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1.
BMC Bioinformatics ; 20(Suppl 12): 322, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216979

RESUMO

BACKGROUND: Cell size is a key characteristic that significantly affects many aspects of cellular physiology. There are specific control mechanisms during cell cycle that maintain the cell size within a range from generation to generation. Such control mechanisms introduce substantial variabilities to important properties of the cell cycle such as growth and division. To quantitatively study the effect of such variability in progression through cell cycle, detailed stochastic models are required. RESULTS: In this paper, a new hybrid stochastic model is proposed to study the effect of molecular noise and size control mechanism on the variabilities in cell cycle of the budding yeast Saccharomyces cerevisiae. The proposed model provides an accurate, yet computationally efficient approach for simulation of an intricate system by integrating the deterministic and stochastic simulation schemes. The developed hybrid stochastic model can successfully capture several key features of the cell cycle observed in experimental data. In particular, the proposed model: 1) confirms that the majority of noise in size control stems from low copy numbers of transcripts in the G1 phase, 2) identifies the size and time regulation modules in the size control mechanism, and 3) conforms with phenotypes of early G1 mutants in exquisite detail. CONCLUSIONS: Hybrid stochastic modeling approach can be used to provide quantitative descriptions for stochastic properties of the cell cycle within a computationally efficient framework.


Assuntos
Ciclo Celular , Modelos Biológicos , Saccharomyces cerevisiae/citologia , Fase G1 , Regulação Fúngica da Expressão Gênica , Mutação/genética , Fenótipo , Ploidias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Processos Estocásticos
2.
Nat Commun ; 10(1): 2878, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253789

RESUMO

Brassica napus, an allotetraploid crop, is hypothesized to be a hybrid from unknown varieties of Brassica rapa and Brassica oleracea. Despite the economic importance of B. napus, much is unresolved regarding its phylogenomic relationships, genetic structure, and diversification. Here we conduct a comprehensive study among diverse accessions from 183 B. napus (including rapeseed, rutabaga, and Siberian kale), 112 B. rapa, and 62 B. oleracea and its wild relatives. Using RNA-seq of B. napus accessions, we define the genetic diversity and sub-genome variance of six genetic clusters. Nuclear and organellar phylogenies for B. napus and its progenitors reveal varying patterns of inheritance and post-formation introgression. We discern regions with signatures of selective sweeps and detect 8,187 differentially expressed genes with implications for B. napus diversification. This study highlights the complex origin and evolution of B. napus providing insights that can further facilitate B. napus breeding and germplasm preservation.


Assuntos
Brassica napus/genética , Brassica napus/metabolismo , Ploidias , Regulação da Expressão Gênica de Plantas , Genômica , Organelas , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Tubérculos , Polimorfismo de Nucleotídeo Único , RNA de Plantas/genética , Análise de Sequência de RNA , Transcriptoma
3.
BMC Evol Biol ; 19(1): 130, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221097

RESUMO

BACKGROUND: Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller's Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller's Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction. RESULTS: In the present study, we generate and analyze intraindividual cloned data of a nuclear and a mitochondrial gene of sexual, fissiparous and facultative wild populations of the species Dugesia subtentaculata. We find that most individuals, independently of their reproductive strategy, are mosaics. However, the intraindividual haplotype and nucleotide diversity of fissiparous and facultative individuals is significantly higher than in sexual individuals, with no signs of Muller's Ratchet. Finally, we also find that this high intraindividual genetic diversity of fissiparous and facultative individuals is composed by different combinations of ancestral and derived haplotypes of the species. CONCLUSIONS: The intraindividual analyses of genetic diversity point out that fissiparous reproduction leaves a very special genetic footprint in individuals, characterized by mosaicism combined with the Meselson effect (named in the present study as the mosaic Meselson effect). Interestingly, the different intraindividual combinations of ancestral and derivate genetic diversity indicate that haplotypes generated during periods of fissiparous reproduction can be also transmitted to the progeny through sexual events, resulting in offspring showing a wide range of genetic diversity and putatively allowing purifying selection to act at both intraindividual and individual level. Further investigations, using Dugesia planarians as model organisms, would be of great value to delve into this new model of genetic evolution by the combination of fission and sex.


Assuntos
Planárias/genética , Planárias/fisiologia , Animais , Evolução Molecular , Variação Genética , Haplótipos , Ploidias , Reprodução , Reprodução Assexuada
4.
J Plant Res ; 132(4): 461-471, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115709

RESUMO

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.


Assuntos
Ploidias , Isolamento Reprodutivo , Tabaco/genética , Cromossomos de Plantas/genética , Cruzamentos Genéticos , DNA Intergênico/genética , Citometria de Fluxo , Flores/anatomia & histologia , Germinação/genética , Filogenia , Folhas de Planta/anatomia & histologia , Análise de Sequência de DNA , Tabaco/anatomia & histologia , Tabaco/fisiologia
5.
Bioresour Technol ; 285: 121332, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30999194

RESUMO

The aim of this work was to study salt stress effects on DNA content and oil production processes integrating harvesting, lipid accumulation and oil extraction. Salt-induced enlargement of Parachlorella kessleri cells, with increasing content of DNA and neutral lipid were found. The 34.77% neutral lipid content and biomass concentration of 0.83 g L-1 were obtained after 7 days of salt treatment, compared with that of 13.57% and 0.89 g L-1 cultivated under normal condition. Sedimentation efficiency increased markedly from 15% to 90% due to the cell enlargement. Disruption fraction and the recovery rate of total lipids of wet cells under salt stress were significantly higher than that of normal conditions (100% and 82.4% for salt stress vs.76.8% and 51.1% for normal conditions). This work demonstrated that salt-induced increase in cell size and DNA content was an effective strategy for the enhancement of oil production, microalgae harvesting and oil extraction.


Assuntos
Clorófitas , Microalgas , Biomassa , Lipídeos , Ploidias
6.
MBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015323

RESUMO

Homologous chromosome number (ploidy) has diversified among bacteria, archaea, and eukaryotes over evolution. In bacteria, model organisms such as Escherichia coli possess a single chromosome encoding the entire genome during slow growth. In contrast, other bacteria, including cyanobacteria, maintain multiple copies of individual chromosomes (polyploid). Although a correlation between ploidy level and cell size has been observed in bacteria and eukaryotes, it is poorly understood how replication of multicopy chromosomes is regulated and how ploidy level is adjusted to cell size. In addition, the advantages conferred by polyploidy are largely unknown. Here we show that only one or a few multicopy chromosomes are replicated at once in the cyanobacterium Synechococcus elongatus and that this restriction depends on regulation of DnaA activity. Inhibiting the DnaA intrinsic ATPase activity in S. elongatus increased the number of replicating chromosomes and chromosome number per cell but did not affect cell growth. In contrast, when cell growth rate was increased or decreased, DnaA level, DnaA activity, and the number of replicating chromosomes also increased or decreased in parallel, resulting in nearly constant chromosome copy number per unit of cell volume at constant temperature. When chromosome copy number was increased by inhibition of DnaA ATPase activity or reduced culture temperature, cells exhibited greater resistance to UV light. Thus, it is suggested that the stepwise replication of the genome enables cyanobacteria to maintain nearly constant gene copy number per unit of cell volume and that multicopy chromosomes function as backup genetic information to compensate for genomic damage.IMPORTANCE Polyploidy has evolved many times across the kingdom of life. The relationship between cell growth and chromosome replication in bacteria has been studied extensively in monoploid model organisms such as Escherichia coli but not in polyploid organisms. Our study of the polyploid cyanobacterium Synechococcus elongatus demonstrates that replicating chromosome number is restricted and regulated by DnaA to maintain a relatively stable gene copy number/cell volume ratio during cell growth. In addition, our results suggest that polyploidy confers resistance to UV, which damages DNA. This compensatory polyploidy is likely necessitated by photosynthesis, which requires sunlight and generates damaging reactive oxygen species, and may also explain how polyploid bacteria can adapt to extreme environments with high risk of DNA damage.


Assuntos
Cromossomos/metabolismo , Replicação do DNA , Ploidias , Synechococcus/crescimento & desenvolvimento , Synechococcus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dosagem de Genes , Synechococcus/enzimologia
7.
Genetics ; 212(1): 141-152, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30902809

RESUMO

Parental imbalances in the endosperm leading to impaired development and eventual hybrid seed failure are common causes of postzygotic isolation in flowering plants. Endosperm sensitivity to parental dosage is reflected by canonical phenotypes of "parental excess" in reciprocal interploid crosses. Moreover, parental-excess traits are also evident in many homoploid interspecific crosses, potentially reflecting among-lineage variation in "effective ploidy" driven by endosperm properties. However, the genetic basis of effective ploidy is unknown and genome-wide expression perturbations in parental-excess endosperms from homoploid crosses have yet to be reported. The tomato clade (Solanum section Lycopersicon), encompassing closely related diploids with partial-to-complete hybrid seed failure, provides outstanding opportunities to study these issues. Here, we compared replicated endosperm transcriptomes from six crosses within and among three wild tomato lineages. Strikingly, strongly inviable hybrid crosses displayed conspicuous, asymmetric expression perturbations that mirror previously characterized parental-excess phenotypes. Solanum peruvianum, the species inferred to have evolved higher effective ploidy than the other two, drove expression landscape polarization between maternal and paternal roles. This global expression divergence was mirrored in functionally important gene families such as MADS-box transcription factors and E3 ubiquitin ligases, and revealed differences in cell cycle tuning that match phenotypic differences in developing endosperm and mature seed size between reciprocal crosses. Our work starts to uncover the complex interactions between expression divergence, parental conflict, and hybrid seed failure that likely contributed to plant diversity.


Assuntos
Cruzamentos Genéticos , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento , Ploidias , Solanum/genética , Endosperma/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Solanum/crescimento & desenvolvimento
8.
Planta ; 249(6): 1875-1887, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30864014

RESUMO

MAIN CONCLUSION: A set of reliable SSR markers were developed for Ziziphus mauritiana. The genetic relationship of Z. mauritiana germplasms was generally consistent with their geographical origin, and low diversity in the maternal lineage was revealed. Ziziphus mauritiana, known as Indian jujube, is an important fruit crop that is native to southern Asia and eastern Africa. There is a variety of germplasm resources, and particularly many new cultivars were selected and introduced into wide tropical regions in recent years. However, there are few practical molecular markers for cultivar authentication and genetic analysis. In this study, we developed 55 polymorphic nuclear SSR markers based on restriction-site associated DNA sequences and transcriptome sequencing. We selected 14 robust nSSR markers for further analysis of 117 Z. mauritiana accessions from four countries (45 from China, 39 from Vietnam, 25 from Pakistan and 8 from Myanmar). In total, 137 alleles were detected and DNA fingerprints for each accession were constructed. Cluster analysis based on the unweighted pair group method with arithmetic mean displayed that most accessions clustered consistently with their geographic origin. In addition, there was common and high degree polyploidization based on nSSR and flow cytometry analyses. Only two of the 50 SSR loci in noncoding regions from the chloroplast genome had polymorphisms, and 5 haplotypes in total were identified among the 117 accessions. Haplotype C with 89 accessions was the most dominant haplotype and presented in four countries. This indicates low diversity in the maternal lineage of tested Z. mauritiana germplasm. Our research provides reliable marker resources for cultivar authentication and new insights into the genetic diversity, polyploidization and domestication of Z. mauritiana.


Assuntos
Variação Genética , Genoma de Planta/genética , Repetições de Microssatélites/genética , Ziziphus/genética , Alelos , Análise por Conglomerados , Domesticação , Marcadores Genéticos/genética , Ploidias , Polimorfismo Genético/genética
9.
Genome ; 62(4): 243-252, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30785785

RESUMO

Sweet potato is one of the most important crops worldwide; however, basic research in this crop is limited. In this study, we aimed to construct a detailed karyotype of six species of Ipomoea (hexaploid Ipomoea batatas and five related species, namely, one tetraploid, I. tabascana and four diploids, I. splendor-sylvae, I. trifida, I. tenuissima, and I. × leucantha) and understand the relationship among these species. Two satellite repeats (viz., Itf_1 and Itf_2) were identified from the diploid I. trifida genome sequence using RepeatExplorer on Galaxy. Together with the ribosomal DNA (rDNA), although without distinguishable chromosomes, a detailed karyotype was constructed for the six species. Our results showed a similar karyotype between I. tenuissima and I. × leucantha, indicating their close relationship. The signal distribution pattern of Itf_1, 45S rDNA combination, detected only in I. trifida, I. tabascana, and I. batatas, implied their close relationships. The chromosomes carrying 5S rDNA could be conserved among the six species as they always carried the Itf_2 signals, which generated a similar signal distribution pattern. The results enabled a detailed comparative cytogenetic analysis, providing valuable information to understand the relationship among these species and help assemble the genome sequence of the six species of Ipomoea.


Assuntos
DNA de Plantas , Ipomoea/genética , Cariótipo , Sequências Repetitivas de Ácido Nucleico , Cromossomos de Plantas , DNA Ribossômico , Ipomoea batatas/genética , Ploidias , Análise de Sequência de DNA
10.
Methods Mol Biol ; 1920: 111-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737689

RESUMO

Metazoan animals are typically diploid, possessing two sets of a chromosome in the somatic cells of an organism. In naturally diploid species, alteration from the endogenous diploid state is usually embryonic lethal. However, the ability to experimentally manipulate ploidy of animal embryos has fundamental as well as applied biology advantages. In this chapter we describe experimental procedures to convert normally diploid zebrafish embryos into haploid or tetraploid states. We also describe methodologies to verify the ploidy of embryos and the utility of ploidy manipulation in expediting the isolation of mutations using both forward and reverse genetic strategies in zebrafish.


Assuntos
Desenvolvimento Embrionário/genética , Engenharia Genética , Testes Genéticos , Ploidias , Peixe-Zebra/genética , Animais , Diploide , Técnicas de Cultura Embrionária , Embrião não Mamífero , Feminino , Fertilização In Vitro , Estudos de Associação Genética , Engenharia Genética/métodos , Haploidia , Hibridização in Situ Fluorescente , Masculino , Mutação , Poliploidia , Característica Quantitativa Herdável , Espermatozoides/metabolismo , Espermatozoides/efeitos da radiação
11.
Mol Immunol ; 107: 123-131, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30738249

RESUMO

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Megacariócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2/genética , Subunidade p45 do Fator de Transcrição NF-E2/imunologia , Selectina-P/genética , Selectina-P/imunologia , Ploidias , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Acetato de Tetradecanoilforbol/farmacologia
12.
Plant Reprod ; 32(1): 63-75, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30719569

RESUMO

KEY MESSAGE: Application of a low-input chromatin profiling method, CUT&RUN, to FACS-purified Arabidopsis endosperm nuclei generates parental-specific genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility. Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. In Arabidopsis, profiling the epigenetic landscape of endosperm on a genome-wide scale is challenging due to its small size, mode of development and close association with maternal tissue. Here, we applied a low-input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using limited numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN make it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable further study of tissue- or cell-type-specific epigenomes in Arabidopsis and other plant species.


Assuntos
Arabidopsis/metabolismo , Cromatina/metabolismo , Endosperma/metabolismo , Núcleo Celular/metabolismo , Epigênese Genética , Citometria de Fluxo , Ploidias , Reprodutibilidade dos Testes
13.
Proc Natl Acad Sci U S A ; 116(11): 5182-5187, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30792353

RESUMO

Floret fertility is a key determinant of the number of grains per inflorescence in cereals. During the evolution of wheat (Triticum sp.), floret fertility has increased, such that current bread wheat (Triticum aestivum) cultivars set three to five grains per spikelet. However, little is known regarding the genetic basis of floret fertility. The locus Grain Number Increase 1 (GNI1) is shown here to be an important contributor to floret fertility. GNI1 evolved in the Triticeae through gene duplication. The gene, which encodes a homeodomain leucine zipper class I (HD-Zip I) transcription factor, was expressed most abundantly in the most apical floret primordia and in parts of the rachilla, suggesting that it acts to inhibit rachilla growth and development. The level of GNI1 expression has decreased over the course of wheat evolution under domestication, leading to the production of spikes bearing more fertile florets and setting more grains per spikelet. Genetic analysis has revealed that the reduced-function allele GNI-A1 contributes to the increased number of fertile florets per spikelet. The RNAi-based knockdown of GNI1 led to an increase in the number of both fertile florets and grains in hexaploid wheat. Mutants carrying an impaired GNI-A1 allele out-yielded WT allele carriers under field conditions. The data show that gene duplication generated evolutionary novelty affecting floret fertility while mutations favoring increased grain production have been under selection during wheat evolution under domestication.


Assuntos
Fertilidade/genética , Flores/genética , Flores/fisiologia , Genes Homeobox , Mutação/genética , Triticum/genética , Triticum/fisiologia , Alelos , Clonagem Molecular , Evolução Molecular , Flores/anatomia & histologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ploidias , Locos de Características Quantitativas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triticum/anatomia & histologia
14.
Planta ; 249(4): 953-973, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30715560

RESUMO

MAIN CONCLUSION: Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.


Assuntos
Biotecnologia , Edição de Genes , Plantas Medicinais/genética , Reatores Biológicos , Biotecnologia/métodos , Edição de Genes/métodos , Engenharia Genética/métodos , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Medicinais/metabolismo , Ploidias , Técnicas de Cultura de Tecidos
15.
Genome Biol ; 20(1): 27, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30722791

RESUMO

BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed. RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects. CONCLUSION: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.


Assuntos
Sistemas CRISPR-Cas , Variação Estrutural do Genoma , Genômica/métodos , Sequenciamento Completo do Genoma , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Ploidias , Software
16.
J Assist Reprod Genet ; 36(4): 629-636, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30617927

RESUMO

PURPOSE: This paper aims to investigate the efficacy of IVF with preimplantation genetic testing for aneuploidy (PGT-A), using only best-scoring blastocysts from young (≤ 35 years) infertile patients undergoing single blastocyst frozen embryo transfers (FET). METHOD: In this randomized controlled trial (RCT) registered 29 March 2017, 302 infertile patient-couples eligible to participate underwent autologous ICSI blastocyst freeze-all cycles. Two-hundred and twenty patient-couples satisfied the inclusion criteria (i.e., female age ≤ 35 years, two-day 5 ≥ 2BB blastocysts) and were randomized to either the PGT-A (PGT-A group, n = 109) selection arm or morphology score (morphology group, n = 111) selection arm. In both arms, the highest ranking (by morphological score) blastocysts were selected for FET. RESULTS: Of the 109 best-scoring blastocysts that underwent PGT-A, 80 were predicted to be euploid (73.4%) and were transferred in FET (euploid subgroup). There was no statistical difference in LB rate between the euploid subgroup and morphology group (56.3% vs 58.6%, odds ratio 0.91 (95% CI 0.51-1.63), p = 0.750). In a multiple logistic regression, the transfer of euploid blastocysts was not found to be a significant predictor of LB when adjusting for female age, infertility duration, antral follicle count, and blastocyst quality, with the independent odds expressed as 0.91 (95% CI 0.50-1.66, p = 0.760). CONCLUSION: In young (≤ 35 years) infertile patients with at least two ≥ 2BB blastocysts, PGT-A blastocyst selection does not result in an enhanced LB rate, with the evidence suggesting that the effectivity of PGT-A may be limited by the effectivity of TE biopsy. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03095053.


Assuntos
Desenvolvimento Embrionário/genética , Infertilidade/genética , Ploidias , Transferência de Embrião Único/métodos , Adulto , Aneuploidia , Biópsia , Blastocisto/citologia , Blastocisto/metabolismo , Índice de Massa Corporal , Técnicas de Cultura Embrionária , Implantação do Embrião/fisiologia , Feminino , Fertilização In Vitro , Testes Genéticos , Humanos , Infertilidade/patologia , Gravidez , Taxa de Gravidez , Diagnóstico Pré-Implantação/métodos
17.
Fertil Steril ; 111(4): 753-762.e1, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30683589

RESUMO

OBJECTIVE: To develop and validate Raman metabolic footprint analysis to determine chromosome euploidy and aneuploidy in embryos fertilized in vitro. DESIGN: Retrospective study. SETTING: Academic hospital. PATIENT(S): Unselected assisted reproductive technology population. INTERVENTION(S): To establish the analysis protocol, spent embryo culture medium samples with known genetic outcomes from 87 human embryos were collected and measured with the use of Raman spectroscopy. Individual Raman spectra were analyzed to find biologic components contributing to either euploidy or aneuploidy. To validate the protocol via machine-learning algorithms, additional 1,107 Raman spectra from 123 embryo culture media (61 euploidy and 62 aneuploidy) were analyzed. MAIN OUTCOME MEASURE(S): Raman-based footprint profiling of spent culture media and preimplantation genetic testing for aneuploidy (PGT-A). RESULT(S): Mean-centered Raman spectra and principal component analysis showed differences in the footprints of euploid and aneuploid embryos growing in culture medium. Significant differences in Raman bands associated with small RNAs and lipids were also observed. Stacking classification based on k-nearest-neighbor, random forests, and extreme-gradient-boosting algorithms achieved an overall accuracy of 95.9% in correctly assigning either euploidy or aneuploidy based on Raman spectra, which was validated by PGT-A sequencing results. CONCLUSION(S): This study suggests that chromosomal abnormalities in embryos should lead to changes of metabolic footprints in embryo growth medium that can be detected by Raman spectroscopy. The ploidy status of embryos was analyzed by means of Raman-based footprint profiling of spent culture media and was consistent with PGT-A testing performed by next-generation sequencing.


Assuntos
Aneuploidia , Meios de Cultura/química , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Diagnóstico Pré-Implantação/métodos , Análise Espectral Raman/métodos , Células Cultivadas , Aberrações Cromossômicas , Meios de Cultura/metabolismo , Análise Citogenética/métodos , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/metabolismo , Fertilização In Vitro , Ensaios de Triagem em Larga Escala , Humanos , Aprendizado de Máquina , Metaboloma , Modelos Biológicos , Ploidias , Valor Preditivo dos Testes , Estudos Retrospectivos
18.
Genes Genomics ; 41(4): 445-458, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30610620

RESUMO

INTRODUCTION: The phosphatidylethanolamine-binding protein (PEBP) gene family plays a crucial role in seed germination, reproductive transformation, and other important developmental processes in plants, but its distribution in Gossypium genomes or species, evolutionary properties, and the fates of multiple duplicated genes remain unclear. OBJECTIVES: The primary objectives of this study were to elucidate the distribution and characteristics of PEBP genes in Gossypium, as well as the evolutionary pattern of duplication and deletion, and functional differentiation of PEBPs in plants. METHODS: Using the PEBP protein sequences in Arabidopsis thaliana as queries, blast alignment was carried out for the identification of PEBP genes in four sequenced cotton species. Using the primers designed according to the PEBP genome sequences, PEBP genes were cloned from 15 representative genomes of Gossypium genus, and the gene structure, CDS sequence, protein sequence and properties were predicted and phylogenetic analysis was performed. Taking PEBP proteins of grape as reference, grouping of orthologous gene, analysis of phylogeny and divergence of PEBPs in nine species were conducted to reconstruct the evolutionary pattern of PEBP genes in plants. RESULTS: We identified and cloned 160 PEBPs from 15 cotton species, and the phylogenetic analysis showed that the genes could be classified into the following three subfamilies: MFT-like, FT-like and TFL1-like. There were eight single orthologous group (OG) members in each diploid and 16 double OG members in each tetraploid. An analysis of the expression and selective pressure indicated that expression divergence and strong purification selection within the same OG presented in the PEBP gene family. CONCLUSION: An evolutionary pattern of duplication and deletion of the PEBP family in the evolutionary history of Gossypium was suggested, and three pairs of genes resulted from different whole-genome duplication events.


Assuntos
Evolução Molecular , Gossypium/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Gossypium/classificação , Filogenia , Proteínas de Plantas/metabolismo , Ploidias , Seleção Genética , Fatores de Transcrição/metabolismo
19.
Nutrients ; 11(2)2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30678169

RESUMO

The high global demand of wheat and its subsequent consumption arise from the physicochemical properties of bread dough and its contribution to the protein intake in the human diet. Gluten is the main structural complex of wheat proteins and subjects affected by celiac disease (CD) cannot tolerate gluten protein. Within gluten proteins, α-gliadins constitute the most immunogenic fraction since they contain the main T-cell stimulating epitopes (DQ2.5-glia-α1, DQ2.5-glia-α2, and DQ2.5-glia-α3). In this work, the celiac immunotoxic potential of α-gliadins was studied within Triticeae: diploid, tetraploid, and hexaploid species. The abundance and immunostimulatory capacity of CD canonical epitopes and variants (with one or two mismatches) in all α-gliadin sequences were determined. The results showed that the canonical epitopes DQ2.5-glia-α1 and DQ2.5-glia-α3 were more frequent than DQ2.5-glia-α2. A higher abundance of canonical DQ2.5-glia-α1 epitope was found to be associated with genomes of the BBAADD, AA, and DD types; however, the abundance of DQ2.5-glia-α3 epitope variants was very high in BBAADD and BBAA wheat despite their low abundance in the canonical epitope. The most abundant substitution was that of proline to serine, which was disposed mainly on the three canonical DQ2.5 domains on position 8. Interestingly, our results demonstrated that the natural introduction of Q to H at any position eliminates the toxicity of the three T-cell epitopes in the α-gliadins. The results provided a rational approach for the introduction of natural amino acid substitutions to eliminate the toxicity of three T-cell epitopes, while maintaining the technological properties of commercial wheats.


Assuntos
Aegilops/química , Doença Celíaca/imunologia , Epitopos/genética , Variação Genética , Gliadina/imunologia , Triticum/química , Aegilops/genética , Aegilops/imunologia , Sequência de Aminoácidos , Proliferação de Células , Criança , Gliadina/genética , Humanos , Leucócitos Mononucleares/fisiologia , Ploidias , Linfócitos T , Triticum/genética , Triticum/imunologia
20.
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