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Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.