Transcriptome profile analysis of young floral buds of fertile and sterile plants from the self-pollinated offspring of the hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus

Abstract

Background: The fertile and sterile plants were derived from the self-pollinated offspring of the F1 hybrid between the novel restorer line NR1 and the Nsa CMS line in Brassica napus. To elucidate gene expression and regulation caused by the A and C subgenomes of B. napus, as well as the alien chromosome and cytoplasm from Sinapis arvensis during the development of young floral buds, we performed a genome-wide high-throughput transcriptomic sequencing for young floral buds of sterile and fertile plants.

Results: In this study, equal amounts of total RNAs taken from young floral buds of sterile and fertile plants were sequenced using the Illumina/Solexa platform. After filtered out low quality data, a total of 2,760,574 and 2,714,441 clean tags were remained in the two libraries, from which 242,163 (Ste) and 253,507 (Fer) distinct tags were obtained. All distinct sequencing tags were annotated using all possible CATG+17-nt sequences of the genome and transcriptome of Brassica rapa and those of Brassica oleracea as the reference sequences, respectively. In total, 3231 genes of B. rapa and 3371 genes of B. oleracea were detected with significant differential expression levels. GO and pathway-based analyses were performed to determine and further to understand the biological functions of those differentially expressed genes (DEGs). In addition, there were 1089 specially expressed unknown tags in Fer, which were neither mapped to B. oleracea nor to B. rapa, and these unique tags were presumed to arise basically from the added alien chromosome of S. arvensis. Fifteen genes were randomly selected and their expression levels were confirmed by quantitative RT-PCR, and fourteen of them showed consistent expression patterns with the digital gene expression (DGE) data.

Conclusions: A number of genes were differentially expressed between the young floral buds of sterile and fertile plants. Some of these genes may be candidates for future research on CMS in Nsa line, fertility restoration and improved agronomic traits in NR1 line. Further study of the unknown tags which were specifically expressed in Fer will help to explore desirable agronomic traits from wild species.

Figure 1

Comparation of floral morphology between sterile and fertile plants.A, young buds of sterile and fertile plants (left, fertile; right, sterile); B, Anthers corresponding to young buds (left, sterile; right, fertile); C, the cross section of fertile anthers; D, the cross section of sterile anthers; E, mature flowers of sterile and fertile plants (left, fertile; right, sterile); F, Anthers corresponding to mature flowers (left, fertile; right, sterile). Scale bars = 50 μm.

Figure 2

Differentially expressed tags and corresponding genes in Ste and Fer.A, Differentially expressed tags in Ste and Fer. The “x” axis represents fold-change of differentially expressed unique tags in two libraries. The “y” axis represents the number of unique tags (log10). Differentially accumulating unique tags with a 5-fold difference between libraries are shown in the red region (97.66%). The blue (0.24%) and green (2.07%) regions represent unique tags that are up- and down-regulated for more than 5 folds in the Fer and Ste libraries, respectively. B, Comparision of gene expression levels between the two libraries. For comparing gene expression levels between the two libraries, each library was normalized to 1 million tags. Red dots represent transcripts more prevalent in Fer library, green dots show those present at a lower frequency in Fer and blue dots indicate transcripts that did not change significantly. The parameters “FDR <0.001” and “log2 Ratio ≥ 1” were used as the threshold to judge the significance of gene expression difference.

Figure 3

Changes in gene expression in Fer and Ste libraries. Numbers of up-regulated, down-regulated, specific to Fer and specific to Ste genes were summarized.

Figure 4

Histogram showing Gene Ontology functional analysis of DEGs. The frequency of GO terms was analyzed using GO Slim Assignment. The y-axis and x-axis indicate the names of clusters and the ratio of each cluster, respectively.

Figure 5

The summary enriched pathways of DEGs in B. rapa and B. oleracea.

Figure 6

Histogram showing GO analysis of DEGs which specific to Fer or Ste in B. rapa.

Figure 7

Overview of CMS in sterile plants and fertility restoration in fertile plants. Expression of mitochondrial CMS genes led to dysfunction of mitochondria. Products of nuclear restorer genes suppressed the effects of CMS genes. The state of the mitochondria was signaled to nuclear target genes and resulted in male sterile or fertile flower.

Figure 8

Real-time PCR validations of tag-mapped genes. Relative level, (2−^△CT△CT); TPM, log2 Ratio (Fer/Ste).