Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs).

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.

In silico integration of disease resistance QTL, genes and markers with the Brassica juncea physical map.

Brassica juncea (AABB), Indian mustard, is a source of disease resistance genes for a wide range of pathogens. The availability of reference genome sequences for B. juncea has made it possible to characterise the genomic structure and distribution of these disease resistance genes. Potentially functional disease resistance genes can be identified by co-localization with genetically mapped disease resistance quantitative trait loci (QTL). Here we identify and characterise disease resistance gene analogs (RGAs), including nucleotide-binding site-leucine-rich repeat (NLR), receptor-like kinase (RLK) and receptor-like protein (RLP) classes, and investigate their association with disease resistance QTL intervals. The molecular genetic marker sequences for four white rust (Albugo candida) disease resistance QTL, six blackleg (Leptosphaeria maculans) disease resistance QTL and BjCHI1, a gene cloned from B. juncea for hypocotyl rot disease, were extracted from previously published studies and used to compare with candidate RGAs. Our results highlight the complications for the identification of functional resistance genes, including the duplicated appearance of genetic markers for several resistance loci, including Ac2(t), AcB1-A4.1, AcB1-A5.1, Rlm6 and PhR2 in both the A and B genomes, due to the presence of homoeologous regions. Furthermore, the white rust loci, Ac2(t) and AcB1-A4.1, mapped to the same position on chromosome A04 and may be different alleles of the same gene. Despite these challenges, a total of nine candidate genomic regions hosting 14 RLPs, 28 NLRs and 115 RLKs were identified. This study facilitates the mapping and cloning of functional resistance genes for applications in crop improvement programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01309-5.

Resistance Gene Analogs in the Brassicaceae: Identification, Characterization, Distribution, and Evolution.

The Brassicaceae consists of a wide range of species, including important Brassica crop species and the model plant Arabidopsis (Arabidopsis thaliana). Brassica spp. crop diseases impose significant yield losses annually. A major way to reduce susceptibility to disease is the selection in breeding for resistance gene analogs (RGAs). Nucleotide binding site-leucine rich repeats (NLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are the main types of RGAs; they contain conserved domains and motifs and play specific roles in resistance to pathogens. Here, all classes of RGAs have been identified using annotation and assembly-based pipelines in all available genome annotations from the Brassicaceae, including multiple genome assemblies of the same species where available (total of 32 genomes). The number of RGAs, based on genome annotations, varies within and between species. In total 34,065 RGAs were identified, with the majority being RLKs (21,691), then NLRs (8,588) and RLPs (3,786). Analysis of the RGA protein sequences revealed a high level of sequence identity, whereby 99.43% of RGAs fell into several orthogroups. This study establishes a resource for the identification and characterization of RGAs in the Brassicaceae and provides a framework for further studies of RGAs for an ultimate goal of assisting breeders in improving resistance to plant disease.

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.

Characterization of disease resistance genes in the Brassica napus pangenome reveals significant structural variation.

Methods based on single nucleotide polymorphism (SNP), copy number variation (CNV) and presence/absence variation (PAV) discovery provide a valuable resource to study gene structure and evolution. However, as a result of these structural variations, a single reference genome is unable to cover the entire gene content of a species. Therefore, pangenomics analysis is needed to ensure that the genomic diversity within a species is fully represented. Brassica napus is one of the most important oilseed crops in the world and exhibits variability in its resistance genes across different cultivars. Here, we characterized resistance gene distribution across 50 B. napus lines. We identified a total of 1749 resistance gene analogs (RGAs), of which 996 are core and 753 are variable, 368 of which are not present in the reference genome (cv. Darmor-bzh). In addition, a total of 15 318 SNPs were predicted within 1030 of the RGAs. The results showed that core R-genes harbour more SNPs than variable genes. More nucleotide binding site-leucine-rich repeat (NBS-LRR) genes were located in clusters than as singletons, with variable genes more likely to be found in clusters. We identified 106 RGA candidates linked to blackleg resistance quantitative trait locus (QTL). This study provides a better understanding of resistance genes to target for genomics-based improvement and improved disease resistance.

Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome.

Brassica oleracea is an important agricultural species encompassing many vegetable crops including cabbage, cauliflower, broccoli and kale; however, it can be susceptible to a variety of fungal diseases such as clubroot, blackleg, leaf spot and downy mildew. Resistance to these diseases is meditated by specific disease resistance genes analogs (RGAs) which are differently distributed across B. oleracea lines. The sequenced reference cultivar does not contain all B. oleracea genes due to gene presence/absence variation between individuals, which makes it necessary to search for RGA candidates in the B. oleracea pangenome. Here we present a comparative analysis of RGA candidates in the pangenome of B. oleracea. We show that the presence of RGA candidates differs between lines and suggests that in B. oleracea, SNPs and presence/absence variation drive RGA diversity using separate mechanisms. We identified 59 RGA candidates linked to Sclerotinia, clubroot, and Fusarium wilt resistance QTL, and these findings have implications for crop breeding in B. oleracea, which may also be applicable in other crops species.

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus.

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

Diversity of fatty acid biosynthesis genes across the soybean pangenome.

Soybean (Glycine max) is a major crop that contributes more than half of global oilseed production. Much research has been directed towards improvement of the fatty acid profile of soybean seeds through marker assisted breeding. Recently published soybean pangenomes, based on thousands of soybean lines, provide an opportunity to identify new alleles that may be involved in fatty acid biosynthesis. In this study, we identify fatty acid biosynthesis genes in soybean pangenomes based on sequence identity with known genes and examine their sequence diversity across diverse soybean collections. We find three possible instances of a gene missing in wild soybean, including FAD8 and FAD2-2D, which may be involved in oleic and linoleic acid desaturation, respectively, although we recommend follow-up research to verify the absence of these genes. More than half of the 53 fatty acid biosynthesis genes identified contained missense variants, including one linked with a previously identified QTL for oil quality. These variants were present in multiple studies based on either short read mappings or alignment of reference grade genomes. Missense variants were found in previously characterized genes including FAD2-1A and FAD2-1B, both of which are involved in desaturation of oleic acid, as well as uncharacterized candidate fatty acid biosynthesis genes. We find that the frequency of missense alleles in fatty acid biosynthesis genes has been reduced significantly more than the global average frequency of missense mutations during domestication, and missense variation in some genes is near absent in modern cultivars. This could be due to the selection for fatty acid profiles in seed, though future work should be conducted towards understanding the phenotypic impacts of these variants.

Application of crop wild relatives in modern breeding: An overview of resources, experimental and computational methodologies.

Global agricultural industries are under pressure to meet the future food demand; however, the existing crop genetic diversity might not be sufficient to meet this expectation. Advances in genome sequencing technologies and availability of reference genomes for over 300 plant species reveals the hidden genetic diversity in crop wild relatives (CWRs), which could have significant impacts in crop improvement. There are many ex-situ and in-situ resources around the world holding rare and valuable wild species, of which many carry agronomically important traits and it is crucial for users to be aware of their availability. Here we aim to explore the available ex-/in- situ resources such as genebanks, botanical gardens, national parks, conservation hotspots and inventories holding CWR accessions. In addition we highlight the advances in availability and use of CWR genomic resources, such as their contribution in pangenome construction and introducing novel genes into crops. We also discuss the potential and challenges of modern breeding experimental approaches (e.g. de novo domestication, genome editing and speed breeding) used in CWRs and the use of computational (e.g. machine learning) approaches that could speed up utilization of CWR species in breeding programs towards crop adaptability and yield improvement.

Copy Number Variation among Resistance Genes Analogues in Brassica napus.

Copy number variations (CNVs) are defined as deletions, duplications and insertions among individuals of a species. There is growing evidence that CNV is a major factor underlining various autoimmune disorders and diseases in humans; however, in plants, especially oilseed crops, the role of CNVs in disease resistance is not well studied. Here, we investigate the genome-wide diversity and genetic properties of CNVs in resistance gene analogues (RGAs) across eight Brassica napus lines. A total of 1137 CNV events (704 deletions and 433 duplications) were detected across 563 RGAs. The results show CNVs are more likely to occur across clustered RGAs compared to singletons. In addition, 112 RGAs were linked to a blackleg resistance QTL, of which 25 were affected by CNV. Overall, we show that the presence and abundance of CNVs differ between lines, suggesting that in B. napus, the distribution of CNVs depends on genetic background. Our findings advance the understanding of CNV as an important type of genomic structural variation in B. napus and provide a resource to support breeding of advanced canola lines.

Mining of Cloned Disease Resistance Gene Homologs (CDRHs) in Brassica Species and Arabidopsis thaliana.

Various diseases severely affect Brassica crops, leading to significant global yield losses and a reduction in crop quality. In this study, we used the complete protein sequences of 49 cloned resistance genes (R genes) that confer resistance to fungal and bacterial diseases known to impact species in the Brassicaceae family. Homology searches were carried out across Brassica napus, B. rapa, B. oleracea, B. nigra, B. juncea, B. carinata and Arabidopsis thaliana genomes. In total, 660 cloned disease R gene homologs (CDRHs) were identified across the seven species, including 431 resistance gene analogs (RGAs) (248 nucleotide binding site-leucine rich repeats (NLRs), 150 receptor-like protein kinases (RLKs) and 33 receptor-like proteins (RLPs)) and 229 non-RGAs. Based on the position and distribution of specific homologs in each of the species, we observed a total of 87 CDRH clusters composed of 36 NLR, 16 RLK and 3 RLP homogeneous clusters and 32 heterogeneous clusters. The CDRHs detected consistently across the seven species are candidates that can be investigated for broad-spectrum resistance, potentially providing resistance to multiple pathogens. The R genes identified in this study provide a novel resource for the future functional analysis and gene cloning of Brassicaceae R genes towards crop improvement.

Whole-Genome DNA Methylation Analysis in Brassica rapa subsp. perviridis in Response to Albugo candida Infection.

DNA methylation is an epigenetic mark associated with several mechanisms in plants including immunity mechanisms. However, little is known about the regulatory role of DNA methylation in the resistance response of Brassica species against fungal diseases. White rust, caused by the fungus Albugo candida, is one of the most widespread and destructive diseases of all the cultivated Brassica species, particularly Brassica rapa L. and Brassica juncea (L.) Czern and Coss. Here, we investigate whole-genome DNA methylation modifications of B. rapa subsp. perviridis in response to white rust. As a result, 233 and 275 differentially methylated regions (DMRs) in the susceptible cultivar "Misugi" and the resistant cultivar "Nanane" were identified, respectively. In both cultivars, more than half of the DMRs were associated with genes (DMR-genes). Gene expression analysis showed that 13 of these genes were also differentially expressed between control and infected samples. Gene ontology enrichment analysis of DMR genes revealed their involvement in various biological processes including defense mechanisms. DMRs were unevenly distributed around genes in susceptible and resistant cultivars. In "Misugi," DMRs tended to be located within genes, while in "Nanane," DMRs tended to be located up and downstream of the genes. However, CG DMRs were predominantly located within genes in both cultivars. Transposable elements also showed association with all three sequence contexts of DMRs but predominantly with CHG and CHH DMRs in both cultivars. Our findings indicate the occurrence of DNA methylation modifications in B. rapa in response to white rust infection and suggest a potential regulatory role of DNA methylation modification in defense mechanisms which could be exploited to improve disease resistance.

Genome-Wide Mining of Disease Resistance Gene Analogs Using Conserved Domains.

The production of legume crop species is severely affected by disease, imposing a significant yield loss annually worldwide. Plant resistance gene analogs (RGAs) play specific roles in plant resistance responses, and their identification and subsequent application in breeding programs help to reduce this yield loss. RGAs contain conserved domains and motifs, which can be used for their identification and classification. Nucleotide-binding site-leucine-rich repeat (NLR), receptor like kinase (RLK), and receptor like protein (RLP) genes are the main types of RGAs. Computational identification and characterization of RGAs has been performed successfully among different plant species. Here, we explain the computational workflow for genome-wide RGA identification in legumes.

Genome-Wide Identification and Evolution of Receptor-Like Kinases (RLKs) and Receptor like Proteins (RLPs) in Brassica juncea.

Brassica juncea, an allotetraploid species, is an important germplasm resource for canola improvement, due to its many beneficial agronomic traits, such as heat and drought tolerance and blackleg resistance. Receptor-like kinase (RLK) and receptor-like protein (RLP) genes are two types of resistance gene analogues (RGA) that play important roles in plant innate immunity, stress response and various development processes. In this study, genome wide analysis of RLKs and RLPs is performed in B. juncea. In total, 493 RLKs (LysM-RLKs and LRR-RLKs) and 228 RLPs (LysM-RLPs and LRR-RLPs) are identified in the genome of B. juncea, using RGAugury. Only 13.54% RLKs and 11.79% RLPs are observed to be grouped within gene clusters. The majority of RLKs (90.17%) and RLPs (52.83%) are identified as duplicates, indicating that gene duplications significantly contribute to the expansion of RLK and RLP families. Comparative analysis between B. juncea and its progenitor species, B. rapa and B. nigra, indicate that 83.62% RLKs and 41.98% RLPs are conserved in B. juncea, and RLPs are likely to have a faster evolution than RLKs. This study provides a valuable resource for the identification and characterisation of candidate RLK and RLP genes.

DNA Methylation: Toward Crop Disease Resistance Improvement.

Crop diseases, in conjunction with climate change, are a major threat to global crop production. DNA methylation is an epigenetic mark and is involved in plants' biological processes, including development, stress adaptation, and genome evolution. By providing a new source of variation, DNA methylation introduces novel direction to both scientists and breeders with its potential in disease resistance enhancement. Here, we discuss the impact of pathogen-induced DNA methylation modifications on a host's transcriptome reprogramming and genome stability, as part of the plant's defense mechanisms. We also highlight the knowledge gaps that need to be investigated for understanding the entire role of DNA methylation in plant pathogen interactions. This will ultimately assist breeders toward improving resistance and decreasing yield losses.

Chalcone synthase genes from milk thistle (Silybum marianum): isolation and expression analysis.

Silymarin is a flavonoid compound derived from milk thistle (Silybum marianum) seeds which has several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; thereby, the identification of CHS encoding genes in milk thistle plant can be of great importance. In the current research, fragments of CHS genes were amplified using degenerate primers based on the conserved parts of Asteraceae CHS genes, and then cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of CHS gene family,SmCHS1 and SmCHS2. Third member, full-length cDNA (SmCHS3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp), encoding 63 and 349 amino acids, respectively. In silico analysis of SmCHS3 sequence contains all the conserved CHS sites and shares high homology with CHS proteins from other plants.Real-time PCR analysis indicated that SmCHS1 and SmCHS3 had the highest transcript level in petals in the early flowering stage and in the stem of five upper leaves, followed by five upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis.