Mapping and Identifying a Candidate Gene (Bnmfs) for Female-Male Sterility through Whole-Genome Resequencing and RNA-Seq in Rapeseed (Brassica napus L.)

Abstract

In oilseed crops, carpel and stamen development play vital roles in pollination and rapeseed yield, but the genetic mechanisms underlying carpel and stamen development remain unclear. Herein, a male- and female-sterile mutant was obtained in offspring of a (Brassica napus cv. Qingyou 14) × (Qingyou 14 × B. rapa landrace Dahuang) cross. Subsequently, F₂-F₉ populations were generated through selfing of the heterozygote plants among the progeny of each generation. The male- and female-sterility exhibited stable inheritance in successive generations and was controlled by a recessive gene. The mutant kept the same chromosome number (2n = 38) as B. napus parent but showed abnormal meiosis for male and female. One candidate gene for the sterility was identified by simple sequence repeat (SSR) and insertion deletion length polymorphism (InDel) markers in F₇-F₉ plants, and whole-genome resequencing with F₈ pools and RNA sequencing with F₉ pools. Whole-genome resequencing found three candidate intervals (35.40-35.68, 35.74-35.75, and 45.34-46.45 Mb) on chromosome C3 in B. napus and candidate region for Bnmfs was narrowed to approximately 1.11-Mb (45.34-46.45 M) by combining SSR and InDel marker analyses with whole-genome resequencing. From transcriptome profiling in 0-2 mm buds, all of the genes in the candidate interval were detected, and only two genes with significant differences (BnaC03g56670D and BnaC03g56870D) were revealed. BnaC03g56870D was a candidate gene that shared homology with the CYP86C4 gene of Arabidopsis thaliana. Quantitative reverse transcription (qRT)-PCR analysis showed that Bnmfs primarily functioned in flower buds. Thus, sequencing and expression analyses provided evidence that BnaC03g56870D was the candidate gene for male and female sterility in the B. napus mutant.

Keywords: Brassica napus L.; RNA-seq; female-male sterility; mutant; whole-genome resequencing.

Figure 1

Flow diagram for the population construction of mutant.

Figure 2

Phenotypic characterization of the sterile and fertile floral buds. (A–K) Phenotype of sterile flower buds. (a–k) Phenotype of fertile flower buds. (A–K) and (a–k) represent the sterile and fertile bud development stages, respectively. Bars = 1 mm

Figure 3

Anther development in fertile plants (A–H) and sterile plants (a–h). PMC, pollen mother cell; Ep, Epidermal cell layer; En, endothelial cell layer; Mi, middle layer; Tp, tapetum layer; YMsp, young microspore; DMsp, degenerated microspore; Msp, microspore; PG, pollen grain. Bars = 50 μm in (A–G) and (a–g); 10 μm in (H) and (h).

Figure 4

Meiosis in the anthers of sterile floral buds. (A–D) Similar chromatin condensation. (E) Darkly stained pseudo-nuclear condensation. (F) Few cells with coagulate pseudo-chromosomes. (G) Abnormal chromosome behavior. Bars = 10 μm.

Figure 5

SNP index and ΔSNP index Manhattan plot graphs. (A) SNP index Manhattan plot graphs of the fertile pool from F₈. (B) SNP index Manhattan plot graphs of the sterile pool from F₈. (C) ΔSNP index Manhattan plot graphs. The line indicates the threshold value.

Figure 6

Identification and validation of the BnaC03g56870D gene on Brassica napus chromosome C03. (A) ΔSNP index graph based on the analysis of fertile and sterile plants identified a candidate gene in interval of 45.34–46.45 Mb on chromosome C03. (B) Linkage analysis using molecular markers confirmed the location of the candidate gene. (C) The location of the BnaC03g56870D by molecular markers, whole-genome sequencing and RNA-Seq.

Figure 7

Quantitative real-time polymerase chain reaction (qRT-PCR) to confirm differential expression in various organs between fertile (F) and sterile (S) plants. The relative expression levels of three genes identified through RNA-Seq analysis and whole-genome resequencing are shown. The gene expression analysis was performed based on eight biological (buds) replicates, three biological (leaves, stems, and roots) replicates and three technical replicates. ^**Significantly different at the 0.01 level using a t-test.