Wild relatives of wheat are a valuable source of genetic variation for crop improvement. Determining the breeding value (ie., average downstream performance) of exotic germplasm is important in selecting which germplasm to integrate into a breeding program. Despite harboring desirable genetic traits, such as disease resistance and climate resilience, assessing the collective breeding value of these traits is challenging due to the influence of various factors like phenology, height, and flowering time, which mask their agronomic potential.

To address this challenge, we devised a CRISPR/Cas-based gene editing approach to recreate adaptive traits in exotic wheat germplasm, thereby unmasking beneficial traits to support pre-breeding. We employed a two-pronged approach to modify genes controlling adaptive and domestication traits in wheat: the first approach involves a combination of gene editing and hybrid wheat technologies, and the second approach involves direct modification of domestication related genes in wild emmer accessions.

We constructed a multiplex gene knockout construct targeting five key genes, RhtB1, PpdA1, VRN2, Q, and Btr1 that regulate plant height, photoperiod response, vernalization requirement, seed threshability and brittle rachis in wheat, respectively. This construct was used to transform an elite durum wheat cultivar, CDC Fortitude, creating the TetraOM tester line.

Subsequent intercrossing of TetraOM and tetraploid emmer wheat accession (TG3487) produced F1 plants, which were assessed for emergence date, plant height, heading date, time to flowering, maturity and spike characteristics. Although the F1 plants exhibited delayed growth, reduced tiller numbers, decreased fertility rates, and considerably prolonged germination periods, we observed limited editing efficiency in the targeted genes, prompting us to explore alternative strategies.

Among these strategies, the VIGS-based gene editing system, particularly targeting the PDS and Q genes in TG3487, exhibited promising outcomes and is currently being tested on other domestication genes. Additionally, direct editing of domestication genes in the TG3487 background, utilizing the JD633 vector, showed successful transformation and regeneration, as evidenced by targeted disruption of the Rht-B1 gene, resulting in phenotypically shorter plants compared to the wild type.

Further advancement involved a multi-guide approach targeting multiple genes (BTR1, Q, PpdA1, VRN2, and Rht-B1) simultaneously within a single construct. Transformation and sequencing analysis of T0 plants demonstrated successful editing at all five target genes.

This breakthrough sets the stage for efficient simultaneous editing of multiple domestication genes in exotic germplasm within a relatively short timeframe, laying a solid foundation for pre-breeding endeavors involving wild relatives of wheat.