Statistics: Methods and Applications.

Summary
The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off-target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.

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The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny

Summary Genome editing with engineered nucleases (GEEN) represents a highly specific and efficient tool for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. Genome editing techniques initiate specifically targeted double strand breaks facilitating DNArepair pathways that lead to base additions or deletions by non-homologous end joining as well as targeted gene replacements or transgene insertions involving homology-directed repair mechanisms. Many of these techniques and the ancillary processes they employ generate phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis; and therefore, they do not readily fit current definitions of genetically engineered or genetically modified used within most regulatory regimes. Addressing ambiguities regarding the regulatory status of genome editing techniques is critical to their application for development of economically useful crop traits. Continued regulatory focus on the process used, rather than the nature of the novel phenotype developed, results in confusion on the part of regulators, product developers, and the public alike and creates uncertainty as of the use of genome engineering tools for crop improvement.

https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=1095&context=agron_pubs

The Regulatory Status of Genome-edited Crops

Enabling plant synthetic biology through genome engineering.

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.

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Use of designer nucleases for targeted gene and genome editing in plants.

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 Genet 9 (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, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. 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.

https://www.pnas.org/content/pnas/112/36/11211.full.pdf

Point mutation impairs centromeric CENH3 loading and induces haploid plants

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.

/pdf/true-breeding-targeted-gene-knock-out-in-barley-using-3v2b0p9did.pdf

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

Allium subgenus Melanocrommyum from Eurasia is taxonomically complicated with different and contradictory taxonomic treatments proposed in the past. A comprehensive study, covering nearly all existing taxonomic groups and their entire geographic distribution, by sequencing the nuclear rDNA internal transcribed spacer region (ITS) confirmed the monophyly of the subgenus, while most traditionally used sections were either para- or polyphyletic. Addition of ten more species and 150 new accessions to that dataset confirmed the earlier results and underlined that multiple rapid radiations occurred within different monophyletic groups of the subgenus. About 40 well separated molecular clades were recognized, and taxonomically discussed and affiliated, but their phylogenetic relations remained often unresolved. The splits of some large "classical" sections was unavoidable. Therefore the sections Asteroprason R. M. FRITSCH and Procerallium R. M. FRITSCH as well as the subsections Humilicognata R. M. Fritsch, Diffusoumbellata R. M. FRITSCH, Keratoprason R. M. FRITSCH and Pharmakoprason R. M. FRITSCH were newly described, the subsections Decipientia (OMELCZUK) R. M. FRITSCH and Stellata (F. O. KHASS. & R. M. FRITSCH) R. M. FRITSCH were raised to sectional level, four subspecies were raised to species level, and the name Allium jaubertii R. M. FRITSCH was created for an illegitimate species name. Altogether 160 species and subspecies were accepted (plus 53 unclear or synonymous names) in the updated conspectus of A. subg. Melanocrommyum. They were affiliated to 20 sections and 22 subsections (sect. Acanthoprason WENDELBO and sect. Melanocrommyum WEBB & BERTHEL. were only subdivided in alliances). Lectotypes of 17 species were designated or validated, and neotypes for A. isfairamicum O. FEDTSCH., A. suworowii REGEL, A. viridiflorum POBED., and epitypes for A. bakhtiaricum REGEL and A. derderianum REGEL were designated.

New classification of Allium L. subg. Melanocrommyum (Webb & Berthel.) Rouy (Alliaceae) based on molecular and morphological characters.

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification.

Allium section Cepa consists of 12 species most of which are used by humans as condiment, vegetable or medicinal plants. Common onion (Allium cepa) and bunching onion (A. fistulosum) are cultivated species while all others are locally collected from the wild. Although common onion is the most important crop within Allium, its wild progenitor and origin are still not clear. We analyzed the phylogeny of Allium section Cepa using sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region of 36 accessions representing eleven species of section Cepa, together with eight outgroup species with phenetic, cladistic, and model-based algorithms. These analyses confirmed section Cepa to be monophyletic and revealed three species groups within the section. These are (i) A. altaicum/A. fistulosum, (ii) A. farctum/A. roylei/A. asarense/A. cepa/A. vavilovii, and (iii) A. galanthum/A. oschaninii/A. praemixtum/A. pskemense. While the first two groups were statistically well supported for the last group support was low, although it resulted in all phylogenetic analyses conducted. Tree and network-based analyses grouped A. cepa within A. vavilovii, indicating the latter to be progenitor of the common onion. However, also an origin of A. cepa through hybridization of A. vavilovii with A. galanthum or A. fistulosum seems possible. We argue that a subdivision of section Cepa in subsections Cepa and Phyllodolon, although possible from our data, as well as the formal description of alliances do not seem reasonable in a small group of species.

Maia Gurushidze

Papers

Point mutation impairs centromeric CENH3 loading and induces haploid plants

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

New classification of Allium L. subg. Melanocrommyum (Webb & Berthel.) Rouy (Alliaceae) based on molecular and morphological characters.

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification.

Phylogenetic relationships of wild and cultivated species of Allium section Cepa inferred by nuclear rDNA ITS sequence analysis