Brassica napus (canola/rapeseed/oilseed rape) is an important commercial oilseed crop in Australia with an annual production of approximately 1.6 million tons of oil. Canola is an important source of edible vegetable oil and has a broad range of industrial purposes. Blackleg disease (stem canker) caused by the fungal pathogen Leptosphaeria maculans, is one of the most devastating diseases in B. napus. This disease causes significant yield losses with an annual average loss of 15 to 48 %, although losses can reach up to 80% worldwide, mainly in Europe, Australia and Canada.
In order to develop an effective strategy to control the blackleg disease, there is a need to identify blackleg resistance genes in Brassica species and understand the genetic interaction between plant resistance genes and the pathogen avirulence genes. Thus, identification of Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) resistance genes is one of the most important objectives of understanding resistance. NBS-LRR resistance genes have been extensively studied because they represent the largest class of disease resistance genes and play a critical role in defending plants from pathogens.
The objective of this study was to perform comprehensive analysis on identification and characterization of NBS-LRR genes in the B. napus genome and to study the synteny and conservation of NBS-LRR genes between Brassica species. In this study, a total of 641, 249 and 443 NBS-LRR encoding genes in B. napus, B. rapa and B. oleracea, respectively, were identified. The comparative analysis between B. napus and its progenitor species indicated that NBS-LRR genes exhibited similar gene structure, genomic location, arrangement in clusters and syntenic relationships. The results provide evidence that there was a selective advantage to maintaining similar features of NBS-LRR genes in B. napus to both B. rapa and B. oleracea following polyploidization. More than 60% of NBS LRR genes from the progenitor species were conserved. The differences in NBS-LRR gene conservation could be attributed to gene losses or selection pressure to offer species-specific or cultivars-specific resistance.
This study found that the NBS-LRR resistance genes are physically clustered and individual genes involved in clusters were more polymorphic and subject to evolutionary process than singleton genes. These clusters, which have been described in many other species, provide a reservoir of genetic variation influenced by tandem duplication and selection pressure. In addition, there was a significant correlation and co-localization between the number of NBS-LRR genes within the disease QTL intervals and the number of genes involved in gene clusters or duplication. This correlation provides evidence that NBS-LRR are distributed and clustered throughout the genome and tends to be linked and associated with disease QTL intervals.
Genetic studies have identified the gene for gene interactions between avirulence (Avr) genes in L. maculans and their corresponding Rlm (Resistance to Leptosphaeria maculans) genes in B. napus. In addition, genetic mapping studies have shown that there are five major resistance genes on chromosome A7: Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9. At present, none of the genetically mapped Rlm genes on chromosome A7 have been sequenced and validated in B. napus.
A total of 12 NBS-LRR and 18 LRR-containing resistance genes were on B. napus chromosome A7 located within the Rlm QTL region of interest. The comparative analysis of these resistance genes between Brassica species confirmed the gene synteny and conservation. However, there was considerable variation; either gene presence/absence or substantial differences in the protein sequence. The comparative analysis allowed making an initial prediction and prioritization for targeting these identified genes for further characterization and validation. In this study, a candidate gene approach, combined with comparative analysis, was exploited for identification of Rlm9 candidate genes in B. napus. The candidate gene approach identified six NBS-LRR and eight LRR-containing genes associated with the Rlm9 QTL region. The NBS-LRR genes were selected for further analysis as highest priority candidate genes.
These results provide the first in-depth molecular characterization of NBS-LRR genes in B. napus providing potential candidate gene for disease resistance trait in B. napus. More importantly, this work has significantly increased our understanding about blackleg resistance in B. napus and major disease resistance genes have been identified.