Chromosome-level genome assembly and population genomic resource to accelerate orphan crop lablab breeding.

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).

Trend, population structure, and trait mapping from 15 years of national varietal trials of UK winter wheat.

There are now a rich variety of genomic and genotypic resources available to wheat researchers and breeders. However, the generation of high-quality and field-relevant phenotyping data which is required to capture the complexities of gene × environment interactions remains a major bottleneck. Historical datasets from national variety performance trials (NVPT) provide sufficient dimensions, in terms of numbers of years and locations, to examine phenotypic trends and study gene × environment interactions. Using NVPT for winter wheat varieties grown in the United Kingdom between 2002 and 2017, we examined temporal trends for eight traits related to yield, adaptation, and grain quality performance. We show a non-stationary linear trend for yield, grain protein content, Hagberg Falling Number (HFN), and days to ripening. Our data also show high environmental stability for yield, grain protein content, and specific weight in UK winter wheat varieties and high environmental sensitivity for HFN. We also show that UK varieties released within this period cluster into four main population groups. Using the historical NVPT data in a genome-wide association analysis, we uncovered a significant marker-trait association peak on wheat chromosome 6A spanning the NAM-A1 gene that have been previously associated with early senescence. Together, our results show the value of utilizing the data routinely collected during national variety evaluation process for examining breeding progress and the genetic architecture of important traits.

Introgression of Maize Lethal Necrosis Resistance Quantitative Trait Loci Into Susceptible Maize Populations and Validation of the Resistance Under Field Conditions in Naivasha, Kenya.

Maize lethal necrosis (MLN), resulting from co-infection by maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) can cause up to 100% yield losses in maize in Africa under serious disease conditions. Maize improvement through conventional backcross (BC) takes many generations but can significantly be shortened when molecular tools are utilized in the breeding process. We used a donor parent (KS23-6) to transfer quantitative trait loci (QTL) for resistance to MLN into nine adapted but MLN susceptible lines. Nurseries were established in Kiboko, Kenya during 2015-2017 seasons and BC3F2 progeny were developed using marker assisted backcrossing (MABC) approach. Six single nucleotide polymorphism (SNP) markers linked to QTL for resistance to MLN were used to genotype 2,400 BC3F2 lines using Kompetitive Allele Specific PCR (KASP) platform. We detected that two of the six QTL had major effects for resistance to MLN under artificial inoculation field conditions in 56 candidate BC3F2 lines. To confirm whether these two QTL are reproducible under different field conditions, the 56 BC3F2 lines including their parents were evaluated in replicated trials for two seasons under artificial MLN inoculations in Naivasha, Kenya in 2018. Strong association of genotype with phenotype was detected. Consequently, 19 superior BC3F2 lines with favorable alleles and showing improved levels of resistance to MLN under artificial field inoculation were identified. These elite lines represent superior genetic resources for improvement of maize hybrids for resistance to MLN. However, 20 BC3F2 lines were fixed for both KASP markers but were susceptible to MLN under field conditions, which could suggest weak linkage between the KASP markers and target genes. The validated two major QTL can be utilized to speed up the breeding process but additional loci need to be identified between the KASP markers and the resistance genes to strengthen the linkage.

Genetic Screening for Mutants with Altered Seminal Root Numbers in Hexaploid Wheat Using a High-Throughput Root Phenotyping Platform.

Roots are the main channel for water and nutrient uptake in plants. Optimization of root architecture provides a viable strategy to improve nutrient and water uptake efficiency and maintain crop productivity under water-limiting and nutrient-poor conditions. We know little, however, about the genetic control of root development in wheat, a crop supplying 20% of global calorie and protein intake. To improve our understanding of the genetic control of seminal root development in wheat, we conducted a high-throughput screen for variation in seminal root number using an exome-sequenced mutant population derived from the hexaploid wheat cultivar Cadenza. The screen identified seven independent mutants with homozygous and stably altered seminal root number phenotypes. One mutant, Cadenza0900, displays a recessive extra seminal root number phenotype, while six mutants (Cadenza0062, Cadenza0369, Cadenza0393, Cadenza0465, Cadenza0818 and Cadenza1273) show lower seminal root number phenotypes most likely originating from defects in the formation and activation of seminal root primordia. Segregation analysis in F2 populations suggest that the phenotype of Cadenza0900 is controlled by multiple loci whereas the Cadenza0062 phenotype fits a 3:1 mutant:wild-type segregation ratio characteristic of dominant single gene action. This work highlights the potential to use the sequenced wheat mutant population as a forward genetic resource to uncover novel variation in agronomic traits, such as seminal root architecture.

Haplotype Analysis of the Pre-harvest Sprouting Resistance Locus Phs-A1 Reveals a Causal Role of TaMKK3-A in Global Germplasm.

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 programs. 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 characterization 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 United Kingdom 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 United Kingdom 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.

The wheat Phs-A1 pre-harvest sprouting resistance locus delays the rate of seed dormancy loss and maps 0.3 cM distal to the PM19 genes in UK germplasm.

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.

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover.

Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.