Discovery of Genomic Regions and Candidate Genes Controlling Root Development Using a Recombinant Inbred Line Population in Rapeseed (Brassica napus L.)

Abstract

Marker-assisted selection enables breeders to quickly select excellent root architectural variations, which play an essential role in plant productivity. Here, ten root-related and shoot biomass traits of a new F₆ recombinant inbred line (RIL) population were investigated under hydroponics and resulted in high heritabilities from 0.61 to 0.83. A high-density linkage map of the RIL population was constructed using a Brassica napus 50k Illumina single nucleotide polymorphism (SNP) array. A total of 86 quantitative trait loci (QTLs) explaining 4.16-14.1% of the phenotypic variances were detected and integrated into eight stable QTL clusters, which were repeatedly detected in different experiments. The codominant markers were developed to be tightly linked with three major QTL clusters, qcA09-2, qcC08-2, and qcC08-3, which controlled both root-related and shoot biomass traits and had phenotypic contributions greater than 10%. Among these, qcA09-2, renamed RT.A09, was further fine-mapped to a 129-kb interval with 19 annotated genes in the B. napus reference genome. By integrating the results of real-time PCR and comparative sequencing, five genes with expression differences and/or amino acid differences were identified as important candidate genes for RT.A09. Our findings laid the foundation for revealing the molecular mechanism of root development and developed valuable markers for root genetic improvement in rapeseed.

Keywords: fine mapping; linkage analysis; major QTL; rapeseed; root development.

Figure 1

Comparison of phenotypic characteristics of two parents, ZS11 and 4D122. The whole plant performance of the two parents was cultured on the 18th day of the three expanding leaves (3 EL). The root scanning images of the two parents were present at 18, 32, and 46 days old with three, seven, and eleven expanding leaves (3EL, 7EL, and 11EL), respectively. The bars are 5 cm. ** and * indicate significance at the 1% and 5% levels of probability, respectively, while ns indicates non-significant difference.

Figure 2

Correlation analysis of captured traits. (A) Correlations of captured traits in each experiment among RILs. For each trait, the frequency distributions of the adjusted means (diagonal), scatterplots (below diagonal), and values of the correlation coefficients (above diagonal) between pairs of traits are shown. (B) Correlations of each captured trait among the four experiments. Red and blue indicate positive and negative correlations, respectively. ***, ** and * denote significance at the 0.1%, 1% and 5% levels of probability among the RILs, respectively.

Figure 2

Correlation analysis of captured traits. (A) Correlations of captured traits in each experiment among RILs. For each trait, the frequency distributions of the adjusted means (diagonal), scatterplots (below diagonal), and values of the correlation coefficients (above diagonal) between pairs of traits are shown. (B) Correlations of each captured trait among the four experiments. Red and blue indicate positive and negative correlations, respectively. ***, ** and * denote significance at the 0.1%, 1% and 5% levels of probability among the RILs, respectively.

Figure 3

Information of the genetic linkage map. (A) The genetic linkage map with 19 linkage groups constructed by 2089 bins. (B) Collin-earity analysis of the genetic distances and physical distances of the markers in the linkage map; different colors represent different chromosomes or linkage groups.

Figure 4

Information of the three major QTL clusters, qcA09-2, qcC08-2, and qcC08-3. Three major QTL clusters with their involved QTLs, LOD curves, and SNP markers at the corresponding linkages. SNPs noted with other names, A9-1, A9-2, A9-3, A9-4, C8-1, C8-2, C8-3, C8-4, and C8-5, were converted into PARMS markers.

Figure 5

Fine mapping of the QTL RT.A09. The genotype and phenotype of recombinants using 11 developed PARMS markers of RT.A09. “A” and “B” represent that the offspring phenotypes of the recombinant line were similar to NIL-A and NIL-B, respectively, of its sister lines. “H” represents that the offspring phenotypes of the recombinant line were separated according to their genotypes.

Figure 6

Relative expression levels of the eleven candidate genes between NIL-A and NIL-B. The mean and the standard deviation were calculated using three biological replicates. ** and * indicate significant differences with p < 0.01 and p < 0.05, respectively.