Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic ...modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.
The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, ...translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation Allshire RC, Karpen GH (2008)Nat Rev Genet9 (12):923–937. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, andArabidopsis thaliana. Haploids were obtained aftercenh3L130F-complementedcenh3-null mutant plants were crossed with wildtypeA. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.
Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can ...be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.
Noncoding chloroplast DNA sequences are widely employed markers in plant species-level phylogenetics and phylogeography. However, chloroplast capture (hybridization) and incomplete sorting of ...ancestral lineages could confound phylogenetic inference using chloroplast DNA. Recently, we studied the phylogenetic of Allium subg. Melanocrommyum based on nuclear rDNA internal transcribed spacer (ITS) sequences and showed that, although the subgenus is monophyletic, most sections are either para- or polyphyletic. To get insights from the chloroplast genome we sequenced the plastid trnL-trnF region in 434 individuals representing 100 species of A. subg. Melanocrommyum and found 74 chloroplast haplotypes. The sequences were analyzed using tree-based (Bayesian and maximum parsimony) and network-based (statistical parsimony network) approaches. The analyses revealed high level of chloroplast haplotype sharing among up to 15 species, as well as presence of several closely related haplotypes within single species. Several characteristics of the data violate the main assumptions of standard tree building methods that is the persistence of ancestral haplotypes and their co-existence with descendant alleles as well as multifurcating relationships among these alleles. The taxon groups inferred from chloroplast trnL-trnF sequence analyses were congruent with the nuclear phylogeny. Thus, bi- and uniparentally inherited datasets strongly contradict the morphology-based taxonomic classification of A. subg. Melanocrommyum. Also, the present study (1) reports a trnF gene duplication in A. subg. Melanocrommyum, (2) infers few putative homoploid hybrid taxa, and (3) shows that natural interspecies hybrids occur rarely in the subgenus. Generally, our data strongly advocate including multiple accessions per species in species-level phylogenetic studies, and show the advantages of networks over tree building methods for analyzing sequences from noncoding chloroplast loci in closely related species.
Allium subgenus
Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory ...taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in
Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.
The Caucasus and Middle East regions are considered to be the primary centre of origin of cultivated grapevine, and, as confirmed by archaeobotanical, archaeological, and cultural evidence, Georgia ...belongs to this earliest centre of winemaking. This study aims to investigate the genetic diversity, population structure and relationships of local autochthonous wine cultivars and wild grapevine, Vitis vinifera subsp. sylvestris. Multiple accessions of 15 Georgian aboriginal cultivars and 42 individuals of wild grapevine from different regions of Georgia and adjacent Turkey were genotyped at 17 nuclear microsatellite loci. A total of 160 alleles were detected with a mean number of 9.41 alleles and the effective number of 4.6 alleles (r) per locus, indicating that the SSRs were highly informative. Despite high genetic diversity, the level of genetic differentiation among defined wild and cultivated populations is low (F ₛₜ = 0,05; P***), which together with the outcome of model-based cluster analyses and genetic assignment methods point to gene flow among wild populations, as well as among cultivated and wild accessions. Besides, the data presented here suggest that local cultivars ‘Saperavi’ and ‘Tavkveri’ are independently derived from different local wild populations, while the majority of Georgian cultivars seem to have a single origin. Overall, the present study takes important steps for better characterization of Georgian cultivated and wild grapevines, and supports Georgia as one of the important centres of grapevine domestication still harbouring valuable genetic resources for grapevine breeding.
Although customized endonucleases transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) are known to be effective agents of mutagenesis in various host plants, ...newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants.
A simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations.
A convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN's functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.
Allium subgenus Melanocrommyum is a species-rich group of perennial onions with uniform karyology, ecology, and breeding system. Thus, the cytological, ecological, and physiological factors often ...correlated with genome size should have negligible effect on genome size variation in Melanocrommyum. We measured DNA content in subgenus Melanocrommyum using flow cytometry based on propidium iodide staining and analyzed the evolution of genome size in a phylogenetic context. The observed 2C genome size variation in 160 accessions of 70 species of the subgenus was high, varying from 26.26–78.73 pg. The significant phylogenetic signal in genome size data suggests that distribution of genome size is in accordance with phylogenetic clades identified by the analysis of nuclear ITS sequences. Estimation of ancestral genome sizes using generalized least squares revealed lineages with increasing as well as decreasing DNA content. We found within-species genome size variation to be mostly below 2.5%. In species where intraspecific genome size differences were in a range of 6–9%, we suggest the existence of cryptic species, as previously inferred by molecular markers. Thus, genome size variation reflects incipient speciation or diversification in Allium subgenus Melanocrommyum. About two-fold differences in DNA content in several Melanocrommyum species indicate the occurrence of diploid and tetraploid cytotypes in these taxa, which for some species has been confirmed by chromosome counts.
Allium subgenus Melanocrommyum is a species-rich group of perennial onions with uniform karyology, ecology, and breeding system. Thus, the cytological, ecological, and physiological factors often ...correlated with genome size should have negligible effect on genome size variation in Melanocrommyum. We measured DNA content in subgenus Melanocrommyum using flow cytometry based on propidium iodide staining and analyzed the evolution of genome size in a phylogenetic context. The observed 2C genome size variation in 160 accessions of 70 species of the subgenus was high, varying from 26.26–78.73 pg. The significant phylogenetic signal in genome size data suggests that distribution of genome size is in accordance with phylogenetic clades identified by the analysis of nuclear ITS sequences. Estimation of ancestral genome sizes using generalized least squares revealed lineages with increasing as well as decreasing DNA content. We found within-species genome size variation to be mostly below 2.5%. In species where intraspecific genome size differences were in a range of 6–9%, we suggest the existence of cryptic species, as previously inferred by molecular markers. Thus, genome size variation reflects incipient speciation or diversification in Allium subgenus Melanocrommyum. About two-fold differences in DNA content in several Melanocrommyum species indicate the occurrence of diploid and tetraploid cytotypes in these taxa, which for some species has been confirmed by chromosome counts.
Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic ...modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.
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