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1.
Proc Natl Acad Sci U S A ; 119(15): e2118879119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377798

RESUMO

SignificancePolyploidy, which occurs in roughly half of all flowering plants and an even higher percentage of grasses, is thought to be a major driver of adaptation. Higher numbers of copies of each gene in polyploid genomes can increase genetic diversity, which could drive shifts in habitat preference, adaptability, and fitness. To test the effects of increased ploidy, we compared genomic diversity, environmental niche, and fitness responses across climatic gradients between tetraploid and octoploid switchgrass. We found that the octoploids contained novel combinations of the ancestral tetraploid genetic diversity, which was linked to the expansion of switchgrass into unsuitable habitats for tetraploid populations. Our experiments revealed evidence of niche divergence, differential fitness, and a generalist-specialist trade-off between cytotypes.


Assuntos
Aclimatação , Panicum , Poliploidia , Aclimatação/genética , Variação Genética , Panicum/genética , Panicum/fisiologia , Tetraploidia
2.
BMC Plant Biol ; 22(1): 176, 2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35387617

RESUMO

BACKGROUND: Autopolyploids, especially artificial lines, provide model systems for understanding the mechanisms of gene dosage effects on trait variation owing to their relatively uniform genetic background. Here, a protocol for in vitro octaploid induction of Populus hopeiensis from leaf blades with colchicine treatment was established through investigation of the effects of different pre-culture durations, colchicine concentrations, and exposure times. RESULTS: We found that pre-culture duration, colchicine concentration, and exposure time had significant effects on the survival rate, shoot regeneration rate, and octaploid induction rate of P. hopeiensis leaf blades. The highest octaploid induction rate (8.61%) was observed when leaf blades pre-cultured for 9 days were treated for 4 days with 100 µM colchicine. The ploidy level of all regenerated plantlets was analyzed by flow cytometry and further confirmed by chromosome counting. A total of 14 octaploids were obtained. The stomatal length, width, and density of leaf blades significantly differed between tetraploid and octaploid plants. Compared with diploid and tetraploid plants, octaploids had a slower growth rate, smaller leaf blade size, and shorter internodes. CONCLUSIONS: We established an effective protocol for inducing octaploids in vitro from autotetraploid P. hopeiensis leaf blades by colchicine treatment.


Assuntos
Populus , Colchicina/farmacologia , Diploide , Folhas de Planta/genética , Populus/genética , Tetraploidia
3.
Open Biol ; 12(4): 210361, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35472287

RESUMO

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.


Assuntos
Sistemas CRISPR-Cas , Giardia lamblia , Edição de Genes/métodos , Giardia lamblia/genética , Humanos , RNA Guia , Tetraploidia
4.
Int J Biol Macromol ; 207: 700-714, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35341886

RESUMO

Plants have evolved a complex and organized response to abiotic stress that involves physiological and metabolic reprogramming, transcription control, epigenetic regulation, and expressions of thousand interacting genes for instance the late embryogenesis abundant (LEA) proteins are expressed in multiple environmental variables during the plant developmental period, and thus play critical role in enhancing drought and salt stress tolerance. A comprehensive molecular and functional characterization of the LEA3 gene was carried out in cotton under abiotic stress conditions in order to elucidate their functions. Seventy eight genes were identified in cotton, and were clustered into six clades moreover; the LEA genes were more upregulated in the tissues of the tetraploid cotton compared to the diploid type. A key gene, Gh_A08G0694 was the most upregulated, and was knocked in tetraploid cotton, the knocked out significantly increased the susceptibility of cotton plants to salinity and drought stresses, moreover, several ABA/stress-associated genes were down regulated. Similarly, overexpression of the key gene, significantly increased tolerance of the overexpressed plants to drought and salinity stress. The key gene is homologous to GhLEA3 protein, found to have strong interaction to key abiotic stress tolerance genes, voltage-dependent anion channel 1 (VDAC1) and glyceraldehyde-3-phosphate dehydrogenase A (gapA).


Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Desenvolvimento Embrionário , Epigênese Genética , Gossypium/genética , Gossypium/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Tetraploidia
5.
Nat Genet ; 54(3): 342-348, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35241824

RESUMO

Potato is the most widely produced tuber crop worldwide. However, reconstructing the four haplotypes of its autotetraploid genome remained an unsolved challenge. Here, we report the 3.1 Gb haplotype-resolved (at 99.6% precision), chromosome-scale assembly of the potato cultivar 'Otava' based on high-quality long reads, single-cell sequencing of 717 pollen genomes and Hi-C data. Unexpectedly, ~50% of the genome was identical-by-descent due to recent inbreeding, which was contrasted by highly abundant structural rearrangements involving ~20% of the genome. Among 38,214 genes, only 54% were present in all four haplotypes with an average of 3.2 copies per gene. Taking the leaf transcriptome as an example, 11% of the genes were differently expressed in at least one haplotype, where 25% of them were likely regulated through allele-specific DNA methylation. Our work sheds light on the recent breeding history of potato, the functional organization of its tetraploid genome and has the potential to strengthen the future of genomics-assisted breeding.


Assuntos
Solanum tuberosum , Tetraploidia , Alelos , Cromossomos , Haplótipos/genética , Melhoramento Vegetal , Solanum tuberosum/genética
6.
Genes (Basel) ; 13(3)2022 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-35327995

RESUMO

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L-1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.


Assuntos
MicroRNAs , Robinia , Sintase do Amido , Perfilação da Expressão Gênica , Ácidos Indolacéticos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Robinia/genética , Robinia/metabolismo , Sintase do Amido/genética , Tetraploidia , beta-Frutofuranosidase/genética
8.
Nature ; 604(7904): 146-151, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35355016

RESUMO

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.


Assuntos
Instabilidade Cromossômica , Dano ao DNA , Duplicação Gênica , Fase S , Tetraploidia , Instabilidade Cromossômica/genética , Replicação do DNA , Humanos , Cariótipo , Mitose , Fase S/genética
9.
Plant Biol (Stuttg) ; 24(3): 502-509, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35246912

RESUMO

Oxalic acid (OA) is a crucial pathogenic factor for Sclerotinia spp. fungi, which is closely related to Botrytis spp. fungi. Whether OA is a pathogenic factor for the causal agent of grey mould in lily, Botrytis elliptica, and the response of lily to OA are poorly understood. To address these questions, lesion tissues and deposition of calcium oxalate (CaOX) and callose were observed in diploid and tetraploid leaves of L. rosthornii after inoculation with B. elliptica. Oxalate oxidase (OXO) activity and the transcript levels of some genes related to OA degradation (LrGLP1, LrGLP2 and LrWRKY4), reactive oxygen species (ROS) production/scavenging systems (LrRBOHD, LrGST, LrPOD and LrAPX1) and pathogen-related protein (PR) synthesis (LrCHI, LrBGL and LrPR10) were compared. After diploid and tetraploid leaves inoculation, lesion tissue and callose and CaOX were separately observed around in guard cells and stomata rather than the epidermis in the infected area. OXO activity was triggered at 2 h post-inoculation (hpi) in both ploidy leaves, and it was higher in the latter from 12-48 hpi. Expression of LrGLP1, LrGLP2, LrRBOHD, LrGST, LrPOD, LrCHI, LrBGL and LrPR10 was higher in tetraploids than in diploids from 24(12)-36(48) hpi. In conclusion, for B. elliptica, OA mainly chelates Ca2+ from the stomata cell wall. The strong capability to degrade OA and higher expression levels of some genes related to ROS accumulation/scavenging and PR synthesis may partially explain the relatively higher grey mould resistance of tetraploid L. rosthornii.


Assuntos
Botrytis , Lilium , Botrytis/metabolismo , Ácido Oxálico/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Tetraploidia
10.
Am J Bot ; 109(2): 259-271, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35137947

RESUMO

PREMISE: Endoreduplication, nonheritable duplication of a nuclear genome, is widespread in plants and plays a role in developmental processes related to cell differentiation. However, neither ecological nor cytological factors influencing intraspecific variation in endoreduplication are fully understood. METHODS: We cultivated plants covering the range-wide natural diversity of diploid and tetraploid populations of Arabidopsis arenosa in common conditions to investigate the effect of original ploidy level on endoreduplication. We also raised plants from several foothill and alpine populations from different lineages and of both ploidies to test for the effect of elevation. We determined the endoreduplication level in leaves of young plants by flow cytometry. Using RNA-seq data available for our populations, we analyzed gene expression analysis in individuals that differed in endoreduplication level. RESULTS: We found intraspecific variation in endoreduplication that was mainly driven by the original ploidy level of populations, with significantly higher endoreduplication in diploids. An effect of elevation was also found within each ploidy, yet its direction exhibited rather regional-specific patterns. Transcriptomic analysis comparing individuals with high vs. low endopolyploidy revealed a majority of differentially expressed genes related to the stress and hormone response and to modifications especially in the cell wall and in chloroplasts. CONCLUSIONS: Our results support the general assumption of higher potential of low-ploidy organisms to undergo endoreduplication and suggest that endoreduplication is further integrated within the stress response pathways for a fine-tune adjustment of the endoreduplication process to their local environment.


Assuntos
Arabidopsis , Arabidopsis/genética , Diploide , Endorreduplicação/genética , Ploidias , Tetraploidia
11.
Int J Mol Sci ; 23(4)2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35216323

RESUMO

Allotetraploid durum wheat is the second most widely cultivated wheat, following hexaploid bread wheat, and is one of the major protein and calorie sources of the human diet. However, durum wheat is encountered with a severe grain yield bottleneck due to the erosion of genetic diversity stemming from long-term domestication and especially modern breeding programs. The improvement of yield and grain quality of durum wheat is crucial when confronted with the increasing global population, changing climate environments, and the non-ignorable increasing incidence of wheat-related disorders. This review summarized the domestication and evolution process and discussed the durum wheat re-evolution attempts performed by global researchers using diploid einkorn, tetraploid emmer wheat, hexaploid wheat (particularly the D-subgenome), etc. In addition, the re-evolution of durum wheat would be promoted by the genetic enrichment process, which could diversify allelic combinations through enhancing chromosome recombination (pentaploid hybridization or pairing of homologous chromosomes gene Ph mutant line induced homoeologous recombination) and environmental adaptability via alien introgressive genes (wide cross or distant hybridization followed by embryo rescue), and modifying target genes or traits by molecular approaches, such as CRISPR/Cas9 or RNA interference (RNAi). A brief discussion of the future perspectives for exploring germplasm for the modern improvement and re-evolution of durum wheat is included.


Assuntos
Grão Comestível/genética , Triticum/genética , Alelos , Cromossomos de Plantas/genética , Cruzamentos Genéticos , Diploide , Domesticação , Genes de Plantas/genética , Humanos , Fenótipo , Locos de Características Quantitativas/genética , Tetraploidia
12.
Gene ; 820: 146200, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35131368

RESUMO

Whitefly inflicts both direct and indirect losses to cotton crop. Whitefly resistant cotton germplasm is a high priority and considered among the best possible solutions to mitigate this issue. In this study, we evaluated cotton leaf curl disease (CLCuD) resistant cotton line Mac7 under whitefly stress. Furthermore, we utilized the already available transcriptome data of Mac7 concerning whitefly stress to elucidate associated mechanisms and identify functionally important genes in cotton. In transcriptomic data analysis, differentially expressed genes (DEGs) were found involved in complex relay pathways, activated on whitefly exposure. The response implicates signalling through resistance genes (R-genes), MAPK, ROS, VQs or RLKs, transcription factors, which leads to the activation of defence responses including, Ca2+messengers, phytohormonal cross-talk, gossypol, flavonoids, PhasiRNA and susceptibility genes (S-genes). The qRT-PCR assay of 10 functionally important genes also showed their involvement in differential responses at 24 and 48 h post whitefly infestation. Briefly, our study helps in understanding the resistant nature of Mac7 under whitefly stress.


Assuntos
Resistência à Doença/genética , Gossypium/genética , Gossypium/metabolismo , Hemípteros , Doenças das Plantas/genética , Transcriptoma , Animais , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Gossypium/imunologia , Tetraploidia
13.
Plant Sci ; 316: 111162, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35151447

RESUMO

Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is a highly efficient tool for disruptive targeted mutagenesis, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, Prime Editing technology has been developed, allowing the simultaneous generation of nucleotide transitions and transversions but also short defined indels. In this study, we report on the successful use of Prime Editing in two plants of interest: the plant model Physcomitrium patens and the tetraploid and highly heterozygous potato (Solanum tuberosum). In both cases editing rates were lower than with other CRISPR-Cas9 based techniques, but we were able to successfully introduce nucleotide transversions into targeted genes, a unique feature of Prime Editing. Additionally, the analysis of potential off-target mutation sites in P. patens suggested very high targeting fidelity in this organism. The present work paves the way for the use Prime Editing in Physcomitrium patens and potato, however highlighting the limitations that need to be overcome for more efficient precision plant breeding.


Assuntos
Solanum tuberosum , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma de Planta , Melhoramento Vegetal , Solanum tuberosum/genética , Tetraploidia
14.
Plant Mol Biol ; 108(4-5): 469-480, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34994920

RESUMO

KEY MESSAGE: Association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content which was verified in diploid and tetraploid potato mapping populations. Potatoes are grown for various purposes like French fries, table potatoes, crisps and for their starch. One of the most important aspects of potato starch is that it contains a high amount of phosphate ester groups which are considered to be important for providing improved functionalization after derivatization processes. Little is known about the variation in phosphate content as such in different potato varieties and thus we studied the genetic diversity for this trait. From other studies it was clear that the phosphate content is controlled by a quantitative trait locus (QTL) underlying the candidate gene α-Glucan Water Dikinase (StGWD) on chromosome 5. We performed direct amplicon sequencing of this gene by Sanger sequencing. Sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 different haplotypes. By assigning tag SNPs to these haplotypes, each of the four alleles present in a cultivar could be deduced and linked to a phosphate content. A high value for intra-individual heterozygosity was observed (Ho = 0.765). The average number of different haplotypes per individual (Ai) was 3.1. Pedigree analysis confirmed that the haplotypes are identical-by-descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotypes originating from introgression of wild potato accessions carrying resistance genes could be traced. Furthermore, association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content varying from 12 nmol PO4/mg starch to 38 nmol PO4/mg starch. These allele effects were verified in diploid and tetraploid mapping populations and offer possibilities to breed and select for this trait.


Assuntos
Fosfatos/metabolismo , Fosfotransferases (Aceptores Pareados)/genética , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Amido/metabolismo , Tetraploidia , Alelos , Variação Genética , Haplótipos , Linhagem , Fosfotransferases (Aceptores Pareados)/metabolismo , Polimorfismo de Nucleotídeo Único
15.
Mol Plant ; 15(3): 520-536, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35026436

RESUMO

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.


Assuntos
Solanum tuberosum , Tetraploidia , Alelos , Cromossomos , Melhoramento Vegetal , Proteoma/genética , Solanum tuberosum/genética , Transcriptoma/genética
16.
New Phytol ; 234(4): 1185-1194, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35064679

RESUMO

The origin of sweetpotato, a hexaploid species, is poorly understood, partly because the identity of its tetraploid progenitor remains unknown. In this study, we identify, describe and characterize a new species of Ipomoea that is sweetpotato's closest tetraploid relative known to date and probably a direct descendant of its tetraploid progenitor. We integrate morphological, phylogenetic, and genomic analyses of herbarium and germplasm accessions of the hexaploid sweetpotato, its closest known diploid relative Ipomoea trifida, and various tetraploid plants closely related to them from across the American continent. We identify wild autotetraploid plants from Ecuador that are morphologically distinct from Ipomoea batatas and I. trifida, but monophyletic and sister to I. batatas in phylogenetic analysis of nuclear data. We describe this new species as Ipomoea aequatoriensis T. Wells & P. Muñoz sp. nov., distinguish it from hybrid tetraploid material collected in Mexico; and show that it likely played a direct role in the origin of sweetpotato's hexaploid genome. This discovery transforms our understanding of sweetpotato's origin.


Assuntos
Ipomoea batatas , Ipomoea , Genoma de Planta , Ipomoea/genética , Ipomoea batatas/genética , Filogenia , Tetraploidia
17.
Plant Physiol ; 188(4): 1917-1930, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35088855

RESUMO

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.


Assuntos
Lycopersicon esculentum , Solanum , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma de Planta/genética , Lycopersicon esculentum/genética , Melhoramento Vegetal , Protoplastos , Regeneração , Solanum/genética , Tetraploidia
18.
Int J Mol Sci ; 23(2)2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35055085

RESUMO

Autophagy is an indispensable biological process and plays crucial roles in plant growth and plant responses to both biotic and abiotic stresses. This study systematically identified autophagy-related proteins (ATGs) in wheat and its diploid and tetraploid progenitors and investigated their genomic organization, structure characteristics, expression patterns, genetic variation, and regulation network. We identified a total of 77, 51, 29, and 30 ATGs in wheat, wild emmer, T. urartu and A. tauschii, respectively, and grouped them into 19 subfamilies. We found that these autophagy-related genes (ATGs) suffered various degrees of selection during the wheat's domestication and breeding processes. The genetic variations in the promoter region of Ta2A_ATG8a were associated with differences in seed size, which might be artificially selected for during the domestication process of tetraploid wheat. Overexpression of TaVAMP727 improved the cold, drought, and salt stresses resistance of the transgenic Arabidopsis and wheat. It also promoted wheat heading by regulating the expression of most ATGs. Our findings demonstrate how ATGs regulate wheat plant development and improve abiotic stress resistance. The results presented here provide the basis for wheat breeding programs for selecting varieties of higher yield which are capable of growing in colder, drier, and saltier areas.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Triticum/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Biomarcadores , Biologia Computacional/métodos , Diploide , Perfilação da Expressão Gênica , Fenótipo , Filogenia , Polimorfismo de Nucleotídeo Único , Mapeamento de Interação de Proteínas , Estresse Fisiológico , Tetraploidia
19.
BMC Plant Biol ; 22(1): 45, 2022 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-35065606

RESUMO

BACKGROUND: The genus Erianthus, which belongs to the "Saccharum complex", includes C4 warm-season grasses. Erianthus species are widely distributed throughout Southeast Asia, East Asia and South Asia. Erianthus arundinaceus (Retz.) Jeswiet is highly adaptable to the environment, has a high percentage of dry matter, and is highly productive. Recently, this species has attracted attention as a novel bioenergy crop and as a breeding material for sugarcane improvement. Such interest in E. arundinaceus has accelerated the collection and conservation of its genetic resources, mainly in Asian countries, and also evaluation of morphological, agricultural, and cytogenetic features in germplasm collections. In Thailand, genetic resources of E. arundinaceus have been collected over the past 20 years and their phenotypic traits have been evaluated. However, the genetic differences and relatedness of the germplasms are not fully understood. RESULTS: A set of 41 primer pairs for nuclear simple sequence repeats (SSRs) developed from E. arundinaceus were used to assess the genetic diversity of 121 Erianthus germplasms collected in Thailand; of these primer pairs, 28 detected a total of 316 alleles. A Bayesian clustering approach with these alleles classified the accessions into four main groups, generally corresponding to the previous classification based on phenotypic analysis. The results of principal coordinate analysis and phylogenetic analysis of the 121 accessions on the basis of the SSR markers showed the same trend as Bayesian clustering, whereas sequence variations of three non-coding regions of chloroplast DNA revealed eight haplotypes among the accessions. The analysis of genetic structure and phylogenetic relationships, however, found some accessions whose classification contradicted the results of previous phenotypic classification. CONCLUSIONS: The molecular approach used in this study characterized the genetic diversity and relatedness of Erianthus germplasms collected across Thailand. This knowledge would allow efficient maintenance and conservation of the genetic resources of this grass and would help to use Erianthus species as breeding materials for development of novel bioenergy crops and sugarcane improvement.


Assuntos
Variação Genética , Filogenia , Poaceae/genética , Poliploidia , Cloroplastos/genética , Haplótipos/genética , Repetições de Microssatélites , Tetraploidia , Tailândia
20.
Am J Hematol ; 97(1): 18-29, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34677878

RESUMO

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação em Linhagem Germinativa , Síndromes Mielodisplásicas/genética , Neutropenia/congênito , Septinas/genética , Linhagem Celular , Células Cultivadas , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Humanos , Recém-Nascido , Masculino , Síndromes Mielodisplásicas/complicações , Neutropenia/complicações , Neutropenia/genética , Tetraploidia
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