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1.
Planta ; 254(4): 68, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34498163

RESUMO

MAIN CONCLUSION: In this review, we have focused on the CRISPR/Cas9 technology for improving the agronomic traits in plants through point mutations, knockout, and single base editing, and we highlighted the recent progress in plant metabolic engineering. CRISPR/Cas9 technology has immense power to reproduce plants with desired characters and revolutionizing the field of genome engineering by erasing the barriers in targeted genome editing. Agriculture fields are using this advance genome editing tool to get the desired traits in the crops plants such as increase yield, improve product quality attributes, and enhance resistance against biotic and abiotic stresses by identifying and editing genes of interest. This review focuses on CRISPR/Cas-based gene knockout for trait improvement and single base editing to boost yield, quality, stress tolerance, and disease resistance traits in crops. Use of CRISPR/Cas9 system to facilitate crop domestication and hybrid breeding are also touched. We summarize recent developments and up-gradation of delivery mechanism (nanotechnology and virus particle-based delivery system) and progress in multiplex gene editing. We also shed lights in advances and challenges of engineering the important metabolic pathways that contain a variety of dietary metabolites and phytochemicals. In addition, we endorsed substantial technical hurdles and possible ways to overcome the unpredictability of CRISPR/Cas technology for broader application across various crop species. We speculated that by making a strong interconnection among all genomic fields will give a gigantic bunt of knowledge to develop crop expressing desired traits.


Assuntos
Sistemas CRISPR-Cas , Melhoramento Vegetal , Agricultura , Sistemas CRISPR-Cas/genética , Genoma de Planta , Plantas Geneticamente Modificadas/genética , Tecnologia
2.
BMC Plant Biol ; 21(1): 418, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517837

RESUMO

BACKGROUND: Bread wheat (Triticum aestivum L.) is one of the most widely consumed cereal crops, but its complex genome makes it difficult to investigate the genetic effect on important agronomic traits. Genome-wide association (GWA) analysis is a useful method to identify genetic loci controlling complex phenotypic traits. With the RNA-sequencing based gene expression analysis, putative candidate genes governing important agronomic trait can be suggested and also molecular markers can be developed. RESULTS: We observed major quantitative agronomic traits of wheat; the winter survival rate (WSR), days to heading (DTH), days to maturity (DTM), stem length (SL), spike length (SPL), awn length (AL), liter weight (LW), thousand kernel weight (TKW), and the number of seeds per spike (SPS), of 287 wheat accessions from diverse country origins. A significant correlation was observed between the observed traits, and the wheat genotypes were divided into three subpopulations according to the population structure analysis. The best linear unbiased prediction (BLUP) values of the genotypic effect for each trait under different environments were predicted, and these were used for GWA analysis based on a mixed linear model (MLM). A total of 254 highly significant marker-trait associations (MTAs) were identified, and 28 candidate genes closely located to the significant markers were predicted by searching the wheat reference genome and RNAseq data. Further, it was shown that the phenotypic traits were significantly affected by the accumulation of favorable or unfavorable alleles. CONCLUSIONS: From this study, newly identified MTA and putative agronomically useful genes will help to study molecular mechanism of each phenotypic trait. Further, the agronomically favorable alleles found in this study can be used to develop wheats with superior agronomic traits.


Assuntos
Genótipo , Fenótipo , Locos de Características Quantitativas , Sementes/anatomia & histologia , Sementes/genética , Triticum/anatomia & histologia , Triticum/genética , Grão Comestível/anatomia & histologia , Grão Comestível/genética , Variação Genética , Genoma de Planta , Estudo de Associação Genômica Ampla
3.
BMC Plant Biol ; 21(1): 419, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517842

RESUMO

BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Interações Hospedeiro-Patógeno/genética , Vírus de Plantas/genética , Soja/genética , Soja/virologia , Viroses/genética , Produtos Agrícolas/genética , Produtos Agrícolas/virologia , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Genoma de Planta , Mutagênese , RNA Guia , RNA de Plantas , RNA Viral
4.
BMC Plant Biol ; 21(1): 417, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34507551

RESUMO

BACKGROUND: The future productivity of wheat (T. aestivum L.) as the most grown crop worldwide is of utmost importance for global food security. Thousand kernel weight (TKW) in wheat is closely associated with grain architecture-related traits, e.g. kernel length (KL), kernel width (KW), kernel area (KA), kernel diameter ratio (KDR), and factor form density (FFD). Discovering the genetic architecture of natural variation in these traits, identifying QTL and candidate genes are the main aims of this study. Therefore, grain architecture-related traits in 261 worldwide winter accessions over three field-year experiments were evaluated. RESULTS: Genome-wide association analysis using 90K SNP array in FarmCPU model revealed several interesting genomic regions including 17 significant SNPs passing false discovery rate threshold and strongly associated with the studied traits. Four of associated SNPs were physically located inside candidate genes within LD interval e.g. BobWhite_c5872_589 (602,710,399 bp) found to be inside TraesCS6A01G383800 (602,699,767-602,711,726 bp). Further analysis reveals the four novel candidate genes potentially involved in more than one grain architecture-related traits with a pleiotropic effects e.g. TraesCS6A01G383800 gene on 6A encoding oxidoreductase activity was associated with TKW and KA. The allelic variation at the associated SNPs showed significant differences betweeen the accessions carying the wild and mutated alleles e.g. accessions carying C allele of BobWhite_c5872_589, TraesCS6A01G383800 had significantly higher TKW than the accessions carying T allele. Interestingly, these genes were highly expressed in the grain-tissues, demonstrating their pivotal role in controlling the grain architecture. CONCLUSIONS: These results are valuable for identifying regions associated with kernel weight and dimensions and potentially help breeders in improving kernel weight and architecture-related traits in order to increase wheat yield potential and end-use quality.


Assuntos
Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , Sementes/anatomia & histologia , Sementes/genética , Triticum/anatomia & histologia , Triticum/genética , Grão Comestível/anatomia & histologia , Grão Comestível/genética , Variação Genética , Genoma de Planta , Estudo de Associação Genômica Ampla , Locos de Características Quantitativas
5.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2703-2718, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472290

RESUMO

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.


Assuntos
Cromossomos de Plantas , Genoma de Planta , Hibridização Genômica Comparativa , Genoma de Planta/genética , Genômica , Triticum/genética
6.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445524

RESUMO

The family of B-box (BBX) transcription factors contains one or two B-BOX domains and sometimes also features a highly conserved CCT domain, which plays important roles in plant growth, development and stress response. Nevertheless, no systematic study of the BBX gene family in Iris germanica L. has been undertaken. In this study, a set of six BBX TF family genes from I. germanica was identified based on transcriptomic sequences, and clustered into three clades according to phylogenetic analysis. A transient expression analysis revealed that all six BBX proteins were localized in the nucleus. A yeast one-hybrid assay demonstrated that IgBBX3 has transactivational activity, while IgBBX1, IgBBX2, IgBBX4, and IgBBX5 have no transcriptional activation ability. The transcript abundance of IgBBXs in different tissues was divided into two major groups. The expression of IgBBX1, IgBBX2, IgBBX3 and IgBBX5 was higher in leaves, whereas IgBBX4 and IgBBX6 was higher in roots. The stress response patterns of six IgBBX were detected under phytohormone treatments and abiotic stresses. The results of this study lay the basis for further research on the functions of BBX gene family members in plant hormone and stress responses, which will promote their application in I. germanica breeding.


Assuntos
Regulação da Expressão Gênica de Plantas , Genoma de Planta , Iris (Planta)/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Iris (Planta)/genética , Iris (Planta)/crescimento & desenvolvimento , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Estresse Fisiológico , Fatores de Transcrição/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445115

RESUMO

The SWEET (Sugars Will Eventually be Exported Transporter) proteins are a novel family of sugar transporters that play key roles in sugar efflux, signal transduction, plant growth and development, plant-pathogen interactions, and stress tolerance. In this study, 22 ClaSWEET genes were identified in Citrullus lanatus (Thunb.) through homology searches and classified into four groups by phylogenetic analysis. The genes with similar structures, conserved domains, and motifs were clustered into the same groups. Further analysis of the gene promoter regions uncovered various growth, development, and biotic and abiotic stress responsive cis-regulatory elements. Tissue-specific analysis showed most of the genes were highly expressed in male flowers and the roots of cultivated varieties and wild cultivars. In addition, qRT-PCR results further imply that ClaSWEET proteins might be involved in resistance to Fusarium oxysporum infection. Moreover, a significantly higher expression level of these genes under various abiotic stresses suggests its multifaceted role in mediating plant responses to drought, salt, and low-temperature stress. The genome-wide characterization and phylogenetic analysis of ClaSWEET genes, together with the expression patterns in different tissues and stimuli, lays a solid foundation for future research into their molecular function in watermelon developmental processes and responses to biotic and abiotic stresses.


Assuntos
Transporte Biológico/genética , Citrullus/genética , Genoma de Planta/genética , Família Multigênica/genética , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Açúcares/metabolismo , Fusarium/genética , Regulação da Expressão Gênica de Plantas/genética , Estudo de Associação Genômica Ampla/métodos , Filogenia , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética
8.
Gene ; 802: 145866, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352297

RESUMO

Exploring the molecular identities and the genetic diversity of a plant species is crucial in figuring out the evolutionary pressure of genes as well as in molecular breeding application. Nineteen chloroplast genomes of Panicum species in the National Center for Biotechnology Information database were downloaded and analyzed. The base composition, the effective number of codons, the relative synonymous codon usage, the codon bias index and the codon adaptation index of all genes in all chloroplast genomes, as well as the correlation coefficient among them, were calculated and discussed. The correspondence analysis and the clustering characteristics among nineteen genomes base on the relative synonymous codon usage values of nineteen chloroplast genomes were calculated and analyzed. In order to figuring out the evolutionary diversity of certain genes, the codon usage pattern of forty-one typical genes were separately counted and compared. Summations of their standard deviations were considered to evaluate their genetic diversities. The results of codon usage pattern showed that all genes were obvious AU-rich ones in chloroplast genomes of Panicum species, revealing that the natural selection was the main factor that influenced their evolutionary process. The correspondence and clustering analysis among nineteen chloroplast genomes showed that the overall evolutionary differences among them were not significant. However, the analysis on the genetic diversity of tyical genes showed that the degrees of diversity are different, and that the shorter sequences are more prone to instability. These findings would improve our understanding on the evolution of chloroplast genomes of Panicum species and be useful for further study on their evolutionary phenomenon.


Assuntos
Uso do Códon , Genoma de Cloroplastos , Panicum/genética , Evolução Molecular , Variação Genética , Genoma de Planta
9.
Nat Commun ; 12(1): 4979, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404804

RESUMO

Relative contributions of pre-existing vs de novo genomic variation to adaptation are poorly understood, especially in polyploid organisms. We assess this in high resolution using autotetraploid Arabidopsis arenosa, which repeatedly adapted to toxic serpentine soils that exhibit skewed elemental profiles. Leveraging a fivefold replicated serpentine invasion, we assess selection on SNPs and structural variants (TEs) in 78 resequenced individuals and discover significant parallelism in candidate genes involved in ion homeostasis. We further model parallel selection and infer repeated sweeps on a shared pool of variants in nearly all these loci, supporting theoretical expectations. A single striking exception is represented by TWO PORE CHANNEL 1, which exhibits convergent evolution from independent de novo mutations at an identical, otherwise conserved site at the calcium channel selectivity gate. Taken together, this suggests that polyploid populations can rapidly adapt to environmental extremes, calling on both pre-existing variation and novel polymorphisms.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Alelos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Genoma de Planta , Poliploidia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Alcaloides de Triptamina e Secologanina/metabolismo , Solo/química
10.
Science ; 373(6555): 655-662, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353948

RESUMO

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.


Assuntos
Genoma de Planta , Anotação de Sequência Molecular , Zea mays/genética , Centrômero/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Metilação de DNA , Resistência à Doença/genética , Genes de Plantas , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Herança Multifatorial/genética , Fenótipo , Doenças das Plantas , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Tetraploidia , Transcriptoma , Sequenciamento Completo do Genoma
11.
Science ; 373(6556): 792-796, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385396

RESUMO

Molecular time trees indicating that embryophytes originated around 500 million years ago (Ma) during the Cambrian are at odds with the record of fossil plants, which first appear in the mid-Silurian almost 80 million years later. This time gap has been attributed to a missing fossil plant record, but that attribution belies the case for fossil spores. Here, we describe a Tremadocian (Early Ordovician, about 480 Ma) assemblage with elements of both Cambrian and younger embryophyte spores that provides a new level of evolutionary continuity between embryophytes and their algal ancestors. This finding suggests that the molecular phylogenetic signal retains a latent evolutionary history of the acquisition of the embryophytic developmental genome, a history that perhaps began during Ediacaran-Cambrian time but was not completed until the mid-Silurian (about 430 Ma).


Assuntos
Evolução Biológica , Carofíceas , Embriófitas , Fósseis , Carofíceas/anatomia & histologia , Carofíceas/classificação , Carofíceas/genética , Embriófitas/anatomia & histologia , Embriófitas/classificação , Embriófitas/genética , Genoma de Planta , Sedimentos Geológicos , Filogenia , Esporos , Austrália Ocidental
12.
BMC Plant Biol ; 21(1): 378, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399685

RESUMO

BACKGROUND: Understanding mechanisms of sugar accumulation and composition is essential to determining fruit quality and maintaining a desirable balance of sugars in plant storage organs. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. Japanese pear has extensive variation in individual sugar contents in mature fruit. Increasing total sugar content and that of individual high-sweetness sugars is a major target of breeding programs. The objective of this study was to identify quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits. RESULTS: We evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. In particular, a SNP located close to acid invertase gene PPAIV3 on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on accumulation of sucrose and glucose, respectively. We used 'Golden Delicious' doubled haploid 13 (GDDH13), an apple reference genome, to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat of early responsive to dehydration (ERD6)-like sugar transporter genes that might play a role in the phenotypes observed. CONCLUSIONS: SNPs associated with individual sugar accumulation were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. The candidate genes for the QTLs are conserved across Pyrinae genomes, which will be useful for further fruit quality studies in Rosaceae. The accuracies of genomic selection for sucrose, fructose, and glucose with genomic best linear unbiased prediction (GBLUP) were relatively high (0.67-0.75), suggesting that it would be possible to select individuals having high-sweetness fruit with high sucrose and fructose contents and low glucose content.


Assuntos
Genoma de Planta , Pyrus/química , Pyrus/genética , Açúcares/análise , Mapeamento Cromossômico , Cromossomos de Plantas , Frutas/genética , Estudo de Associação Genômica Ampla , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas
13.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
14.
BMC Plant Biol ; 21(1): 363, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376154

RESUMO

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma de Planta , Pinus/genética , DNA de Plantas , Mutação INDEL , RNA Guia , Ribonucleoproteínas/genética
15.
BMC Plant Biol ; 21(1): 371, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34384382

RESUMO

BACKGROUND: The Orchid family is the largest families of the monocotyledons and an economically important ornamental plant worldwide. Given the pivotal role of this plant to humans, botanical researchers and breeding communities should have access to valuable genomic and transcriptomic information of this plant. Previously, we established OrchidBase, which contains expressed sequence tags (ESTs) from different tissues and developmental stages of Phalaenopsis as well as biotic and abiotic stress-treated Phalaenopsis. The database includes floral transcriptomic sequences from 10 orchid species across all the five subfamilies of Orchidaceae. DESCRIPTION: Recently, the whole-genome sequences of Apostasia shenzhenica, Dendrobium catenatum, and Phalaenopsis equestris were de novo assembled and analyzed. These datasets were used to develop OrchidBase 4.0, including genomic and transcriptomic data for these three orchid species. OrchidBase 4.0 offers information for gene annotation, gene expression with fragments per kilobase of transcript per millions mapped reads (FPKM), KEGG pathways and BLAST search. In addition, assembled genome sequences and location of genes and miRNAs could be visualized by the genome browser. The online resources in OrchidBase 4.0 can be accessed by browsing or using BLAST. Users can also download the assembled scaffold sequences and the predicted gene and protein sequences of these three orchid species. CONCLUSIONS: OrchidBase 4.0 is the first database that contain the whole-genome sequences and annotations of multiple orchid species. OrchidBase 4.0 is available at http://orchidbase.itps.ncku.edu.tw/.


Assuntos
Bases de Dados Genéticas , Orchidaceae/genética , Genoma de Planta
16.
BMC Plant Biol ; 21(1): 374, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388987

RESUMO

BACKGROUND: Cadmium (Cd) is a toxic heavy metal that is harmful to the environment and human health. Cd pollution threatens the cultivation of rice (Oryza sativa L.) in many countries. Improving rice performance under Cd stress could potentially improve rice productivity. RESULTS: In this study, 9 growth traits of 188 different cultivated rice accessions under normal and Cd stress conditions were found to be highly variable during the seedling stage. Based on ~3.3 million single nucleotide polymorphisms (SNPs), 119 Cd-mediated growth response (CGR) quantitative trait loci (QTL) were identified by a genome-wide association study (GWAS), 55 of which have been validated by previously reported QTL and 64 were new CGR loci. Combined with the data from the GWAS, transcriptome analysis, gene annotations from the gene ontology (GO) Slim database, and annotations and functions of homologous genes, 148 CGR candidate genes were obtained. Additionally, several reported genes have been found to play certain roles in CGRs. Seven Cd-related cloned genes were found among the CGR genes. Natural elite haplotypes/alleles in these genes that increased Cd tolerance were identified by a haplotype analysis of a diverse mini core collection. More importantly, this study was the first to uncover the natural variations of 5 GST genes that play important roles in CGRs. CONCLUSION: The exploration of Cd-resistant rice germplasm resources and the identification of elite natural variations related to Cd-resistance will help improve the tolerance of current major rice varieties to Cd, as well as provide raw materials and new genes for breeding Cd-resistant varieties.


Assuntos
Cádmio/farmacologia , Genes de Plantas , Oryza/crescimento & desenvolvimento , Oryza/genética , Poluentes do Solo/farmacologia , Alelos , Perfilação da Expressão Gênica , Genoma de Planta , Estudo de Associação Genômica Ampla , Oryza/efeitos dos fármacos , Desenvolvimento Vegetal/genética , Polimorfismo de Nucleotídeo Único
17.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
18.
BMC Plant Biol ; 21(1): 400, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454435

RESUMO

BACKGROUNDS: Pomegranate is an excellent tree species with nutritional, medicinal, ornamental and ecological values. Studies have confirmed that SPL factors play an important role in floral transition and flower development. RESULTS: Used bioinformatics methods, 15 SPL (SQUAMOSA promoter-binding protein-like) genes were identified and analyzed from the 'Taishanhong' pomegranate (P. granatum L.) genome. Phylogenetic analysis showed that PgSPLs were divided into six subfamilies (G1 ~ G6). PgSPL promoter sequences contained multiple cis-acting elements associated with abiotic stress or hormonal response. Based on the transcriptome data, expression profiles of different tissues and different developmental stages showed that PgSPL genes had distinct temporal and spatial expression characteristics. The expression analysis of miR156 in small RNA sequencing results showed that miR156 negatively regulated the expression of target genes. qRT-PCR analysis showed that the expression levels of PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 in leaves were significantly higher than those in buds and stems (p < 0.05). The expression levels of PgSPL5, PgSPL12 and PgSPL13 in flower buds were significantly higher than that in leaves and stems (p < 0.05). The full-length of coding sequence of PgSPL5 and PgSPL13 were obtained by homologous cloning technology. The full length of PgSPL5 is 1020 bp, and PgSPL13 is 489 bp, which encodes 339 and 162 amino acids, respectively. Further investigation revealed that PgSPL5 and PgSPL13 proteins were located in the nucleus. Exogenous plant growth regulator induction experiments showed that PgSPL5 was up-regulated in leaves and stems. PgSPL13 was up-regulated in leaves and down-regulated in stems. When sprayed with 6-BA, IBA and PP333 respectively, PgSPL5 and PgSPL13 were up-regulated most significantly at P2 (bud vertical diameter was 5.1 ~ 12.0 mm) stage of bisexual and functional male flowers. CONCLUSIONS: Our findings suggested that PgSPL2, PgSPL3, PgSPL6, PgSPL11 and PgSPL14 played roles in leaves development of pomegranate. PgSPL5, PgSPL12 and PgSPL13 played roles in pomegranate flower development. PgSPL5 and PgSPL13 were involved in the response process of different plant hormone signal transduction in pomegranate development. This study provided a robust basis for further functional analyses of SPL genes in pomegranate.


Assuntos
Flores/crescimento & desenvolvimento , Flores/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Romã (Fruta)/crescimento & desenvolvimento , Romã (Fruta)/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genoma de Planta , Estudo de Associação Genômica Ampla , Família Multigênica , Filogenia , Análise de Sequência
19.
BMC Genomics ; 22(1): 632, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461821

RESUMO

BACKGROUND: As an important group of the multidrug efflux transporter family, the multidrug and toxic compound extrusion (MATE) family has a wide range of functions and is distributed in all kingdoms of living organisms. However, only two MATE genes in apple have been analyzed and genome-wide comprehensive analysis of MATE family is needed. RESULTS: In this study, a total of 66 MATE (MdMATE) candidates encoding putative MATE transporters were identified in the apple genome. These MdMATE genes were classified into four groups by phylogenetic analysis with MATE genes in Arabidopsis. Synteny analysis reveals that whole genome duplication (WGD) and segmental duplication events played a major role in the expansion of MATE gene family in apple. MdMATE genes show diverse expression patterns in different tissues/organs and developmental stages. Analysis of cis-regulatory elements in MdMATE promoter regions indicates that the function of MdMATE genes is mainly related to stress response. Besides, the changes of gene expression levels upon different pathogen infections reveal that MdMATE genes are involved in biotic stress response. CONCLUSIONS: In this work, we systematically identified MdMATE genes in apple genome using a set of bioinformatics approaches. Our comprehensive analysis provided valuable resources for improving disease resistance in apple and further functional characterization of MATE genes in other species.


Assuntos
Arabidopsis , Malus , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Malus/genética , Malus/metabolismo , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
20.
BMC Res Notes ; 14(1): 343, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461984

RESUMO

OBJECTIVE: Soybean is an important plant used for food, feed and many industrial purposes. Interest in soybean breeding is growing in Central Europe, including Poland. A very large number of soybean accessions are stored in gene banks, but less than 1% of them have been used for breeding. Here, we present genotypic data as well as phenotypic data on plant and seed performance, including seed chlorophyll fluorescence traits, and on yield components within a collection of soybean accessions that are conserved in the Polish Gene Bank at the Plant Breeding and Acclimatization Institute-National Research Institute. RESULTS: The materials used consisted of sub-collections: 79 Polish genotypes, including old traditional cultivars, 24 Canadian, 21 American, 21 Swedish and 31 from Central and Eastern European Countries, 9 from France and 6 from Japan. In total, 9602 high quality SNPs were derived from DArTseq, a method utilising GBS technology. GWAS, performed with the BLINK model, revealed that a total of 41 significant SNPs were mapped for days to flowering, flower colour, plant height, days to pod formation, 100 seed weight, pod colour, seeds and hilum colour and steady-state chlorophyll fluorescence under light (Ft_Lss). This is the first report about the diversity of traditional old Polish soybean cultivars.


Assuntos
Estudo de Associação Genômica Ampla , Soja , Canadá , Genoma de Planta , Melhoramento Vegetal , Polônia , Soja/genética , Estados Unidos
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