Functional and evolutionary study of MLO gene family in the regulation of Sclerotinia stem rot resistance in Brassica napus L

Abstract

Background: Oilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus.

Results: Here, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops.

Conclusion: Our study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops.

Keywords: Brassica napus L.; Evolution; Gene expression; Genome-wide association studies; MLO; Sclerotinia stem rot; Transcriptome.

Fig. 1

GWAS for SSR in B. napus and haplotypes analysis. A, Manhattan plot of GWAS. The dashed horizontal line means the Bonferroni-adjusted significance threshold (P = 1.799 × 10^–8). B, Location of SNP loci associated with SSR resistance and pairwise LD between SNPs. SNP of BnMLO2_2 in 17.35–17.45 Mb, the dots above the red dotted line denote the significantly associated SNPs. C, Gene structure and SNP variation in the promoter of BnMLO2_2. D, Haplotypes based on the SNPs combination in the promoter. E, The corresponding phenotypes of the three haplotypes

Fig. 2

Cis-acting regulatory elements identification of BnMLO2_2 and SSR resistance identification of B. napus. A, All cis-acting regulatory elements that were identified in three haplotypes. B, Single cis-acting regulatory elements and their location in the promoter. C, Phenotype of SSR resistance identification of the three haplotypes at 36 hpi and 48 hpi. D, The expression level of BnMLO2_2 in accessions of the three haplotypes. E, Statistical analysis of phenotype at 48 hpi in the SSR resistance identification experiment. F, Phenotype of Arabidopsis transgenic plants and WT in the inoculation experiment at 36 hpi and 48 hpi. G, Statistical analysis of lesion length in the inoculation experiment of Arabidopsis transgenic plants and WT. H, Statistical analysis of phenotype in Arabidopsis WT and transgenic lines at 36 hpi and 48 hpi in the inoculation experiment. “**” means p < 0.05 in ANOVA test

Fig. 3

Expression pattern of BnMLO2 members and their expression level in S and R cultivar lines. A, The expression level of 7 BnMLO2 members in 35 tissues or stages of B. napus cultivar line ZS11 from BnTIR. B, The expression level of seven BnMLO2 genes in 888-5 and M083 leaf before S. sclerotiorum inoculation, the expression of BnMLO2_2 in R line was higher than that in S line. C, The expression level of seven BnMLO2 genes in 888-5 and M083 leaf at 24 hpi with S. sclerotiorum inoculation, the expression of BnMLO2_2 in R line was also higher than that in S line

Fig. 4

Sequence alignment and SSR resistance identification in Arabidopsis transgenic lines. A, Protein conserved domain of AtMLO2 and BnMLO2_2. B, Sequence alignment of BnMLO2_2 and AtMLO2, red letters indicate the same sequence. C, The phenotype of Arabidopsis WT, mlo2, and transgenic lines at different times after inoculation. D, Relative expression of AtMLO2 in Arabidopsis WT, mlo2, and transgenic lines. E, Statistical analysis of phenotype in Arabidopsis WT, mlo2, and transgenic lines at 24 hpi, 36 hpi, and 48 hpi in the inoculation experiment. F, Cell death staining of Arabidopsis WT, mlo2, and transgenic lines at 36 hpi. G, Relative expression of AtPR1 in Arabidopsis WT, mlo2, and transgenic lines at 0 hpi and 24 hpi. “*” means p < 0.05 in ANOVA test; “**” means p < 0.01 in ANOVA test

Fig. 5

Evolution of MLO genes in Brassicaceae and the phylogenetic and syntenic relationship between A and C subgenome. A, The evolution of MLOs from Arabidopsis to B. napus. Orthologous gene identification was based on genomic alignments between Arabidopsis, B. rapa, B. oleracea, and B. napus. Black lines indicate the positional relationship of MLO genes. B, The phylogenetic relationship of MLO genes in Brassicaceae. A total of 123 protein sequences in Arabidopsis, B. rapa, B. oleracea, and B. napus were used to construct the phylogenetic relationship tree, different color represents different groups. C, Syntenic relationship between A and C subgenome. The circle consisted of different colors showing different chromosomes and the physical distance (Mb). Grey lines represent syntenic sequences, and the highlighted red lines indicated syntenic gene pairs of BnMLOs

Fig. 6

Gene structure and conserved domains of BnMLO genes. A, The phylogenetic tree of AtMLO and BnMLO proteins, in which four clades were classified. B, Gene structure of 15 AtMLO and 57 BnMLO genes. The untranslated regions (UTR) and exons are indicated by red and green boxes, respectively, the introns are shown with black lines. C, Gene conserved domains of 15 AtMLO and 57 BnMLO proteins. MLO and MLO family conserved domains are represented in green and yellow boxes. The scale label is displayed at the bottom of the figure. kb: kilobase pairs; aa: amino acid