New crops have the potential to improve the climate resilience of agriculture, to supply biobased materials to reduce fossil oil use and CO2-emissions, and to enable a transition to plant-based protein diets. However, these crops have not been fully domesticated (i.e., "wild" traits like seed dehiscence, antinutritional factors (ANF), and seed dormancy are still present) and the lack of investment in their genetic improvement leaves them as "emerging and underexposed crops". This project aims to fast-track the use of modern breeding tools in two emerging and underexposed crops: 1) quinoa, a species with high abiotic stress tolerance to salinity and drought, and gluten-free grains with a high and balanced content of essential amino acids; and 2) white lupin, a robust leguminous species adapted to temperate climates that exhibits protein and oil contents comparable to those of soy. This project will speed-up the process of creation of new genetic variation and varieties in these crops. Mutant populations will be created, and gene editing tools (CRISPR) will be developed to target antinutritional factors (i.e., saponin in quinoa and alkaloids in lupin). Other targets include enhancing the quality of primary and secondary products (protein content, starch quality, straw quality). Key genes in the pathways of these traits are known, and some examples in these crops already have been created by WR using reverse genetics screens. Also, forward mutation screens will be used to find mutants with either increased or impaired abiotic stress tolerance (drought and salt) to find genes underlying resilience to abiotic stresses. Finally, mutation breeding and gene editing will be used to create new possibilities for making F1-hybrids. Two routes to creating F1-hybrid systems will be targeted: 1) finding lines with delayed male flowering to be used as maternal lines in an F1, but which can be multiplied by selfing; and 2) simplifying the use of cytoplasmic male sterility (CMS) in cases where CMS is available, but restorer/maintainer lines are lacking. In quinoa, restorer gene loci are known, and knockout lines of those genes will be tested for suitability as maintainer lines. The core innovation in this project to be developed for these new crops is the development of classical mutation breeding and gene editing CRISPR/CAS9 strategies in combination with advanced phenotyping, sequencing technologies, and (heuristic) gene function knowledge. Bioinformatics tools for the analysis of sequence data to find rare mutations and to predict gene function knowledge will also be a key innovation of the project. The plant materials from mutation breeding can be used within the legal framework of the European Union. To facilitate the practical use of mutants produced with CRISPR/CAS9, we aim to develop a transient expression of CRISPR/CAS9 that leads to mutants but not to integration of transgenic elements into the genome of the plant.

This project contributes to the development of generic tools for genetic research and breeding of two emerging and underexposed crops (quinoa and white lupin), and therefore is at the heart of priority 51. The project also contributes to other priorities in the field of more climate change resilient crops, circular economy (transition from fossil to biobased) and in the field of the transition to plant-based proteins. The two emerging crops will produce plant protein of
high quality.

The project will allow to follow up on significant progress made in the last few years in the field of
genomics, QTL-mapping, mutation breeding and advanced gene editing and to make the use of the
tools possible in the two emerging crops, quinoa and lupin. Currently, the full complement of mutation populations and transient expression systems for CRISPR/CAS9 mutation induction is not
yet available. The project will also develop dedicated forward/reverse genetic and bioinformatics
methods for these species. Collectively, these tools (mutant populations, CRISPR/CAS9 transient
expression system, bioinformatics methods) will be used to create new useful genetic diversity and
new genotypes for use in breeding with superior traits. In quinoa both EMS mutation breeding and
CRISPR/CAS9 are targeted, but in lupin - as the results are intended to be used in an organic farming market in the end - CRISPR/CAS9 will not be used.
Furthermore, the technologies that will be developed in this project can be applied to other crops, so classifies under generic technology and tool development.

WP1 Selection for target genes for mutation breeding and gene editing

A) Find the molecular-genetic background of natural mutants by bulked segregant sequencing analysis in populations segregating for domestication traits in lupin (alkaloid content, disease tolerance and traits conferring tolerance to abiotic stress).
B) Select target genes for mutation breeding to modify target processes based on bioinformatics tools that can predict gene functions (for metabolic enzymes, or their regulatory elements, promoters and transcription factors) (quinoa and lupin)
WP2. Create new genetic diversity for domestication traits for which genetic variation is lacking due to the short breeding history in these emerging crops. Both classical mutation breeding (chemical) and advanced gene editing tools (CRISPR/CAS9) will be used and developed:
2a. Develop EMS mutation strategies and populations in lupin and quinoa
2b. Set up CRISPR/CAS9 tools in quinoa (target of transient expression of CRISP system).
WP3. Forward genetic screens to discover QTL and genes involved in seed yield and abiotic stress tolerance by evaluation of mutant populations (lupin and quinoa).
WP4. Reverse genetics to validate target genes for domestication traits and create the new variants. Targets in quinoa include 1) transcription factor (BHLH25) regulating the pathway to saponins (bitter taste) and 2) restorer gene for CMS in quinoa (to make maintainer lines) and a mitochondrial mutation to make new CMS towards a de novo F1-hybrid system. Targets in lupin include alkaloid content.

Envisioned results:
1. A population in white lupin segregating for yield, alkaloid content (grain and leaves), tolerance to anthracnose (starting point for breeding non-alkaloid, resistant varieties by Radicle Crops and Lekker Lupine) (WP2a)
2. New EMS mutants in quinoa and white lupin for gene function analysis and for breeding programmes of Radicle Crops, Lekker Lupine and Wageningen Research) (WP2a)
3. Knowledge on gene function will result in possibilities to search existing genetic variation for functional alleles for these genes in large collections of quinoa and white lupin. Accessions with such functional genetic diversity can be used in the breeding programmes of partners (WP1-WP4)
4. The CRISPR/CAS9 methods for new crops can provide a fast way of targeting multiple gene targets in a single event. This can speed up gene function analysis. The knowledge obtained can then be used to modify target genes through classical mutation breeding to fast-track new varieties in line with current legislation (WP2b)
5. The transient expression system of CRISPR/CAS9 will provide a method for making CRISPR mutants without introgression of transgenic elements (i.e., and therefore functionally equivalent to EMS mutants). This can help the acceptance of CRISPR mutants. Marketing by Radicle Crops of CRISPR mutants will prove easier and technology will be a service product of Plantik (WP4)
6. Bio-informatic tools developed for the two example crops will also be useful for other new and emerging crops (e.g., hemp, industrial oil crops, biomass crops). The partner Plantik can use the new tools in their business of providing bio-informatics services (WP1)

Detailed expected deliverables:

WP1A Find the molecular-genetic background of natural mutants by bulked segregant sequencing analysis (lupin)
Year 1:
Cross contrasting parents (alkaloid, disease tolerance, yield)
Self F1 to obtain >800 F2 seeds
Year 2:
Self F2- plants to obtain > 700 F3 families
Collect leaf material of > 700 F2 plants
Evaluate alkaloid content of the seed of the F3 families
Make bulks of leaves of low and high alkaloid genotypes and isolate DNA
Year 3:
100x sequencing of the two bulks and parents, bioinformatics
Identify genes with mutations in the genome interval where the bulks differ in sequence
Year 4:
Scientific reporting

WP1B Select target genes for mutation breeding to modify targets processes based on bioinformatics tools that can predict gene functions
Year 1:
Finding target genes for alkaloid system including modifying genes
Finding target genes for saponin route (validation of heuristic system)
Finding target genes for restoration of fertility genes
Fine tuning the bio-informatics pipelines for mutant discovery

Year 2:
Finding target genes for alkaloid system including modifying genes
Finding target genes for saponin route (validation of heuristic system)
Finding target genes for restoration of fertility genes
Fine tuning the bio-informatics pipelines for mutant discovery

Year 3:
Using the mutant discovery pipelines
Making improved heuristic tools for finding target genes

Year 4:
Using the mutant discovery pipeline
Making improved heuristic tools for finding target genes
Scientific reporting

WP2. Create new genetic diversity for domestication traits for which genetic variation is lacking
Year 1:
Make EMS population of lupin and quinoa: grow 1,000 mutated M1-seeds each
Harvest 10 seeds per M1 family and multiply to 12,000 M2 seeds per crop
Year 2:
Sow 12,000 M2 seeds for each crop and harvest 12,000 M3 families
Collect leaf material of the M2 plants per M1 parent to obtain 1,000 leaf/DNA samples
Finalize set up CRISPR/Cas9 tools in quinoa (target of transient expression of CRISPR system) and produce target gene constructs
Year 4:
Scientific reporting

WP3. Forward genetic screens to discover QTL and genes
Year 3:
Grow 12,000 M3 families in field at mild and severe drought stress: find knockout genotypes
Whole genome sequence analysis of 6 phenotypically selected mutants per crop and the wild type lines
Find the mutations where the mutants differ and use heuristic tools of Plantik to filter possible candidate genes
Year 4:
Whole genome sequence analysis of 6 phenotypically selected mutants per crop and the wild type lines
Find the mutations where the mutants differ and use heuristic tools of Plantik to filter possible candidate genes
Scientific reporting

WP4. Reverse genetics to validate target genes for domestication traits
Year 3:
Find EMS knockout of alkaloid gene in mutant population in lupin
Find EMS knockout of restorer of fertility gene in quinoa
Maker CRISPR mutant lines knockout of the BHLH15 saponin gene
Make CRISPR mutant lines with knockout of restorer of fertility gene
Make CRISPR mutant lines with mitochondrial gene giving pollen sterility in a maintainer background
Year 4:
Multiply alkaloid knockout genotype
Multiply knockout of Rf gene genotype
Scientific reporting
Each year a project meeting will be held in the period May/June. The goal of this meeting is to discuss the progress of the project with all the Consortium members and to show the actual field/greenhouse and labtrials. At the annual meeting progress will be reviewed and if delays in the delivery of certain result occur, it will be decided how timelines will be adjusted or how effort increased.