Taro (Colocasia esculenta) is a food staple widely cultivated in the humid tropics of Asia, Africa, Pacific and the Caribbean. One of the greatest threats to taro production is Taro Leaf Blight caused by the oomycete pathogen Phytophthora colocasiae. Here we describe a de novo taro genome assembly and use it to analyze sequence data from a Taro Leaf Blight resistant mapping population. The genome was assembled from linked-read sequences (10x Genomics; ∼60x coverage) and gap-filled and scaffolded with contigs assembled from Oxford Nanopore Technology long-reads and linkage map results. The haploid assembly was 2.45 Gb total, with a maximum contig length of 38 Mb and scaffold N50 of 317,420 bp. A comparison of family-level (Araceae) genome features reveals the repeat content of taro to be 82%, >3.5x greater than in great duckweed (Spirodela polyrhiza), 23%. Both genomes recovered a similar percent of Benchmarking Universal Single-copy Orthologs, 80% and 84%, based on a 3,236 gene database for monocot plants. A greater number of nucleotide-binding leucine-rich repeat disease resistance genes were present in genomes of taro than the duckweed, ∼391 versus ∼70 (∼182 and ∼46 complete). The mapping population data revealed 16 major linkage groups with 520 markers, and 10 quantitative trait loci (QTLs) significantly associated with Taro Leaf Blight disease resistance. The genome sequence of taro enhances our understanding of resistance to TLB, and provides markers that may accelerate breeding programs. This genome project may provide a template for developing genomic resources in other understudied plant species.

Culex mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a conservation threat to insular bird species. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex has been lagging compared to other disease vectors, with only a few reports testing the functionality of CRISPR in these species. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that when altered, lead to visible, homozygous-viable phenotypes. We further validate our approach on two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. Lastly, we generate a double-mutant line, demonstrating the possibility of combining multiple such mutations in a single individual. Our work provides a platform of five validated loci that could be used for targeted mutagenesis for research in Culex quinquefasciatus aimed at the development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.

Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex quinquefasciatus has been lagging compared to other disease vectors. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Culex quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.

Bacteria from the genusXanthomonasare prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing expression of host susceptibility (S) genes.Xanthomonas phaseolipv.manihotis(Xpm) causes bacterial blight on the staple food crop, cassava. The Xpm effector, TAL20, induces ectopic expression of the S gene,MeSWEET10a, a sugar transporter that contributes to cassava bacterial blight susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to theMeSWEET10aTAL20 effector binding site and/or coding sequence. In several of the regenerated lines,MeSWEET10aexpression was no longer induced byXpmand in these cases, we observed reduced cassava bacterial blight disease symptoms post Xpm infection.MeSWEET10ais expressed in cassava flowers. Therefore, we investigated flower development and reproductive function in anMeSWEET10amutant line. We found that theMeSWEET10amutant produced phenotypically wildtype cassava flowers and viable F1 seed. Thus, blockingMeSWEET10ainduction is a viable strategy for decreasing cassava susceptibility to CBB.