Project description:Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.

Project description:BackgroundTaihangia rupestris, an andromonoecious plant species, bears both male and hermaphroditic flowers within the same individual. However, the establishment and development of male and hermaphroditic flowers in andromonoecious Taihangia remain poorly understood, due to the limited genetic and sequence information. To investigate the potential molecular mechanism in the regulation of Taihangia flower formation, we used de novo RNA sequencing to compare the transcriptome profiles of male and hermaphroditic flowers at early and late developmental stages.ResultsFour cDNA libraries, including male floral bud, hermaphroditic floral bud, male flower, and hermaphroditic flower, were constructed and sequenced by using the Illumina RNA-Seq method. Totally, 84,596,426 qualified Illumina reads were obtained and then assembled into 59,064 unigenes, of which 24,753 unigenes were annotated in the NCBI non-redundant protein database. In addition, 12,214, 7,153, and 8,115 unigenes were assigned into 53 Gene Ontology (GO) functional groups, 25 Clusters of Orthologous Group (COG) categories, and 126 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. By pairwise comparison of unigene abundance between the samples, we identified 1,668 differential expressed genes (DEGs), including 176 transcription factors (TFs) between the male and hermaphroditic flowers. At the early developmental stage, we found 263 up-regulated genes and 436 down-regulated genes expressed in hermaphroditic floral buds, while 844 up-regulated genes and 314 down-regulated genes were detected in hermaphroditic flowers at the late developmental stage. GO and KEGG enrichment analyses showed that a large number of DEGs were associated with a wide range of functions, including cell cycle, epigenetic processes, flower development, and biosynthesis of unsaturated fatty acid pathway. Finally, real-time quantitative PCR was conducted to validate the DEGs identified in the present study.ConclusionIn this study, transcriptome data of this rare andromonoecious Taihangia were reported for the first time. Comparative transcriptome analysis revealed the significant differences in gene expression profiles between male and hermaphroditic flowers at early and late developmental stages. The transcriptome data of Taihangia would be helpful to improve the understanding of the underlying molecular mechanisms in regulation of flower formation and unisexual flower establishment in andromonoecious plants.

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