Identification of QTLs for resistance to sclerotinia stem rot and BnaC.IGMT5.a as a candidate gene of the major resistant QTL SRC6 in Brassica napus

Abstract

Stem rot caused by Sclerotinia sclerotiorum in many important dicotyledonous crops, including oilseed rape (Brassica napus), is one of the most devastating fungal diseases and imposes huge yield loss each year worldwide. Currently, breeding for Sclerotinia resistance in B. napus, as in other crops, can only rely on germplasms with quantitative resistance genes. Thus, the identification of quantitative trait locus (QTL) for S. sclerotiorum resistance/tolerance in this crop holds immediate promise for the genetic improvement of the disease resistance. In this study, ten QTLs for stem resistance (SR) at the mature plant stage and three QTLs for leaf resistance (LR) at the seedling stage in multiple environments were mapped on nine linkage groups (LGs) of a whole genome map for B. napus constructed with SSR markers. Two major QTLs, LRA9 on LG A9 and SRC6 on LG C6, were repeatedly detected across all environments and explained 8.54-15.86% and 29.01%-32.61% of the phenotypic variations, respectively. Genotypes containing resistant SRC6 or LRA9 allele showed a significant reduction in disease lesion after pathogen infection. Comparative mapping with Arabidopsis and data mining from previous gene profiling experiments identified that the Arabidopsis homologous gene of IGMT5 (At1g76790) was related to the SRC6 locus. Four copies of the IGMT5 gene in B. napus were isolated through homologous cloning, among which, only BnaC.IGMT5.a showed a polymorphism between parental lines and can be associated with the SRC6. Furthermore, two parental lines exhibited a differential expression pattern of the BnaC.IGMT5.a gene in responding to pathogen inoculation. Thus, our data suggested that BnaC.IGMT5.a was very likely a candidate gene of this major resistance QTL.

Figure 1. Stem resistance (SR) of the two parental lines, Hua 5 and J7005, and the HJ-DH population.

(A) Disease lesion on the stem of Hua5 and J7005 at 7 dpi (left panel) and 30 dpi (right panel). Arrows indicate the lesion boundaries on the stem. (B) Lesion length of Hua5 and J7005 at 7 dpi. * and ** indicates a significant difference at P<0.05 and P<0.01 levels, respectively. (C) Frequency distributions of the lesion length in the HJ-DH population in Wuhan, 2009–2010 (left), Wuhan, 2010–2011(middle) and Huanggang, 2010–2011 (right). Arrows indicate the mean lesion length of the parental lines.

Figure 2. Leaf resistance (LR) of the two parental lines, Hua 5 and J7005, and the HJ-DH population.

(A) Disease lesion on the leaf of Hua5 and J7005 at 2 dpi. (B) Lesion area of Hua5 and J7005 at 2 dpi. * and ** indicates a significant difference at P<0.05 and P<0.01 levels, respectively. (C) Frequency distributions of the lesion area in the HJ-DH population in the season of 2010–2011 (left) and in 2011–2012 (right). Arrows indicate the mean lesion size of the parental lines.

Figure 3. QTLs for LR and SR mapped on the HJ-DH genetic linkage map.

The bar to the left of the LG indicates the 1-LOD confidence interval for the QTL and the triangle indicates the QTL peak position.

Figure 4. Effects of the major QTL SRC6 and LRA9 on SR and LR.

Each group (AA and BB) for the DH population is classified according to the genotype of the makers (BEN327 for SRC6, BGR23 for LRA9) that closely linked to the peaks of the major QTLs. * and ** indicate a significant difference between AA and BB group at P<0.05 and P<0.01 levels, respectively.

Figure 5. Comparative map of LG C6 of B. napus with B. oleracea and Arabidopsis.

Column A presents the linkage map of LG C6 of the HJ-DH population. The confidence interval of SRC6 is shown in color. Column B is the conserved blocks identified in B. napus, which is labeled according to Schranz et al. and colored differently based on the Arabidopsis (At) chromosome positions defined by Parkin et al. . Column C lists the homologous colinear loci in B. oleracea chromosome 7 (BoC7) corresponding to SSR markers in LG C6. The number designates the physical position in B. oleracea chromosome with the size of amplification fragments. Column D lists the genes encoding homologous loci in A. thaliana. Column E presents the modified LG C6 after adding BnaC.IGMT5.a on the map.

Figure 6. Molecular cloning of IGMT5 genes in three Brassica species and induced expression of BnaC.IGMT5.a after inoculation with Scelrotinia pathogen.

(A) Phylogenetic analysis of IGMT5 genes in B. rapa, B. oleracea and B. napus. Neighbor joining tree is presented based on the deduced amino acid sequences of IGMT5 genes in three Brassica species. Bootstrap values (1,000 replications) are shown at each branch as a percentage. A branch length scale bar is shown beneath each tree. (B) BnaC.IGMT5.a expression is induced in Hua 5 after inoculation with Sclerotinia pathogen, but not induced in J7005. RT-PCR analysis was conducted with RNAs from pooled tissues after inoculations at each time point. BnActin was used as an internal control. (C) BnaC.IGMT5.a is associated with resistant phenotype. PCR products amplified from the copy-specific marker of BnaC.IGMT5.a are presented. Lane 1–10 are the samples from most resistant lines and lane 11–20 the most susceptible lines from the HJ-DH population. (D) RT-PCR analysis of BnaC.IGMT5.a in the ten most resistant lines (lane 1–10) and ten most susceptible lines (lane 11–20) at 72 hpi.