CRISPR-Based Genome Editing Tools: An Accelerator in Crop Breeding for a Changing Future.

Genome editing is an important strategy to maintain global food security and achieve sustainable agricultural development. Among all genome editing tools, CRISPR-Cas is currently the most prevalent and offers the most promise. In this review, we summarize the development of CRISPR-Cas systems, outline their classification and distinctive features, delineate their natural mechanisms in plant genome editing and exemplify the applications in plant research. Both classical and recently discovered CRISPR-Cas systems are included, detailing the class, type, structures and functions of each. We conclude by highlighting the challenges that come with CRISPR-Cas and offer suggestions on how to tackle them. We believe the gene editing toolbox will be greatly enriched, providing new avenues for a more efficient and precise breeding of climate-resilient crops.

Candidate Rlm6 resistance genes against Leptosphaeria. maculans identified through a genome-wide association study in Brassica juncea (L.) Czern.

KEY MESSAGE: One hundred and sixty-seven B. juncea varieties were genotyped on the 90K Brassica assay (42,914 SNPs), which led to the identification of sixteen candidate genes for Rlm6. Brassica species are at high risk of severe crop loss due to pathogens, especially Leptosphaeria maculans (the causal agent of blackleg). Brassica juncea (L.) Czern is an important germplasm resource for canola improvement, due to its good agronomic traits, such as heat and drought tolerance and high blackleg resistance. The present study is the first using genome-wide association studies to identify candidate genes for blackleg resistance in B. juncea based on genome-wide SNPs obtained from the Illumina Infinium 90 K Brassica SNP array. The verification of Rlm6 in B. juncea was performed through a cotyledon infection test. Genotyping 42,914 single nucleotide polymorphisms (SNPs) in a panel of 167 B. juncea lines revealed a total of seven SNPs significantly associated with Rlm6 on chromosomes A07 and B04 in B. juncea. Furthermore, 16 candidate Rlm6 genes were found in these regions, defined as nucleotide binding site leucine-rich-repeat (NLR), leucine-rich repeat RLK (LRR-RLK) and LRR-RLP genes. This study will give insights into the blackleg resistance in B. juncea and facilitate identification of functional blackleg resistance genes which can be used in Brassica breeding.

Advancing designer crops for climate resilience through an integrated genomics approach.

Climate change and exponential population growth are exposing an immediate need for developing future crops that are highly resilient and adaptable to changing environments to maintain global food security in the next decade. Rigorous selection from long domestication history has rendered cultivated crops genetically disadvantaged, raising concerns in their ability to adapt to these new challenges and limiting their usefulness in breeding programmes. As a result, future crop improvement efforts must rely on integrating various genomic strategies ranging from high-throughput sequencing to machine learning, in order to exploit germplasm diversity and overcome bottlenecks created by domestication, expansive multi-dimensional phenotypes, arduous breeding processes, complex traits and big data.

Quantitative Trait Loci for Heat Stress Tolerance in Brassica rapa L. Are Distributed across the Genome and Occur in Diverse Genetic Groups, Flowering Phenologies and Morphotypes.

Heat stress events during flowering in Brassica crops reduce grain yield and are expected to increase in frequency due to global climate change. We evaluated heat stress tolerance and molecular genetic diversity in a global collection of Brassica rapa accessions, including leafy, rooty and oilseed morphotypes with spring, winter and semi-winter flowering phenology. Tolerance to transient daily heat stress during the early reproductive stage was assessed on 142 lines in a controlled environment. Well-watered plants of each genotype were exposed to the control (25/15 °C day/night temperatures) or heat stress (35/25 °C) treatments for 7 d from the first open flower on the main stem. Bud and leaf temperature depression, leaf conductance and chlorophyll content index were recorded during the temperature treatments. A large genetic variation for heat tolerance and sensitivity was found for above-ground biomass, whole plant seed yield and harvest index and seed yield of five pods on the main stem at maturity. Genetic diversity was assessed on 212 lines with 1602 polymorphic SNP markers with a known location in the B. rapa physical map. Phylogenetic analyses confirmed two major genetic populations: one from East and South Asia and one from Europe. Heat stress-tolerant lines were distributed across diverse geographic origins, morphotypes (leafy, rooty and oilseed) and flowering phenologies (spring, winter and semi-winter types). A genome-wide association analysis of heat stress-related yield traits revealed 57 SNPs distributed across all 10 B. rapa chromosomes, some of which were associated with potential candidate genes for heat stress tolerance.

The details are in the genome-wide SNPs: Fine scale evolution of the Malaysian weedy rice.

Weedy rice (Oryza spp.) is a major nuisance to rice farmers from all over the world. Although the emergence of weedy rice in East Malaysia on the island of Borneo is very recent, the threat to rice yield has reached an alarming stage. Using 47,027 genotyping-by-sequencing (GBS)-derived SNPs and candidate gene analysis of the plant architecture domestication gene TAC1, we assessed the genetic variations and evolutionary origin of weedy rice in East Malaysia. Our findings revealed two major evolutionary paths for genetically distinct weedy rice types. Whilst the cultivar-like weedy rice are very likely to be the weedy descendant of local coexisting cultivars, the wild-like weedy rice appeared to have arisen through two possible routes: (i) accidental introduction from Peninsular Malaysia weedy rice populations, and (ii) weedy descendants of coexisting cultivars. The outcome of our genetic analyses supports the notion that Sabah cultivars and Peninsular Malaysia weedy rice are the potential progenitors of Sabah weedy rice. Similar TAC1 haplotypes were shared between Malaysian cultivated and weedy rice populations, which further supported the findings of our GBS-SNP analyses. These different strains of weedy rice have convergently evolved shared traits, such as seeds shattering and open tillers. A comparison with our previous simple-sequence repeat-based population genetic analyses highlights the strength of genome-wide SNPs, including detection of admixtures and low-level introgression events. These findings could inform better strategic management for controlling the spread of weedy rice in the region.

The Use of Genetic and Gene Technologies in Shaping Modern Rapeseed Cultivars (Brassica napus L.).

Since their domestication, Brassica oilseed species have undergone progressive transformation allied with the development of breeding and molecular technologies. The canola (Brassica napus) crop has rapidly expanded globally in the last 30 years with intensive innovations in canola varieties, providing for a wider range of markets apart from the food industry. The breeding efforts of B. napus, the main source of canola oil and canola meal, have been mainly focused on improving seed yield, oil quality, and meal quality along with disease resistance, abiotic stress tolerance, and herbicide resistance. The revolution in genetics and gene technologies, including genetic mapping, molecular markers, genomic tools, and gene technology, especially gene editing tools, has allowed an understanding of the complex genetic makeup and gene functions in the major bioprocesses of the Brassicales, especially Brassica oil crops. Here, we provide an overview on the contributions of these technologies in improving the major traits of B. napus and discuss their potential use to accomplish new improvement targets.

Understanding Host-Pathogen Interactions in Brassica napus in the Omics Era.

Brassica napus (canola/oilseed rape/rapeseed) is an economically important crop, mostly found in temperate and sub-tropical regions, that is cultivated widely for its edible oil. Major diseases of Brassica crops such as Blackleg, Clubroot, Sclerotinia Stem Rot, Downy Mildew, Alternaria Leaf Spot and White Rust have caused significant yield and economic losses in rapeseed-producing countries worldwide, exacerbated by global climate change, and, if not remedied effectively, will threaten global food security. To gain further insights into the host-pathogen interactions in relation to Brassica diseases, it is critical that we review current knowledge in this area and discuss how omics technologies can offer promising results and help to push boundaries in our understanding of the resistance mechanisms. Omics technologies, such as genomics, proteomics, transcriptomics and metabolomics approaches, allow us to understand the host and pathogen, as well as the interaction between the two species at a deeper level. With these integrated data in multi-omics and systems biology, we are able to breed high-quality disease-resistant Brassica crops in a more holistic, targeted and accurate way.

Genotyping for Species Identification and Diversity Assessment Using Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq).

Genotyping-by-sequencing (GBS) is a powerful approach for studying the genetic diversity of legume species. By using restriction enzymes or other methods to generate a reduced representation of the genome for sequencing, GBS can provide genome-wide single nucleotide polymorphisms (SNP) for diversity analysis at high throughput and low cost. Here we describe a novel double-digest restriction site-associated DNA sequencing (ddRAD-seq) approach. We also describe the downstream bioinformatic analysis of the sequencing data, including alignment to a reference genome, de novo assembly, SNP calling, phylogenetic analysis, and structure analysis.

When West Meets East: The Origins and Spread of Weedy Rice Between Continental and Island Southeast Asia.

Weedy crop relatives are among the world's most problematic agricultural weeds, and their ability to rapidly evolve can be enhanced by gene flow from both domesticated crop varieties and wild crop progenitor species. In this study, we examined the role of modern commercial crop cultivars, traditional landraces, and wild relatives in the recent emergence and proliferation of weedy rice in East Malaysia on the island of Borneo. This region of Malaysia is separated from the Asian continent by the South China Sea, and weedy rice has become a major problem there more recently than on the Malaysian peninsular mainland. Using 24 polymorphic SSR loci and genotype data from the awn-length domestication gene An-1, we assessed the genetic diversity, population structure and potential origins of East Malaysian weeds; 564 weedy, cultivated and wild rice accessions were analyzed from samples collected in East Malaysia, Peninsular Malaysia and neighboring countries. While there is considerable evidence for contributions of Peninsular Malaysian weed ecotypes to East Malaysian populations, we find that local crop cultivars and/or landraces from neighboring countries are also likely contributors to the weedy rice infestations. These findings highlight the implications of genetic admixture from different cultivar source populations in the spread of weedy crop relatives and the urgent need for preventive measurements to maintain sustainable crop yields.

Current Status and Challenges in Identifying Disease Resistance Genes in Brassica napus.

Brassica napus is an economically important crop across different continents including temperate and subtropical regions in Europe, Canada, South Asia, China and Australia. Its widespread cultivation also brings setbacks as it plays host to fungal, oomycete and chytrid pathogens that can lead to serious yield loss. For sustainable crop production, identification of resistance (R) genes in B. napus has become of critical importance. In this review, we discuss four key pathogens affecting Brassica crops: Clubroot (Plasmodiophora brassicae), Blackleg (Leptosphaeria maculans and L. biglobosa), Sclerotinia Stem Rot (Sclerotinia sclerotiorum), and Downy Mildew (Hyaloperonospora parasitica). We first review current studies covering prevalence of these pathogens on Brassica crops and highlight the R genes and QTL that have been identified from Brassica species against these pathogens. Insights into the relationships between the pathogen and its Brassica host, the unique host resistance mechanisms and how these affect resistance outcomes is also presented. We discuss challenges in identification and deployment of R genes in B. napus in relation to highly specific genetic interactions between host subpopulations and pathogen pathotypes and emphasize the need for common or shared techniques and research materials or tighter collaboration between researchers to reconcile the inconsistencies in the research outcomes. Using current genomics tools, we provide examples of how characterization and cloning of R genes in B. napus can be carried out more effectively. Lastly, we put forward strategies to breed resistant cultivars through introgressions supported by genomic approaches and suggest prospects that can be implemented in the future for a better, pathogen-resistant B. napus.