Deploying Genome Editing Tools for Dissecting the Biology of Nut Trees

Abstract

Walnuts are among the most important nut crops grown in temperate regions of the world. Commercial production in California, and increasingly worldwide, relies on only few clonally grafted scion genotypes, particularly "Chandler," and more recently clonally propagated disease-resistant rootstocks. Diseases, nematodes, insect pests, abiotic stresses, and other nutritional and environmental factors, can reduce walnut productivity and quality, affecting grower profitability. The California Walnut Breeding Program at UC Davis has developed and released scion cultivars and rootstocks to help address some of these problems. Sequencing of the walnut genome is expected to speed walnut breeding by facilitating development of molecular markers that can be linked to phenotypic traits, and by reducing the time needed to screen new genotypes. Nonetheless, conventional breeding of tree crops is still a long-term proposition. Here we describe a toolkit that utilizes the CRISPR-Cas9 system to enable rapid and precise editing of the genomes of currently grown commercial clonal walnut genotypes. A computational tool was developed to aid selection of guide RNAs targeting specific genomic sites of this crop and to identify potential off-target sites. As a proof-of-concept, the gene encoding phytoene desaturase (PDS) of walnut was targeted, disrupting its expression. A dominant visual phenotype associated with the successful editing of this gene was observed in non-pigmented "albino" shoots obtained from in vitro cultures. This toolkit can now be adapted to other important tree crop members of the order Fagales, which includes other valuable nut and tree species such as pecans, butternuts, chestnuts, hickory nuts, hazelnut, wingnut, and oaks.

First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

“…To determine whether the CRISPR/Cas9 system may be suitable for gene editing in C. sativa , we used the pds gene as a target. As this gene is related to chlorophyll biosynthesis (Walawage et al, 2019 ; Wang et al, 2020 ), an albino phenotype of somatic embryos is expected, which would allow easier discrimination of mutated embryos. This visual marker has also been applied in defining editing methods in other woody species such as cassava (Odipio et al, 2017 ), coffee (Breitler et al, 2018 ), and walnut (Walawage et al, 2019 ).…”

“…As this gene is related to chlorophyll biosynthesis (Walawage et al, 2019 ; Wang et al, 2020 ), an albino phenotype of somatic embryos is expected, which would allow easier discrimination of mutated embryos. This visual marker has also been applied in defining editing methods in other woody species such as cassava (Odipio et al, 2017 ), coffee (Breitler et al, 2018 ), and walnut (Walawage et al, 2019 ). Besides the proof-of-concept studies, the CRISPR/Cas9 system has been applied to obtain disease-resistant fruit trees (Bewg et al, 2018 ).…”

“…Breitler et al (2018) pointed out that the loss of function of pds gene causes a reduction in photosynthetic pigments that provoke the low germination rates. Similarly, Walawage et al (2019) found in walnut that pds gene is essential for proper plant growth. As expected, chestnut-transformed somatic embryos showed shoots displaying the typical albino phenotype, but a stunted phenotype with shorter internodes and small leaves was also observed (Figures 4A,B).…”

“…This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by the homology-directed recombination or to target and modify the transcription. In addition, CRISPR/Cas9 is inexpensive, simple, and highly flexible allowing the rapid target plant genome editing (Walawage et al, 2019 ). The development of new genome editing technologies in plant breeding fostered a growing interest for in vitro culture and regeneration protocols, which represent a major bottleneck in the application of these techniques in many plant species of agricultural and industrial interest.…”

“…So far, reports of genome editing in tree species are still limited (Bewg et al, 2018 ). Nowadays, gene editing has been only reported in a few fruit tree species such as coffee (Breitler et al, 2018 ), apple (Nishitani et al, 2016 ), grape (Wang et al, 2018 ), cocoa (Fister et al, 2018 ), and walnut (Walawage et al, 2019 ), while in forest species, gene editing has been only achieved in poplar (Fan et al, 2015 ).…”