CRISPR-Cas9-mediated genome editing in apple and grapevine.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

Selection of reference genes suitable for normalization of qPCR data under abiotic stresses in bioenergy crop Arundo donax L.

Suitable reference gene selection in qRT-PCR is a key pre-requisite to produce reliable data in gene expression analyses. In this study, novel primers for six commonly used reference genes (AC1, TLF, Act2, TUB α, EF-1α and GAPDH) plus two new candidates (pDUF221 and RPN6) were designed and comparatively tested for expression stability under abiotic stresses (osmotic, heavy metal and heat shock) in shoot, root and their combination of Arundo donax L., a raising non-food energy crop. Expression stability rankings from the most to the least stable gene in each condition and in two tissues (young shoots and roots) were generated with geNorm, NormFinder and BestKeeper programs. All programs provided similar rankings and, strikingly, in most cases identified one of the new candidates, RPN6, as the most suitable reference gene. This novel set of reliable references allows to choose either the best combination of reference genes across multiple stress/organ conditions or to select condition-specific genes that can improve the quality of qRT-PCR analysis. This work provides a solid basis for the functional characterization of A. donax, by enabling accurate quantification of the transcriptional responsiveness under a series of common stress conditions of any gene of interest in this promising biomass/bioenergy species.

Dissection of early transcriptional responses to water stress in Arundo donax L. by unigene-based RNA-seq.

BACKGROUND: Arundo donax L. (Poaceae) is considered one of the most promising energy crops in the Mediterranean region because of its high biomass yield and low input requirements, but to date no information on its transcriptional responses to water stress is available. RESULTS: We obtained by Illumina-based RNA-seq the whole root and shoot transcriptomes of young A. donax plants subjected to osmotic/water stress with 10 and 20 % polyethylene glycol (PEG; 3 biological replicates/organ/condition corresponding to 18 RNA-Seq libraries), and identified a total of 3034 differentially expressed genes. Blast-based mining of stress-related genes indicated the higher responsivity of roots compared to shoots at the early stages of water stress especially under the milder PEG treatment, with a majority of genes responsive to salt, oxidative, and dehydration stress. Analysis of gene ontology terms underlined the qualitatively different responses between root and shoot tissues. Among the most significantly enriched metabolic pathways identified using a Fisher's exact test with FDR correction, a crucial role was played in both shoots and roots by genes involved in the signaling cascade of abscisic acid. We further identified relatively large organ-specific differences in the patterns of drought-related transcription factor AP2-EREBP, AUX/IAA, MYB, bZIP, C2H2, and GRAS families, which may underlie the transcriptional reprogramming differences between organs. Through comparative analyses with major Poaceae species based on Blast, we finally identified a set of 53 orthologs that can be considered as a core of evolutionary conserved genes important to mediate water stress responses in the family. CONCLUSIONS: This study provides the first characterization of A. donax transcriptome in response to water stress, thus shedding novel light at the molecular level on the mechanisms of stress response and adaptation in this emerging bioenergy species. The inventory of early-responsive genes to water stress identified could constitute useful markers of the physiological status of A. donax and be a basis for the improvement of its productivity under water limitation. The full water-stressed A. donax transcriptome is available for Blast-based homology searches through a dedicated web server (http://ecogenomics.fmach.it/arundo/).