Genetic basis for dosage sensitivity in Arabidopsis thaliana

Aneuploidy, the relative excess or deficiency of specific chromosome types, results in gene dosage imbalance. Plants can produce viable and fertile aneuploid individuals, while most animal aneuploids are inviable or developmentally abnormal. The swarms of aneuploid progeny produced by Arabidopsis triploids constitute an excellent model to investigate the mechanisms governing dosage sensitivity and aneuploid syndromes. Indeed, genotype alters the frequency of aneuploid types within these swarms. Recombinant inbred lines that were derived from a triploid hybrid segregated into diploid and tetraploid individuals. In these recombinant inbred lines, a single locus, which we call SENSITIVE TO DOSAGE IMBALANCE (SDI), exhibited segregation distortion in the tetraploid subpopulation only. Recent progress in quantitative genotyping now allows molecular karyotyping and genetic analysis of aneuploid populations. In this study, we investigated the causes of the ploidy-specific distortion at SDI. Allele frequency was distorted in the aneuploid swarms produced by the triploid hybrid. We developed a simple quantitative measure for aneuploidy lethality and using this measure demonstrated that distortion was greatest in the aneuploids facing the strongest viability selection. When triploids were crossed to euploids, the progeny, which lack severe aneuploids, exhibited no distortion at SDI. Genetic characterization of SDI in the aneuploid swarm identified a mechanism governing aneuploid survival, perhaps by buffering the effects of dosage imbalance. As such, SDI could increase the likelihood of retaining genomic rearrangements such as segmental duplications. Additionally, in species where triploids are fertile, aneuploid survival would facilitate gene flow between diploid and tetraploid populations via a triploid bridge and prevent polyploid speciation. Our results demonstrate that positional cloning of loci affecting traits in populations containing ploidy and chromosome number variants is now feasible using quantitative genotyping approaches.     

Genetic stability of human endometrial mesenchymal stem cells assessed with morphological and molecular karyotyping

The aim of this study was to monitor the genetic stability of endometrial mesenchymal stem cells (eMSCs) by G-banding and molecular karyotyping. We evaluated the sensitivity of each method to assess the genetic stability of eMSCs. G-banding karyotyping performed on passages 6 and 15 showed that more than 80% cells had normal karyotype. Random karyotypic changes were found in a small part of the cell population: aneuploidy, isochromosomes, chromosome breakages, interchromosomal association. Molecular karyotyping carried out on the 6th and 14th passages revealed genomic stability, except for in the case of chromosomes 7 and 14. Microduplications 7q36.3 (62 kb) and 14q11.2 (165kb) were found in these chromosomes. We interpreted these aberrations as being derived from the donor of these cells. The morphological and molecular karyotyping complemented each other. Using these methods, we can analyze karyotypic stability at different levels of the genomic organization.     

Epigenetic processes in a tetraploid mammal

Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome.     

Recent progress and challenges in population genetics of polyploid organisms: an overview of current state‐of‐the‐art molecular and statistical tools

Despite the importance of polyploidy and the increasing availability of new genomic data, there remain important gaps in our knowledge of polyploid population genetics. These gaps arise from the complex nature of polyploid data (e.g. multiple alleles and loci, mixed inheritance patterns, association between ploidy and mating system variation). Furthermore, many of the standard tools for population genetics that have been developed for diploids are often not feasible for polyploids. This review aims to provide an overview of the state‐of‐the‐art in polyploid population genetics and to identify the main areas where further development of molecular techniques and statistical theory is required. We review commonly used molecular tools (amplified fragment length polymorphism, microsatellites, Sanger sequencing, next‐generation sequencing and derived technologies) and their challenges associated with their use in polyploid populations: that is, allele dosage determination, null alleles, difficulty of distinguishing orthologues from paralogues and copy number variation. In addition, we review the approaches that have been used for population genetic analysis in polyploids and their specific problems. These problems are in most cases directly associated with dosage uncertainty and the problem of inferring allele frequencies and assumptions regarding inheritance. This leads us to conclude that for advancing the field of polyploid population genetics, most priority should be given to development of new molecular approaches that allow efficient dosage determination, and to further development of analytical approaches to circumvent dosage uncertainty and to accommodate ‘flexible’ modes of inheritance. In addition, there is a need for more simulation‐based studies that test what kinds of biases could result from both existing and novel approaches.     

Estimation of allele frequencies in polyploids under certain patterns of inheritance

Allele frequencies have long been studied by biologists interested in evolution and speciation. More recently, with the application of molecular markers in human DNA profiling we have also seen the need for reliable population allele frequency estimates for making probabilistic inferences. There is now interest in applying the same DNA profiling technology to identification of plant varieties. HortResearch maintains a large germplasm of horticultural plant species. It is becoming evident that accurate identification of these accessions through DNA fingerprinting is essential for effective utilisation and maintenance of this germplasm. Microsatellites are the markers of choice for this fingerprinting. However, such markers do not reveal the dosage of alleles in a polyploid. Polyploidy is common amongst horticultural plants. Estimating allele frequencies in a polyploid population is, therefore, complicated because of some marker genotypes being phenotypically indistinguishable. For example, in a tetraploid, with four alleles at a locus showing polysomic inheritance, although 35 genotypes are possible, these will fall into only 15 marker phenotypic classes. Furthermore 'null' individuals are rarely detected in polyploids. Furthermore, some polyploids can be cryptic exhibiting disomy, instead of the polysomic inheritance. We will discuss the implications of these factors and present an EM-type algorithm for estimating allele frequencies of a polyploid population under certain patterns of inheritance. The method will be demonstrated on simulated data. We also discuss the nature of some of the additional problems that may be encountered with estimating allele frequencies in polyploids for which other solutions still need to be developed.     

Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts

The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.     

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

 The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids. Received: 4 January 1999 / Accepted: 4 January 1999     

Population genetic structure of diploid sexual and polyploid apomictic hawthorns (Crataegus; Rosaceae) in the Pacific Northwest

Polyploidy and gametophytic apomixis are two important and associated processes in plants. Many hawthorn species are polyploids and can reproduce both sexually and apomictically. However, the population genetic structure of these species is poorly understood. Crataegus douglasii is represented exclusively by self-compatible tetraploid pseudogamous apomicts across North America, whereas Crataegus suksdorfii found in the Pacific Northwest is known to include self-incompatible diploid sexuals as well as polyploid apomicts. We compare population structure and genetic variability in these two closely related taxa using microsatellite and chloroplast sequence markers. Using 13 microsatellite loci located on four linkage groups, 251 alleles were detected in 239 individuals sampled from 15 localities. Within-population multilocus genotypic variation and molecular diversity are greatest in diploid sexuals and lowest in triploid apomicts. Apart from the isolation of eastern North American populations of C. douglasii , there is little evidence of isolation by distance in this taxon. Genetic diversity in western populations of C. douglasii suggests that gene flow is frequent, and that colonization and establishment are often successful. In contrast, local populations of C. suksdorfii are more markedly differentiated. Gene flow appears to be limited primarily by distance in diploids and by apomixis and self-compatibility in polyploids. We infer that apomixis and reproductive barriers between cytotypes are factors that reduce the frequency of gene flow among populations, and may ultimately lead to allopatric speciation in C. suksdorfii . Our findings shed light on evolution in woody plants that show heterogeneous ploidy levels and reproductive systems.     

Partitioning of genetic variation across the genome using multimarker methods in a wild bird population

The underlying basis of genetic variation in quantitative traits, in terms of the number of causal variants and the size of their effects, is largely unknown in natural populations. The expectation is that complex quantitative trait variation is attributable to many, possibly interacting, causal variants, whose effects may depend upon the sex, age and the environment in which they are expressed. A recently developed methodology in animal breeding derives a value of relatedness among individuals from high‐density genomic marker data, to estimate additive genetic variance within livestock populations. Here, we adapt and test the effectiveness of these methods to partition genetic variation for complex traits across genomic regions within ecological study populations where individuals have varying degrees of relatedness. We then apply this approach for the first time to a natural population and demonstrate that genetic variation in wing length in the great tit (Parus major) reflects contributions from multiple genomic regions. We show that a polygenic additive mode of gene action best describes the patterns observed, and we find no evidence of dosage compensation for the sex chromosome. Our results suggest that most of the genomic regions that influence wing length have the same effects in both sexes. We found a limited amount of genetic variance in males that is attributed to regions that have no effects in females, which could facilitate the sexual dimorphism observed for this trait. Although this exploratory work focuses on one complex trait, the methodology is generally applicable to any trait for any laboratory or wild population, paving the way for investigating sex‐, age‐ and environment‐specific genetic effects and thus the underlying genetic architecture of phenotype in biological study systems.     

QF-PCR筛查男性不育患者Y染色体无精子症因子微缺失

为评估定量荧光PCR(Quantitative fluorescent polymerase chain reaction, QF-PCR)技术在快速筛查无精子症因子(Azoospermia factor, AZF)微缺失中的应用, 文章对1218例非梗阻性无精子症、少精子症的男性不育患者, 采用多重QF-PCR结合毛细管电泳技术, 检测Y染色体长臂AZF区9个序列标签位点(Sequence tagged site, STS)以及性染色体短臂的AMEL(Amelogenin)和SRY(Sex-determining region of Y chromosome)位点, 辅以常规染色体G显带方法进行核型分析。结果显示, 1218例患者中105例可见AZF区微缺失(8.62%), 其中AZFc区缺失(67.62%)最常见, 其次为AZFb,c区缺失(20.95%); AZFb区缺失(7.62%)和AZFa区缺失(3.81%)则较少见; 另有5例患者为AZFa,b,c区缺失合并AMEL-Y缺失, 提示可能缺少Y染色体, 经核型分析验证为46,XX(性反转)。105例AZF区微缺失患者的染色体核型分析显示染色体异常16例, 其中“Yqh-”12例。根据AMEL-X/AMEL-Y比值, 可见1218例患者中86例可能存在性染色体异常, 经核型分析验证, 68例为性染色体非整倍体。多重QF-PCR技术, 一个反应即能检测样本的多个位点, 并可提示性染色体是否存在异常, 有助于男性不育患者尽早明确病因, 也为后续的检查和治疗提供依据。     

Polyploid origins in a circumpolar complex in Draba (Brassicaceae) inferred from cloned nuclear DNA sequences and fingerprints

Polyploid evolution has been of major importance in the arctic flora, but rarely addressed on the full circumpolar scale. Herein we study the allopolyploid Draba lactea and its close allies, which form a taxonomically intricate arctic-alpine complex including diploids, tetraploids, and hexaploids. Based on samples from the entire circumpolar area, we inferred the origins of polyploids in this complex using cloned DNA sequences from two nuclear regions (one intron from a gene encoding a second largest subunit in the RNA polymerase family, RPD2, and the ribosomal internal transcribed spacer region, ITS) and DNA fingerprints (random amplified polymorphic DNAs, RAPDs). Although D. lactea and all other polyploids examined in Draba are genetic alloploids showing fixed heterozygosity, the data obtained in the present study suggest that each of the polyploids analyzed here may have originated from a single diploid lineage: hexaploid D. lactea via tetraploid D. lactea from the D. palanderiana lineage (not from the D. fladnizensis and D. nivalis lineages as previously hypothesized), the tetraploid D. turczaninovii from the D. fladnizensis lineage, the tetraploid D. porsildii from the D. lonchocarpa lineage, and a tetraploid here named Draba spB from the D. nivalis lineage. Draba lactea has probably originated several times in the Beringian area, and it is not necessary to invoke complex origins based on a combination of different species lineages as previously suggested.     

Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicaudatus: a review

This paper assesses the present state of the art of ploidy manipulation in the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae). Diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.     

Genetic diversity in diploid vs. tetraploid Rorippa amphibia (Brassicaceae)

The frequency of polyploidy increases with latitude in the Northern Hemisphere, especially in deglaciated, recently colonized areas. The cause or causes of this pattern are largely unknown, but a greater genetic diversity of individual polyploid plants due to a doubled genome and/or a hybrid origin is seen as a likely factor underlying selective advantages related to life in extreme climates and/or colonization ability. A history of colonization in itself, as well as a recent origin, and possibly a limited number of polyploidization events would all predict less genetic diversity in polyploids than in diploids. The null hypothesis of higher gene diversity in polyploids has to date hardly been quantified and is here tested in self-incompatible Rorippa amphibia (Brassicaceae). The species occurs in diploid and tetraploid forms and displays clear geographical polyploidy in Europe. On the basis of eight microsatellite loci it can be concluded that the level of gene diversity is higher in tetraploids than in diploids, to an extent that is expected under neutral evolution when taking into account the larger effective population size in the doubled cytotype. There is thus no evidence for reduced genetic diversity in the tetraploids. The evidence presented here may mean that the tetraploids' origin is not recent, has not been affected by bottlenecks and/or that tetraploids were formed multiple times while an effect of introgression may also play a role.     

植物多倍体基因组的形成与进化

多倍化是植物进化变异的自然现象,也是促进植物发生进化改变的重要力量。在被子植物中,约 70％的种类在进化史中曾发生过一次或多次多倍化的过程。目前的研究结果表明,自然界绝大多数多倍体是通过未减数配子的融合而形成的,并且很多多倍体种是通过多次独立的多倍化过程而重复发生的。由多倍化所导致的重复基因在多倍体基因组中可能有三种不同的命运,即：保持原有的功能、基因沉默或分化并执行新的功能。多倍化以后,重复基因组的进化动态则主要表现在染色体重排和“染色体二倍化”、不同基因组之间的相互渗透、以及核-质之间的相互作用等方面。     

The BOY NAMED SUE Quantitative Trait Locus Confers Increased Meiotic Stability to an Adapted Natural Allopolyploid of Arabidopsis

Whole-genome duplication resulting from polyploidy is ubiquitous in the evolutionary history of plant species. Yet, polyploids must overcome the meiotic challenge of pairing, recombining, and segregating more than two sets of chromosomes. Using genomic sequencing of synthetic and natural allopolyploids of Arabidopsis thaliana and Arabidopsis arenosa, we determined that dosage variation and chromosomal translocations consistent with homoeologous pairing were more frequent in the synthetic allopolyploids. To test the role of structural chromosomal differentiation versus genetic regulation of meiotic pairing, we performed sequenced-based, high-density genetic mapping in F2 hybrids between synthetic and natural lines. This F2 population displayed frequent dosage variation and deleterious homoeologous recombination. The genetic map derived from this population provided no indication of structural evolution of the genome of the natural allopolyploid Arabidopsis suecica, compared with its predicted parents. The F2 population displayed variation in meiotic regularity and pollen viability that correlated with a single quantitative trait locus, which we named BOY NAMED SUE, and whose beneficial allele was contributed by A. suecica. This demonstrates that an additive, gain-of-function allele contributes to meiotic stability and fertility in a recently established allopolyploid and provides an Arabidopsis system to decipher evolutionary and molecular mechanisms of meiotic regularity in polyploids.     

Other tetraploid species and conspecific diploids as sources of genetic variation for an autotetraploid

? Premise of the study: Most plants are polyploid and have more than two copies of the genome. The evolutionary success of polyploids is often attributed to their potential to harbor increased genetic variation, but it is poorly understood how polyploids can attain such variation. Because of their formation bottleneck, newly formed tetraploids start out with little variation. Tetraploids may attain genetic variation through a combination of new mutations, recurrent formation, and gene exchange with diploid ancestors or related tetraploid species. We explore the role of gene exchange and introgression in autotetraploid Rorippa amphibia, a species that harbors more genetic variation than its diploid ancestors. ? Methods: We crossed autotetraploid R. amphibia to diploid conspecifics and tetraploid R. sylvestris and backcrossed resulting F(1) hybrids. We used flow cytometry to determine the ploidy of all progeny. ? Key results: Tetraploids of R. amphibia and R. sylvestris were interfertile; F(1) hybrids were fertile and could backcross. Crosses between diploids and tetraploids yielded a small number of viable, often tetraploid progeny. This indicates that unreduced gametes can facilitate gene flow from diploids to tetraploids. We detected a frequency of unreduced gametes of around 2.7 per 1000, which was comparable between diploids and tetraploids. ? Conclusions: Introgression from tetraploid R. sylvestris provides a realistic source of variation in autotetraploid R. amphibia. Only in a scenario where other compatible partners are absent, for example immediately after tetraploidization, gene flow through unreduced gametes from diploids could be an important source of genetic variation for tetraploids.     

Genetics of early miscarriage

A miscarriage is the most frequent complication of a pregnancy. Poor chromosome preparations, culture failure, or maternal cell contamination may hamper conventional karyotyping. Techniques such as chromosomal comparative genomic hybridization (chromosomal‐CGH), array-comparative genomic hybridization (array-CGH), fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase chain reaction (QF-PCR) enable us to trace submicroscopic abnormalities. We found the prevalence of chromosome abnormalities in women facing a single sporadic miscarriage to be 45% (95% CI: 38–52; 13 studies, 7012 samples). The prevalence of chromosome abnormalities in women experiencing a subsequent miscarriage after preceding recurrent miscarriage proved to be comparable: 39% (95% CI: 29–50; 6 studies 1359 samples). More chromosome abnormalities are detected by conventional karyotyping compared to FISH or MLPA only (chromosome region specific techniques), and the same amount of abnormalities compared to QF-PCR (chromosome region specific techniques) and chromosomal‐CGH and array-CGH (whole genome techniques) only. Molecular techniques could play a role as an additional technique when culture failure or maternal contamination occurs: recent studies show that by using array-CGH, an additional 5% of submicroscopic chromosome variants can be detected. Because of the small sample size as well as the unknown clinical relevance of these molecular aberrations, more and larger studies should be performed of submicroscopic chromosome abnormalities among sporadic miscarriage samples. For recurrent miscarriage samples molecular technique studies are relatively new. It has often been suggested that miscarriages are due to chromosomal abnormalities in more than 50%, but the present review has determined that chromosomal and submicroscopic genetic abnormalities on average are prevalent in maximally half of the miscarriage samples. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

The dynamics of aneuploidy in an induced tetraploid population of Lolium multiflorum x Lolium perenne

Summary Populations of induced polyploids invariably contain a substantial proportion of aneuploid individuals. A model is described which can predict the level of aneuploidy in successive generations of a closed population of Lolium tetraploids. The results demonstrate clearly that the proportion of aneuploid individuals increases sharply for two to three generations and then stabilizes at a level determined by the gametic output of the euploid plants. A change in the gametic output of aneuploid individuals has a relatively small effect on the final level of aneuploidy reached.     

Induction of tetraploid derivatives of maize inbred lines by nitrous oxide gas treatment

Maize (Zea mays L.) is a model organism for various genetic and physiological studies. Induction of autotetraploid lines from elite inbred lines is valuable for investigating gene dosage effects on the molecular level. We applied nitrous oxide gas at the time of fertilization (30-36 h after pollination) for 20 h on maize inbred line Oh43. The nitrous oxide gas treatment between pressures of 600-1000 kPa proved to be effective in inducing tetraploids. The treatment also significantly increased the rates of germless and shriveled kernels. Twelve inbred lines were treated with nitrous oxide gas for 20 h at either 800 or 900 kPa pressures, 30 or 36 h after pollination. Although tetraploid or tetraploid class aneuploid plants from 9 of 12 inbreds tested were successfully generated, only six genotypes produced progenies. The successful tetraploid inbred lines were from the A188, B73, H99, Oh43, Stock 6, and W22 genetic backgrounds. Aneuploids, plants with broken chromosomes and chimeras, were also found among the treated materials.