Devani Ravi Suresh, Sinha Sangram, Banerjee Jayeeta, Sinha Rabindra Kumar, Bendahmane Abdelhafid, Banerjee Anjan Kumar
Biology Division, Indian Institute of Science Education and Research (IISER), Pune, Pune, Maharashtra, India.
Department of Botany, Tripura University, Suryamaninagar, Tripura, India.
BMC Plant Biol. 2017 Dec 12;17(1):241. doi: 10.1186/s12870-017-1187-z.
Coccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood.
We have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 'Trinity Transcripts' clustering into 378,860 'Trinity Genes'. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers.
Suppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO-mediated sex modification.
大籽栝楼（ivy gourd）是葫芦科的一种雌雄异株植物，具有异形性染色体。大籽栝楼雄株和雌株的染色体组成分别为22A + XY和22A + XX。此前我们发现，自然界中还存在一种独特的大籽栝楼雌雄同株形式（22A + XX），其花朵形态上为雌雄同体（GyM-H）。此外，在雌株上施用硝酸银（AgNO）可诱导雄蕊发育，从而形成形态上为雌雄同体的花朵（Ag-H），尽管不存在Y染色体。由于缺乏基因组序列，且性连锁基因的鉴定速度缓慢，大籽栝楼的性别表达和修饰尚未得到很好的理解。
我们对早期的雄花、雌花、GyM-H花和Ag-H花以及中期的雄花芽和GyM-H花芽进行了全面的RNA测序研究。使用Trinity进行从头转录组组装，并通过BLAST2GO和Trinotate管道进行注释。组装的转录组由467,233个“Trinity转录本”组成，聚类为378,860个“Trinity基因”。Female_Early_vs_Male_Early、Ag_Early_vs_Female_Early和GyM-H_Middle_vs_Male_Middle比较分别显示出35,694、3574和14,954个差异表达的转录本。此外，对选定候选基因的qRT-PCR分析验证了数字基因表达谱结果。有趣的是，与雄花芽相比，乙烯反应相关基因在雌花芽中上调。此外，我们观察到AgNO处理通过下调乙烯反应转录因子抑制了Ag-H花中的乙烯反应，从而导致雄蕊发育。此外，与雌花芽相比，与雄蕊发育相关的GO术语在早期的雄花、GyM-H花和Ag-H花芽中富集，这支持了雌花花中雄蕊生长停滞的事实。
与雌花芽相比，雄花和Ag-H花中乙烯反应的抑制表明乙烯在雄蕊抑制中可能发挥作用，类似于甜瓜和黄瓜等雌雄同株瓜类。此外，与中期GyM-H花芽相比，与花粉育性相关的GO术语在雄花芽中减少，表明Y染色体对花粉育性的必要性。总体而言，本研究将有助于鉴定新的性别偏向基因，以进一步研究雄蕊停滞、花粉育性和AgNO介导的性别修饰。
