Shallu Thakur’s research while affiliated with University of Florida and other places

Cucurbita crops, which include market types of pumpkin and squash, have unparalleled fruit variation and equally important economic value worldwide. Pumpkin and squash have versatile uses but are most popular in culinary, ornamental, snacking, and seed oil industries. The production of Cucurbita crops is hindered by diseases caused by fungal, oomycetes, and viral pathogens. Host resistance is an important component of integrated disease management for Cucurbita crops and is a major goal for plant breeders. This review addresses the major diseases of Cucurbita, including powdery mildew, downy mildew, Phytophthora rot, and aphid and whitefly transmitted viruses, with an emphasis on germplasm exploitation for the development of resistant cultivars. Resistance to powdery mildew derived from Cucurbita okeechobeensis subsp. martinezii (designated PM‐0) has been extensively deployed in commercial cultivars and was recently mapped on chromosomes 3 and 10 of Cucurbita moschata and Cucurbita pepo, respectively. Resistance to Phytophthora crown rot is present across several Cucurbita species, including Cucurbita lundelliana, C. okeechobeenesis subsp. okeechobeenesis, C. moschata, and C. pepo. Mapping studies have identified significant loci associated with Phytophthora crown rot resistance on chromosomes 4, 11, and 20 of C. moschata and chromosomes 4, 5, 8, 12, 13, 16, and 19 of C. pepo. Sources of resistance to aphid‐transmitted viruses exist in C. moschata, Cucurbita ficifolia, Cucurbita ecuadorensis, Cucurbita martinezii, C. ecuadorensis, Cucurbita maxima, and Cucurbita foetidissima. The availability of DNA markers linked to resistance against zucchini yellow mosaic virus and papaya ringspot virus in C. moschata and C. pepo has facilitated marker‐assisted selection (MAS) in breeding programs. On the other hand, sources of resistance to tomato leaf curl New Delhi virus (ToLCNDV), a major whitefly‐transmitted virus in Cucurbita, have been identified in C. moschata, C. lundelliana, and C. okeechobeensis. A major locus conferring resistance to ToLCNDV in C. moschata was recently mapped on chromosome 8 enabling the application of MAS with a prediction accuracy of 94.3%. Overall, the continued discovery and application of genomic tools for resistance breeding in Cucurbita will accelerate the rate of genetic gain while reducing costs associated with phenotyping.

Phytophthora capsici incites foliar blight, root, fruit, and crown rot in squash (Cucurbita spp.) and limits production worldwide. Resistance to crown rot in C. moschata breeding line #394-1-27-12 is conferred by three dominant genes, but the molecular mechanisms underlying this resistance are poorly understood. In the current study, RNA sequencing was used to investigate transcriptional changes in #394-1-27-12 (resistant) and Butterbush (susceptible) following infection by P. capsici at 12, 24, 48, 72, and 120 h post inoculation (hpi). Overall, the number of differentially expressed genes (DEGs) in Butterbush (2648) exceeded those in #394-1-27-12 (1729), but in both genotypes, the highest number of DEGs was observed at 72 hpi and least at 24 hpi. Our gene ontology (GO) analysis revealed a downregulation of the genes involved in polysaccharide and lignin metabolism in Butterbush but as an upregulation of those associated with regulation of peptidase activity. However, in #394-1-27-12, the downregulated genes were primarily associated with response to stimuli, whereas those upregulated were involved in oxidation-reduction and response to stress. The upregulated genes in #394-1-27-12 included defensin-like proteins, respiratory-burst oxidases, ethylene-responsive transcription factors, cytochrome P450 proteins, and peroxidases. These findings provide a framework for the functional validation of the molecular mechanisms underlying resistance to P. capsici in cucurbits.

Gene editing using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) system has become an important biotechnological tool for studying gene function and improving crops. In the present study, the potential of the system was assessed for squash (Cucurbita pepo subspecies pepo) by targeting phytoene desaturase (PDS) gene using the particle bombardment method. The recombinant pHSE401 vector, carrying two sgRNAs (gRNA1 and gRNA2) specific to the PDS homolog (Cp4.1LG08g06310, CpPDS) under the control of Arabidopsis U6 promoter and the Cas9 protein was developed and bombarded into cotyledonary node explants of squash cv. Black Beauty. The transformation efficiency of 4.5% was observed and all the transformants exhibited albino/bleached phenotype. The CpPDS knockout system generated easily detectable bleached/albino explants within 6–8 weeks. The albino phenotype was confirmed through Sanger sequencing which detected several deletion mutations (single, two and three bp deletion) within the CpPDS-gRNA1 target. However, no mutations were found within the CpPDS-gRNA2 target. This study demonstrated CRISPR/Cas9 as a viable tool for gene editing in squash and provides a platform for the modification of economically important traits in the crop.