Identification of candidate genes for blackleg resistance in the new Brassica juncea canola

Abstract

Brassica species are at the high risk of severe crop loss due to pathogens, especially Leptosphaeria maculans (the causal agent of blackleg). To date, only two blackleg resistance genes, Rlm2 and LepR3, have been cloned, both from Brassica napus, which are allelic variants encoded by theLepR3/Rlm2 locus on chromosome A10. LepR3 confers resistance to avirulence gene AvrLm1, which is also recognised by Rlm1; while Rlm2 interacts with AvrLm2.In this study, seven LepR3/Rlm2 allelic variants have been isolated from 53 Brassica varieties through cloning. Three novel allelic variants have been identified, including B. juncea-like allele lepR3BG2 (lepR3BG2-1 and lepR3BG2-2) encoding 739 amino acids (aa), lepR3BG4 encoding 924 aa and lepR3BG6 (lepR3BG6-1 and lepR3BG6-2) encoding 950 aa. In addition to the previously reported alleles, a total of 10 alleles have been detected in Brassica so far, revealing the diversity of the LepR3/Rlm2 locus. Additionally, no polymorphism has been observed within the LepR3 and Rlm2 genes, respectively, which were isolated from different resistant cultivars. The predicted primary structures of these alleles closely match that of a leucine-rich repeat receptor-like protein (LRR-RLP), and most of them comprise seven domains, except for lepR3BG2 and lepR3BG1 lacking domains D-G. The LRR domains significantly varied in repeat numbers from 24 to 30. Homology modelling of LepR3 and Rlm2 proteins showed a typical non-globular LRR fold, a right-handed super-helix, like the template structures from which it is derived. The combination analysis of primary structure and homology modelling suggested the B-domain of LepR3 and Rlm2 proteins may still contribute to their function; while their LRR motifs in the C-domain serve for ligand specificity; finally, their interaction with signalling partner SOBIR1 and BAK1 requires a C-terminal region. Allele-specific primers have been designed based on the sequences of alleles previously reported and newly found in this study, to rapidly detect the diverse locus of LepR3/Rlm2on chromosome A10. Moreover, the specific markers towards LepR3 and Rlm2 have been applied to confirm the cultivars that contain these resistance genes. The LepR3specific markers also offered a method to verify whether the resistance gene interaction with AvrLm1 is with Rlm1 or LepR3. The results confirm that Rlm1 and LepR3 are two independent resistance loci located on chromosome A07 and A10, respectively.Brassica juncea, an allotetraploid species, is an important germplasm resource for canola improvement, due to its many good agronomic traits, such as heat and drought tolerance and high blackleg resistance. Although the majority of receptor-like kinases (RLKs) and receptor-like proteins (RLPs) genes remain functionally uncharacterised in plants, an increasing number of these genes have been proved to be important in plant innate immunity, stress response and various development processes. Thus, it is important to investigate RLKs and RLPs in B. juncea. In the present study, 493 RLKs (LysM-RLKs and LRR-RLKs) and 228 RLPs (LysM-RLPs and LRR-RLPs) were identified in the genome of B. juncea, 300 and 65 in B. rapa, and 317 and 176 in B. nigra, respectively, using the RGAugury pipeline. The sub-families of LRR-RLKs and LRR-RLPs in B. juncea are found to be larger than those in most reported plants. Only 13.54% RLKs and 11.79% RLPs were observed to be grouped within gene clusters. In B. juncea, 90.17% RLKs and 52.83% RLPs were identified as duplicates, indicating gene duplications significantly contribute to the expansion of RLK and RLP families during the evolution process. Tandem duplications played major roles in RLP duplications, while segmental duplications make limited contributions to RLK and RLP duplications. However, whole-genome duplication was likely responsible for the RLKs expansion. Comparative analysis between B. juncea and its progenitor species indicated 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.Genome-wide association studies (GWAS) are a powerful tool to connect a trait back to its underlying genetics. The present study is the first to conduct GWAS of blackleg resistance in B. juncea using the Infinium 90k Brassica SNP array. The verification of Rlm6 in B. juncea was performed through a cotyledon infection test with L. maculans isolate WAC4110. Out of 42,914 single nucleotide polymorphisms (SNPs) in a panel of 167 B. juncea lines, a total of seven significant SNPs associated with Rlm6 have been identified on chromosomes A07 and B04 in B. juncea. Furthermore, 24 candidate Rlm6 genes were found in the potential regions, defined as nucleotide binding site (NBS), leucine-rich repeat RLK (LRR-RLK), LRR-RLP genes, and RLK-other-receptor. This study will give insights into blackleg resistance in B. junceaand provide direction for B. napus breeding, by introgressing blackleg resistance to B. napus.