Gene expression changes contribute greatly to phenotypic variations in nature. Studying patterns of regulators of gene expression is important to fully understand the molecular mechanism underlying phenotypic variations. In horseshoe bats the cochleae are finely turned to echoes of call frequency. Here, using two recently diverged subspecies of the intermediate horseshoe bat (Rhinolophus affinis hainanus and R. a. himalayanus) with great acoustic variations as the system, we aim to explore relative roles of different regulators of gene expression (differential gene expression, alternative splicing (AS) and long non-coding RNAs (lncRNAs)) in phenotypic variation with a combination of Illumina short-read and Nanopore long-read RNA-seq data from the cochlea. Compared to R. a. hainanus, R. a. himalayanus exhibited much more upregulated differentially expressed genes (DEGs) and multiple of them may play important roles in the maintenance and damage repair of auditory hair cells. We identified 411 differentially expressed lncRNAs and their target DEGs upregulated in R. a. himalayanus were also mainly involved in a protective mechanism for auditory hair cells. Using three different methods of AS analysis, we identified several candidate alternatively spliced genes (ASGs) that expressed different isoforms which may be associated with acoustic divergence of the two subspecies. We observed significantly less overlap than expected between DEGs and ASGs, supporting complementary roles of differential gene expression and AS in generating phenotypic variations. Overall, our study highlights the importance of a combination of short-read and long-read RNA-seq data in examining regulation of gene expression changes responsible for phenotypic variations.

中文翻译：

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长读长和短读长RNA测序数据揭示了不同基因表达调节因子在中间马蹄蝠声学变异中的作用

基因表达的变化极大地导致了自然界的表型变异。研究基因表达调节因子的模式对于充分理解表型变异的分子机制非常重要。马蹄蝠的耳蜗可以精细地转变为呼叫频率的回声。在这里，我们以两个最近分化的中间马蹄蝠亚种（Rhinolophus affinis hainanus 和 R. a. Himalayanus）作为系统，其具有巨大的声学变异，旨在探索不同基因表达调节因子（差异基因表达、选择性剪接）的相对作用。结合来自耳蜗的 Illumina 短读长和 Nanopore 长读长 RNA-seq 数据，研究表型变异中的长非编码 RNA (AS) 和长非编码 RNA (lncRNA)。与 R.a. 相比 海南努斯，R.a. 喜马拉雅山表现出更多上调的差异表达基因（DEG），其中多个可能在听觉毛细胞的维持和损伤修复中发挥重要作用。我们鉴定了 411 个差异表达的 lncRNA 及其在 R. a. 中上调的靶 DEG。喜马拉雅山还主要参与听觉毛细胞的保护机制。使用三种不同的 AS 分析方法，我们鉴定了几个表达不同亚型的候选可变剪接基因 (ASG)，这些亚型可能与两个亚种的声学分歧有关。我们观察到 DEG 和 ASG 之间的重叠明显少于预期，这支持差异基因表达和 AS 在产生表型变异中的互补作用。全面的，