Haploid Clones Research Articles

Wild yeast isolation by middle school students reveals features of North American oak populations of Saccharomyces cerevisiae and Kluyveromyces lactis.

Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO, which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle school students in Denver, Colorado, USA to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. By contrast, we isolated the "dairy yeast", Kluyveromyces lactis, from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state, pointing to high genetic diversity. The outreach activity partnered middle school, high school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus.

Adaptation in Outbred Sexual Yeast is Repeatable, Polygenic and Favors Rare Haplotypes.

We carried out a 200 generation Evolve and Resequence (E&R) experiment initiated from an outbred diploid recombined 18-way synthetic base population. Replicate populations were evolved at large effective population sizes (>105 individuals), exposed to several different chemical challenges over 12 weeks of evolution, and whole-genome resequenced. Weekly forced outcrossing resulted in an average between adjacent-gene per cell division recombination rate of ∼0.0008. Despite attempts to force weekly sex, roughly half of our populations evolved cheaters and appear to be evolving asexually. Focusing on seven chemical stressors and 55 total evolved populations that remained sexual we observed large fitness gains and highly repeatable patterns of genome-wide haplotype change within chemical challenges, with limited levels of repeatability across chemical treatments. Adaptation appears highly polygenic with almost the entire genome showing significant and consistent patterns of haplotype change with little evidence for long-range linkage disequilibrium in a subset of populations for which we sequenced haploid clones. That is, almost the entire genome is under selection or drafting with selected sites. At any given locus adaptation was almost always dominated by one of the 18 founder's alleles, with that allele varying spatially and between treatments, suggesting that selection acts primarily on rare variants private to a founder or haplotype blocks harboring multiple mutations.

Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates.

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

Drug-Resistant Genotypes and Multi-Clonality in Plasmodium falciparum Analysed by Direct Genome Sequencing from Peripheral Blood of Malaria Patients

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.

Systematic screening of nuclear encoded proteins involved in the splicing metabolism of group II introns in yeast mitochondria

Studies of yeast, algae and plants have provided genetic and biochemical evidence that the splicing reaction of organellar localized group II introns either depends on proteins encoded by the introns themselves (‘maturases’) or encoded by other genes of the host organisms. However, only a few of those proteins have been identified to date and characterized in more detail. In order to find new nuclear encoded proteins that assist group II splicing, we screened a complete knockout library of Saccharomyces cerevisiae strain BY4741 consisting of 4878 viable haploid clones. The strain contains a rho + mitochondrial genome with a set of 13 introns including the three group II introns (aI1, aI2, aI5γ) in the gene encoding cytochrome- c-oxidase subunit 1 ( COX1) and the single group II intron (bI1) in the gene encoding cytochrome b ( CYTB). In our screen and initial molecular analysis, we focus on intron aI5γ, the last intron in the COX1 gene.

Mutational Load and the Transition between Diploidy and Haploidy in Experimental Populations of the Yeast Saccharomyces cerevisiae

Mutations were accumulated over hundreds of generations in a mutator strain of yeast in a constant laboratory environment. This ensured that mutations were frequent and that the quality of environment remained unchanged. Mutations were accumulated in asexual populations of diploids but their impact on fitness was tested both for the diploid clones and for haploid clones derived from them. Dozens of harmful and lethal mutations accumulated in diploids, but important phenotypic traits, such as maximum growth rate, did not deteriorate by more than 10%. There were no signs of decline in population size. In strong contrast, the populations of haploids derived from the diploids suffered from high mortality; their density was reduced by more than three orders of magnitude. These findings indicate how ineffective natural selection can be in removing deleterious mutations from populations of clonally reproducing diploids. They also suggest that phenotypic assays of heterozygous diploids may be of little value as indicators of increasing genetic degeneration.

QO Site Deficiency Can Be Compensated by Extragenic Mutations in the Hinge Region of the Iron-Sulfur Protein in the bc1 Complex of Saccharomyces cerevisiae

The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(O)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(O) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 A from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 A from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(O) site and the ISP via the flexible hinge region and that fine-tuning of the Q(O) site catalysis can be achieved by subtle changes in the linker domain of the ISP.

The contribution of haploids, diploids and clones to fine-scale population structure in the seaweed Cladophoropsis membranacea (Chlorophyta).

Local populations of Cladophoropsis membranacea exist as mats of coalesced thalli composed of free-living haploid and diploid plants including clonally reproduced plants of either phase. None of the phases are morphologically distinguishable. We used eight microsatellite loci to explore clonality and fine-scale patch structure in C. membranacea at six sites on the Canary Islands. Mats were always composites of many individuals; not single, large clones. Haploids outnumbered diploids at all sites (from 2:1 to 10:1). In both haploid and diploid plants, genetic diversity was high and there was no significant difference in allele frequencies. Significant heterozygote deficiencies were found in the diploid plants at five out of six sites and linkage disequilibrium was associated with the haploid phase at all sites. Short dispersal distances of gametes/spores and small effective population sizes associated with clonality probably contribute to inbreeding. Spatial autocorrelation analysis revealed that most clones were found within a radius of approximately 60 cm and rarely further than 5 m. Dominance of the haploid phase may reflect seasonal shifts in the relative frequencies of haploids and diploids, but may alternatively reflect superiority of locally adapted and competitively dominant, haploid clones; a strategy that is theoretically favoured in disturbed environments. Although sexual reproduction may be infrequent in C. membranacea, it is sufficient to maintain both life history phases and supports theoretical modelling studies that show that haploid-diploid life histories are an evolutionarily stable strategy.

Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breedingmethods is a long-term effort because of their long generation time. Theproduction of haploid and doubled haploid plants should offer newpossibilities for genetic studies and breeding work. In this study, haploidclones of pear were treated in vitro by oryzalin for chromosomedoubling; the level of ploidy was assessed by flow cytometry. Oryzalinappeared to be an efficient agent for chromosome doubling, the optimalconcentrations range from 200 to 300 μM. For homozygosityassessment, analyses of isozyme markers were carried out, together withmicrosatellite markers PCR-amplified with primers initially developed forapple. The use of isozyme markers confirmed homozygosity of all thedoubled haploid clones except for one. The microsatellite markers can beused earlier than isozymes for checking homozygosity during theprogramme of haploid and doubled haploid clones production. Truedoubled haploid clones of pear were obtained in less than one year andtheir acclimatisation in greenhouse has already started.

Study of RecA-independent homologous recombination and a chromosomal rearrangement in the Escherichia coli strain carrying an extended tandem duplication

A heterozygous tandem duplication in the Escherichia coli deo operon region deoA deoB::Tn5/deoC deoD thr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F- strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec+ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD+ (the DeoD+ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD+ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.

Cells of different ploidy are often present together in Cryptococcus neoformans strains.

We examined the ploidy of C. neoformans strains using both laser scanning cytometry and a fluorescence microscope equipped with a photomultiplier. Haploid strains consisted of normal-sized cells with a haploid DNA amount. In the cell population there were also large-sized cells with a diploid DNA amount. These large cells in haploid strains were isolated using a Skerman micromanipulator, cultivated, and were able to generate diploid clones. Even after only 3-5 transfers on slants, haploid cells were present in all the diploid clones examined. Conversely, haploid clones obtained by single-cell-isolation of normal-sized cells from haploid strains were also shown to contain diploid cells after 3-5 transfers. Fresh haploid isolates from the environment similarly contained diploid cells after 3-5 transfers. Thus, a cellular ploidy shift was shown to occur widely in C. neoformans strains.

3. Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania

Plasmodium falciparum parasites exist as genetically distinct haploid clones in infected people. In the Kilombero valley in south-east Tanzania, at least 85% of the inhabitants of Michenga village harbour more than one clone. Using 2 highly polymorphic unlinked markers, it has been estimated that each infected person harbours between one and 6 P. falciparum clones at any one time, with a mean of 3·5 clones. When mosquitoes acquire gametocytes of 2 different clones in a blood meal, crossing generates recombinant clones differing from their parental genotypes. The inbreeding coefficient of the parasite population has been estimated as 0·33.

Genetics of Cronartium ribicola. IV. Population structure in western North America

Population genetic parameters were estimated for six populations of Cronartium ribicola in western North America from British Columbia to the southern Sierra Nevada, and two outgroups from eastern North America, using isozyme, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers on cultured haploid clones. Diversity was low, with only 8% polymorphism in the 212 markers identified. Each polymorphic locus had only two alleles, except for an RFLP marker in the ribosomal DNA complex with multiple alleles, that resulted from variable numbers of tandem repeats. Expected heterozygosity within populations, estimated from diploid teliospores, was only 0.025. The three types of markers were highly consistent with each other for these parameters. Yet, populations were highly differentiated; the proportion of the total variation attributable to differences among populations was 0.205. Multivariate statistical analysis as well as different clustering algorithms based on contrasting evolutionary assumptions (drift, mutation) all showed similar relationships and differences among populations. Genetic distances were not associated with geographic distances; western populations within a few kilometres of each other were often more distant from each other genetically than they were from eastern populations across the continent. The lack of pattern over the landscape of this metapopulation is consistent with aspects of the life cycle and epidemiological behavior of the pathogen, in which genetic drift appears to play a major role.Key words: white pine blister rust, isozymes, RAPDs, RFLPs, multivariate analysis, genetic drift.

Genetics of Cronartium ribicola. I. Axenic culture of haploid clones

Genetically pure lines of the mononucleate (haploid) stage of Cronartium ribicola can be grown by isolating mycelium from single spots on infected white pine needles or by culturing single basidiospores on defined media preconditioned by cell-free diffusates of massed germinating basidiospores. Although growth is initially slow, cultures can be increased rapidly by alternately subculturing from solid to liquid media and back again indefinitely. The ability to culture pure haploid clones has important implications for studying the genetic structure and sexual behavior of rust populations and the nature of virulence. Keywords: white pine blister rust, sugar pine, virulence.

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

ABSTRACTIn some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy.Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale‐covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.

Applicazione dell'androgenesiin vitroal miglioramento genetico dell'asparago

Abstract The sex genetic determinants of the dioecious species Asparagus offcinalis are codified on the chromosome pair n. 5 and inherited as a monomendelian trait; since the dominant alleal controls the development of androecium, the males are heterozygous and the female homozygous. The male plants of asparagus are know to be superiour respect to the female ones for important yield components, therefore one objectiv of the breeding is the synthesis of all-male hybrids. That is possible by crossing female plants (m/m) with male plaits (M/M) homozygous for sex pair chromosome. The synthes of such kind of males by selfing the rare andromonoecious plants, involves several problems which can be overcome by using the in vitro anther culture technique. At our Institute this technique has been sistematically applied during the last twenty years and allowed to regenerate doubled haploid clones which were evaluated in the field for a minimum period of four years. The best male and female clones were then utilized as parents of all-male F1 hybrids. Following omparative varietal trials in different location for several years, the best F1 all-mal hybrids were identified and released to private seed companies for the production of commercial seed.

Conjugation between unimicronuclear doublets and singlets, and several features of the clones originating from hemikarya of Euplotes woodruffi syngen 1 (ciliophora)

Conjugation of a unimicronuclear doublet with two singlets produced three kinds of exconjugants: an exconjugant singlet cell, with a macronucleus and a micronucleus, originating from a synkaryon (diploid); an exconjugant singlet with a micronucleus and a macronucleus from a hemikaryon (haploid); and an exconjugant doublet consisting of two components, one side bearing a micronucleus and a macronucleus derived from a hemikaryon and the other side with those from a synkaryon. Of the two exconjugant singlets, one died in 64% of all cases and both singlets survived and formed clones in 20% of the cases. Using mainly the latter, the length of the immaturity period and fission rates were compared between diploid and haploid clones. Diploid clones were the same as the control clones in the morphology of their nuclear apparatus. Haploid clones showed faintly stained small micronuclei; They sometimes produced amicronucleates. Exconjugants which were considered to be diploids showed an immaturity period of over 40 fissions and fission rates of over 1.43, while those considered to be haploids often produced clones with short immaturity periods, short clonal life, and low fission rates. The F 1 singlets with short immaturity, when crossed to normal diploids, produced F 2 clones with lower viability and shorter immaturity than those produced by crosses between two normal diploids.

Production of Haploid Sugar Beet (Beta vulgaris L.) by Culturing Unpollinated Ovules

AbstractThe culture of unpollinated ovules is shown to be a suitable system for the production of haploid sugar beet (Beta vulgaris L.). The yield of haploids depended upon the genotype and varied between 0 and 13 % with a mean of 1.0 %. Haploid plants could be produced from approximately 50 % of all genotypes examined.The majority of the haploids isolated (about 90%) maintained the haploid genome level during the in vitro culture and propagation; 10% of the haploid clones showed a spontaneous doubling to the diploid genome level.

Nucleogenesis and stomatogenesis in sexual reproduction of Paramecium tetraurelia may be controlled by chromosomal factors of the germ nucleus (micronucleus)

The nature of control of development of the nucleus and the oral apparatus by the germ nucleus (micronucleus) during sexual reproduction in Paramecium tetraurelia was analyzed by studying nine euploid (mostly haploid) clones. These clones were generated by conjugation between cells lacking micronuclei (amicronucleates) and micronucleates with diploid micronuclei. All except two of the euploid clones were normal in stomatogenesis in asexual reproduction. In contrast, during subsequent sexual reproduction (autogamy), eight of the euploid clones showed correlative abnormalities in nucleogenesis and stomatogenesis. The former involves the generation of gametic nuclei and the postzygotic development of micronuclei and macronuclear anlagen. The latter involves the crucial stomatogenic step of initiation of oral membraneile assembly, as well as the specification of the normal pattern of the oral apparatus. Since during autogamy haploid nuclei underwent meiosis giving rise to aneuploid nuclei and thus genomic imbalance, the present findings support the notion that micronuclear chromosomal factors contribute to both nucleogenesis and stomatogenesis in the sexual cycle.

Effets de rayons γ 60Co sur des vitroplants haploïdes et diploïdes de Gerbera jamesonii

Gerbera jamesonii ‘Super Gerbera’ explants were irradiated in vitro with 60Co γ-rays (8–100 Gy). Some received a single dose, while others received two or three at successive multiplication cycles. Several parameters were measured after 6 weeks of in vitro culture, to compare the effects of irradiation on haploid and diploid clones. Productivity is linked to the ploidy level, whereas total growth is not. Total growth inhibition is proportionate to the given dose. Callus and plantlet growth are either inhibited or stimulated, depending on the type of treatment. Callus growth is stimulated by low doses, which induce the transformation of apices into calli; treatment of successive cycles with low doses (20 and 30 Gy for diploids, 10 and 15 Gy for haploids) ensures better plantlet development. High doses inhibited growth of calli and plantlets up to the 50% level. Variations in morphology and in chlorophyll and anthocyanin content were observed among haploid and diploid irradiated explants. Plantlet production is a good criterion for an overall evaluation. Optimal dosage to ensure 50% growth and multiplication rates is estimated at 26 Gy for diploid clones and 16 Gy for haploid clones.