Comparative Transcriptome Analysis between Fertile and CMS Flower Buds in Wucai (Brassica campestris L.)

Abstract

Background: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq).

Results: Microscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai.

Conclusion: The present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.

Keywords: Cytoplasmic male sterility; Differentially expressed genes; RNA-Seq; Tapetum; Transcriptome analysis; Wucai.

Fig. 1

Morphological characteristics of flowers and microscopic observations of anthers from the sterile and fertile lines of Wucai. a and b, CMS line 12-14A and its maintainer line 12-14B. c, e, g and d, f, h, Inflorescences, flowers, and petals and stamens from the sterile and fertile lines, respectively. i-l, Transverse sections of sterile anthers; abnormal tapetum was formed, and the pollen sacs of sterile anthers did not produce pollen, in the CMS line. m-p, Transverse sections of fertile anthers; normal tapetum and mature anthers developed in the maintainer line. AT, abnormal tapetum; DPG, degenerated pollen grain; E, epidermis; En, endothecium; ML, middle layer; MSp, microspore; PG, pollen grain; T, tapetum. Scale bars in a, b, c and d represent 1 cm; scale bars in e, f, g, h represent 0.5 cm; scale bars in I-P represent 100 μm

Fig. 2

Number of DEGs between sterile and fertile buds. Red indicates upregulated DEGs, and green indicates downregulated DEGs

Fig. 3

Twenty most significantly enriched KEGG pathways

Fig. 4

Heat map analysis of bHLH (a), MYB (b), NAC (c) and WRKY (d) transcription factors. The expression levels shown are based on RPKM data. The color key represents the value of log2(S/F). Red represents high expression, and blue represents low expression. Each row represents a DEG

Fig. 5

qRT-PCR verification of differentially expressed unigenes. S means sterile sample, and F means fertile sample. Relative expression levels were calculated using Actin as an internal control

Fig. 6

Expression of anther and pollen development related genes at different stages using qRT-PCR. S means sterile sample, and F means fertile sample. 1–3 indicate the pollen meiosis stage (bud sizes 0.5–1.5 mm), tetrad stage (1.5–3.0 mm) and uninucleate microspore stage (3.0–4.5 mm) of anther and pollen development, respectively. Relative expression levels were calculated using Actin as an internal control

Fig. 7

The mechanism of FLC affects fertility in Wucai. Black arrows indicate promotion, and inverted T bars indicate repression. Dashed lines indicate that further research is needed. Question marks indicate unknown genes. Red arrows indicate up- or downregulation. AGL18 (Unigene20308), AGAMOUS LIKE 18; AGL104 (CL3910.Contig1, CL3910.Contig2), AGAMOUS LIKE 104; AP3 (CL13421.Contig5), APETALA 3; CO (Unigene36778) CONSTANS; FLC (CL3897.Contig1), Flowering Locus C; FT (CL2127.Contig2), Flowering Locus T; VIN3 (CL8770.Contig1), VERNALIZATION 3; TFs, transcription factors