Identification of genomic regions associated with multi-silique trait in Brassica napus

Abstract

Background: Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217.

Results: Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation.

Conclusions: This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.

Keywords: Association analysis; Brassica napus L.; Multi-silique; Near-isogenic line; Transcriptome sequencing; Whole genome re-sequencing.

Fig. 1

Scheme for constructing the multi-silique population (zws-ms) and the corresponding single-silique NIL population (zws-217)

Fig. 2

Morphological differences between zws-217and zws-ms. a: at the budding stage, zws-ms has inflated, round buds; b: at the full-bloom stage, a normal flower has one pistil and six stamens, while zws-ms has three pistils and nine stamens per floral organ; c: a normal silique in zws-217 and the three-silique trait in zws-ms. These images were taken originally in 2017

Fig. 3

Associated regions calculated using the ED algorithm. Black solid lines represent the fitted values; red dotted lines represent the associated threshold. a: SNP-associated region on ChrA09; b: two SNP-associated regions on ChrC08; the smaller region is indicated by a red arrow; c: InDel-associated region on ChrA09; d: InDel-associated region on ChrC08

Fig. 4

Gene ontology (GO) terms of the genes from the intersecting regions. A total of 2041 genes were divided into three categories: biological processes, cellular components, and molecular functions

Fig. 5

Classified KEGG pathways of the genes from the intersecting regions. The pathways were further classified into five major groups: cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems

Fig. 6

Expression patterns of 39 DEGs located in the two associated regions. The expression levels are given in log₂(FPKM+ 1). FPKM: Fragments per Kilobase Million. T01, T02, and T03: three random samples from zws-ms at the budding stage; T04, T05, and T06: three random samples from zws-217 at the budding stage

Fig. 7

Relative expression levels (folds) of 10 selected genes determined by qPCR. Note: a: BnaA09g45320D; b: BnaC08g40410D; c: BnaA09g45890D; d: BnaC08g41720D; e: BnaC08g42080D; f: BnaC08g40740D; g: BnaA09g45310D; h: BnaA09g47900D; i: BnaC08g39120D; j: BnaC08g41780D