SummaryHomoeologous 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.
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.
Summary
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.
Plant disease-resistance genes play a critical role in providing resistance against pathogens. The largest family of resistance genes are the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) genes. They are classified into two major subfamilies, toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) and coiled-coil (CC)-NBS-LRR (CNL) proteins. We have identified and characterised 641 NBS-LRR genes in Brassica napus, 249 in B. rapa and 443 in B. oleracea. A ratio of 1 : 2 of CNL : TNL genes was found in the three species. Domain structure analysis revealed that 57% of the NBS-LRR genes are typical resistance genes and contain all three domains (TIR/CC, NBS, LRR), whereas the remaining genes are partially deleted or truncated. Of the NBS-LRR genes, 59% were found to be physically clustered, and individual genes involved in clusters were more polymorphic than those not clustered. Of the NBS-LRR genes in B. napus, 50% were identified as duplicates, reflecting a high level of genomic duplication and rearrangement. Comparative analysis between B. napus and its progenitor species indicated that >60% of NBS-LRR genes are conserved in B. napus. This study provides a valuable resource for the identification and characterisation of candidate NBS-LRR genes.
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.
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.
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