The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement. We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19. We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.

The precocious germination of cereal grains before harvest, also known as pre-harvest sprouting, is an important source of yield and quality loss in cereal production. Pre-harvest sprouting is a complex grain defect and is becoming an increasing challenge due to changing climate patterns. Resistance to sprouting is multi-genic, although a significant proportion of the sprouting variation in modern wheat cultivars is controlled by a few major quantitative trait loci, including Phs-A1 in chromosome arm 4AL. Despite its importance, little is known about the physiological basis and the gene(s) underlying this important locus. In this study, we characterized Phs-A1 and show that it confers resistance to sprouting damage by affecting the rate of dormancy loss during dry seed after-ripening. We show Phs-A1 to be effective even when seeds develop at low temperature (13 °C). Comparative analysis of syntenic Phs-A1 intervals in wheat and Brachypodium uncovered ten orthologous genes, including the Plasma Membrane 19 genes (PM19-A1 and PM19-A2) previously proposed as the main candidates for this locus. However, high-resolution fine-mapping in two bi-parental UK mapping populations delimited Phs-A1 to an interval 0.3 cM distal to the PM19 genes. This study suggests the possibility that more than one causal gene underlies this major pre-harvest sprouting locus. The information and resources reported in this study will help test this hypothesis across a wider set of germplasm and will be of importance for breeding more sprouting resilient wheat varieties.

Pre-harvest sprouting (PHS) is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programmes. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 (PM19) genes and the mitogen-activated protein kinase kinase 3 (TaMKK3-A) gene, have been proposed to underlie Phs-A1. To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterisation of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A, and not PM19, is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In UK varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A. Analysis of these haplotypes in a collection of UK and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the study will help inform the choice of germplasm and breeding strategies for the deployment of Phs-A1 resistance into breeding germplasm.

Under-utilised orphan crops hold the key to diversified and climate-resilient food systems. Here, we report on orphan crop genomics using the case of Lablab purpureus (L.) Sweet (lablab) - a legume native to Africa and cultivated throughout the tropics for food and forage. Our Africa-led plant genome collaboration produces a high-quality chromosome-scale assembly of the lablab genome. Our assembly highlights the genome organisation of the trypsin inhibitor genes - an important anti-nutritional factor in lablab. We also re-sequence cultivated and wild lablab accessions from Africa confirming two domestication events. Finally, we examine the genetic and phenotypic diversity in a comprehensive lablab germplasm collection and identify genomic loci underlying variation of important agronomic traits in lablab. The genomic data generated here provide a valuable resource for lablab improvement. Our inclusive collaborative approach also presents an example that can be explored by other researchers sequencing indigenous crops, particularly from low and middle-income countries (LMIC).

Underutilised crops are important for diversifying food systems. The genomics of these crops should not be done in isolation but should align with their breeding and capacity building strategies while leveraging advances made in major crops. Underutilised crops or orphan crops are important for diversifying our food systems towards food and nutrition security. Here, the authors discuss how the development of underutilised crop genomic resource should align with their breeding and capacity building strategies, and leverage advances made in major crops.