main economic and biological indicators and to determine the threshold of resistance to unfavorable conditions of the winter period for possible cultivation in specific conditions of a temperate continental climate. As a result of using the method of modeling damaging factors, apple cultivars that withstood not only the critical temperatures of January, but also especially dangerous frosts after thaws at the end of winter were identified, with the stability of vital tissues at the level of ‘Antonovka’ (a control cultivar), scab immune cultivars (RVi6) ‘Ivanovskoye’, ‘Zdorovie’ and ‘Pamyati Hitrovo’, and immune triploids (3×) ‘Vavilovskoye’, ‘Aleksandr Boyko’, Rozhdestvenskoye’ and ‘Academic Saveliev’. During the whole winter, the winter resistance of summer scab immune triploids, ‘Zhilinskoye’, ‘Maslovskoye’, ‘Yablochny Spas’ and ‘Spasskoye’, was at the level of ‘Antonovka’, but was higher than ‘Melba’. The high precocity of scab-immune cultivars, ’Afrodita’, ‘Ivanovskoye’, ‘Veniaminovskoye’, and ‘Yubiley Moskvy’, and triploids ‘Patriot’ and ‘Rozhdestvenskoye’, have been identified. Triploid cultivars are superior to diploid cultivars and control ‘Antonovka’ in fruit size. According to the complex of characteristics, promising apple cultivars were identified for planting in intensive orchards of the temperate continental climate zones, namely ‘Avgusta’ and ‘Solnyshko’ (summer cultivars), and ‘Aleksandr Boyko’, ‘Vavilovskoye’, ‘Venyaminovskoye’, ‘Ivanovskoye’ and ‘Rozhdestvenskoye’ (winter cultivars).

Tetraploidy induction enhances morphological, physiological and biochemical characteristics of wallflower (Erysimum cheiri (L.) Crantz)

Wallflower (Erysimum cheiri (L.) Crantz is one of the most important bedding flowers and potted plants in the floriculture industry. Its widespread cultivation is hindered due to self-incompatibility and limited breeding. The present study was conducted to investigate the effects of polyploidy induction on different characteristics of wallflower. Seeds were treated with colchicine (0, 0.5, 0.75 and 1%) (w/v) and oryzalin (0, 50, 100 and 150 mgl−1) along various exposure times (24, 36 and 48 h). The highest tetraploidy induction (53%) was seen in 150 mgl−1 oryzalin treatment for 36 h. The chromosome doubled plants indicated increase in trichome number, pollen size, stomata size and number of chloroplasts in the stomata guard cells, while stomata density and pollen number were decreased. The new tetraploid induced wallflowers showed compact growth habit with shorter internodes and roots, while leaves were bigger (60%) and purplish, flowering period was prolonged (65%), and flowers showed greater longevity (41%), diameter (47%) and number (78%), compared to diploid controls. Their anthocyanin and total soluble solids contents were increased by 56% and 140%, respectively. UPLC-MS analysis indicated that phytohormone contents of Ze, SA, JA and ABA were significantly increased in the tetraploid induced plants than diploid controls, while IAA, GA3 and BR were less accumulated. The new ornamental features developed for the first time in wallflower proves that polyploidization is a promising tool to introduce a commercial flowering pot plant cultivar and a new genetic resource for future breeding programs.

Whole genome duplication of wild-type and CINNAMYL ALCOHOL DEHYDROGENASE1-downregulated hybrid poplar reduces biomass yield and causes a brittle apex phenotype in field-grown wild types.

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both greenhouse- and field-grown trees. Strikingly, field-grown WT tetraploids developed a brittle apex phenotype, i.e., their tip broke off just below the apex. In addition, the chromosome doubling altered the biomass composition of field-grown, but not of greenhouse-grown tetraploid poplars. More specifically, the lignin content of field-grown tetraploid poplars was increased at the expense of matrix polysaccharides. This increase in lignin deposition in biomass is likely the cause of the observed brittle apex phenotype, though no major differences in stem anatomy or in mechanical properties could be found between di- and tetraploid WT poplars grown in the field. Finally, without biomass pretreatment, the saccharification efficiency of greenhouse- and field-grown WT diploids was not different from that of tetraploids, whereas that of greenhouse-grown hpCAD tetraploids was higher than that of greenhouse-grown diploids. Upon alkaline pretreatment, the saccharification yield of diploids was similar to that of tetraploids for all genotypes and growth conditions tested. This study showed that a whole genome duplication in hybrid WT and hpCAD poplar did neither result in further improvements in biomass yield, nor in improved biomass composition and, hence, saccharification performance.

Genome-wide characterization and analysis of Golden 2-Like transcription factors related to leaf chlorophyll synthesis in diploid and triploid Eucalyptus urophylla.

Golden 2-Like (GLK) transcription factors play a crucial role in chloroplast development and chlorophyll synthesis in many plant taxa. To date, no systematic analysis of GLK transcription factors in tree species has been conducted. In this study, 40 EgrGLK genes in the Eucalyptus grandis genome were identified and divided into seven groups based on the gene structure and motif composition. The EgrGLK genes were mapped to 11 chromosomes and the distribution of genes on chromosome was uneven. Phylogenetic analysis of GLK proteins between E. grandis and other species provided information for the high evolutionary conservation of GLK genes among different species. Prediction of cis-regulatory elements indicated that the EgrGLK genes were involved in development, light response, and hormone response. Based on the finding that the content of chlorophyll in mature leaves was the highest, and leaf chlorophyll content of triploid Eucalyptus urophylla was higher than that of the diploid control, EgrGLK expression pattern in leaves of triploid and diploid E. urophylla was examined by means of transcriptome analysis. Differential expression of EgrGLK genes in leaves of E. urophylla of different ploidies was consistent with the trend in chlorophyll content. To further explore the relationship between EgrGLK expression and chlorophyll synthesis, co-expression networks were generated, which indicated that EgrGLK genes may have a positive regulatory relationship with chlorophyll synthesis. In addition, three EgrGLK genes that may play an important role in chlorophyll synthesis were identified in the co-expression networks. And the prediction of miRNAs targeting EgrGLK genes showed that miRNAs might play an important role in the regulation of EgrGLK gene expression. This research provides valuable information for further functional characterization of GLK genes in Eucalyptus.

Effects of sex hormone rescue on gametogenesis in allotriploid crucian carp

Sex hormones play an important role in gametogenesis. During the breeding season, sex hormone levels in the sterile allotriploid crucian carp (3nAT) were significantly lower than those in the fertile diploid red crucian carp (2nRCC). Sex hormone rescue was performed to study the relationship between sex hormones and 3nAT sterility. For three months before breeding season, 3nAT females and males were fed the sex hormones estradiol and testosterone, respectively. During breeding season, hormone-rescued 3nAT fish (re-3nAT) showed normal reproductive behavior, and levels of estradiol in female re-3nAT and testosterone in male re-3nAT were similar to those of the diploid control (2nRCC). The vtg gene, which is associated with the hepatic synthesis of vitellogenin in the female, and the genes spef2, tekt1, and ift27, which are associated with the formation of the sperm flagellum, were significantly upregulated in re-3nAT as compared to 3nAT. Under artificially assisted reproduction, re-3nAT individuals produced a few eggs of different sizes and sperm with abnormal structures. However, few individuals had insemination ability. Neither zygotes produced by self-crossing re-3nAT nor those produced by crossing re-3nAT with the haploid gametes produced by 2nRCC exhibited normal embryonic development. Thus, although sex hormone treatment led to the production of abnormal gametes by re-3nAT, viable offspring did not develop, meaning that re-3nAT remained sterile. These results indicated that sex hormones affected gamete maturation but were not the determinant of triploid sterility.

In vitro Induction and Phenotypic Variations of Autotetraploid Garlic (Allium sativum L.) With Dwarfism.

Garlic (Allium sativum L.) is a compelling horticultural crop with high culinary and therapeutic values. Commercial garlic varieties are male-sterile and propagated asexually from individual cloves or bulbils. Consequently, its main breeding strategy has been confined to the time-consuming and inefficient selection approach from the existing germplasm. Polyploidy, meanwhile, plays a prominent role in conferring plants various changes in morphological, physiological, and ecological properties. Artificial polyploidy induction has gained pivotal attention to generate new genotype for further crop improvement as a mutational breeding method. In our study, efficient and reliable in vitro induction protocols of autotetraploid garlic were established by applying different antimitotic agents based on high-frequency direct shoot organogenesis initiated from inflorescence explant. The explants were cultured on solid medium containing various concentrations of colchicine or oryzalin for different duration days. Afterward, the ploidy levels of regenerated plantlets with stable and distinguished characters were confirmed by flow cytometry and chromosome counting. The colchicine concentration at 0.2% (w/v) combined with culture duration for 20 days was most efficient (the autotetraploid induction rate was 21.8%) compared to the induction rate of 4.3% using oryzalin at 60 μmol L–1 for 20 days. No polymorphic bands were detected by simple sequence repeat analysis between tetraploid and diploid plantlets. The tetraploids exhibited a stable and remarkable dwarfness effect rarely reported in artificial polyploidization among wide range of phenotypic variations. There are both morphological and cytological changes including extremely reduced plant height, thickening and broadening of leaves, disappearance of pseudostem, density reduction, and augmented width of stomatal. Furthermore, the level of phytohormones, including, indole propionic acid, gibberellin, brassinolide, zeatin, dihydrozeatin, and methyl jasmonate, was significantly lower in tetraploids than those in diploid controls, except indole acetic acid and abscisic acid, which could partly explain the dwarfness in hormonal regulation aspect. Moreover, as the typical secondary metabolites of garlic, organosulfur compounds including allicin, diallyl disulfide, and diallyl trisulfide accumulated a higher content significantly in tetraploids. The obtained dwarf genotype of autotetraploid garlic could bring new perspectives for the artificial polyploids breeding and be implemented as a new germplasm to facilitate investigation into whole-genome doubling consequences.