The plover neurotranscriptome assembly: transcriptomic analysis in an ecological model species without a reference genome

We assembled a de novo transcriptome of short-read Illumina RNA-Seq data generated from telencephalon and diencephalon tissue samples from the Kentish plover, Charadrius alexandrinus. This is a species of considerable interest in behavioural ecology for its highly variable mating system and parental behaviour, but it lacks genomic resources and is evolutionarily distant from the few available avian draft genome sequences. We assembled and identified over 21 000 transcript contigs with significant expression in our samples, showing high homology to exonic sequences in avian draft genomes. From these, we identified >31 000 high-quality SNPs and > 2500 simple sequence repeats (SSRs). We also analysed expression patterns in our data to identify potential candidate genes related to differences in male and female behaviour, identifying over 200 nonoverlapping putative autosomal transcripts that show significant expression differences between males and females. Gene ontology analysis revealed that female-biased transcripts were significantly enriched for cerebral functions related to learning, cognition and memory, and male-biased transcripts were mostly enriched for terms related to neural function such as neuron projection and synapses. This data set provides one of the first de novo transcriptome assemblies from non-normalized short-read next-generation data and outlines an effective strategy for measuring sequence and expression variability simultaneously without the aid of a reference genome.

我们基于环颈鸻（Charadrius alexandrinus）端脑与间脑组织样本的短读长Illumina RNA测序（RNA-Seq）数据，组装得到了从头转录组（de novo transcriptome）。该物种因其高度多变的交配系统与双亲行为，在行为生态学领域广受关注，但目前其基因组资源匮乏，且与现有的少数鸟类草图基因组序列在进化上亲缘关系较远。我们组装并鉴定出了样本中具有显著表达水平的21000余条转录组重叠群，这些重叠群与鸟类草图基因组中的外显子序列具有高度同源性。基于这些序列，我们鉴定出了31000余个高质量单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点以及2500余个简单序列重复（Simple Sequence Repeats, SSR）标记。我们还对测序数据中的表达模式进行分析，以筛选与雌雄行为差异相关的潜在候选基因，最终鉴定出200余个不重叠的推定常染色体转录本，这些转录本在雌雄个体间呈现显著的表达差异。基因本体论（Gene Ontology, GO）富集分析结果显示，雌性偏向表达的转录本显著富集于与学习、认知及记忆相关的大脑功能通路；而雄性偏向表达的转录本则主要富集于神经元投射、突触等神经功能相关的术语类别。本数据集是首批基于非归一化短读长下一代测序数据的从头转录组组装成果之一，并提出了一种无需参考基因组即可同时检测序列变异与表达变异的有效策略。