New possibilities arise for studies of hybridization: SNP-based markers for the multi-species Daphnia longispina complex derived from transcriptome data

In order to trace population community dynamics and reticulate evolution in hybrid species complexes, long-term comparative studies of natural populations are necessary and thus, tools for fine-scale genetic analyses are needed. In the present study, we developed species-diagnostic SNP markers for hybridizing freshwater crustaceans: the multispecies Daphnia longispina complex. Specifically, we took advantage of transcriptome data from a key species of this hybrid complex, the annotated genome of a related Daphnia species and well-defined reference genotypes from three parental species. Altogether eleven nuclear loci with several species-specific SNP sites were identified in sequence alignments of 22 individuals of three parental species and their hybrids. A PCR-RFLP assay was developed for cost-efficient large population screening by SNP-genotyping. Taxon assignment by RFLP-patterns was nearly perfectly concordant with microsatellite genotyping across several screened populations from Europe. Finally, we were able to amplify two short regions of these loci in formaldehyde-preserved samples dating back to the year 1960. The species-specific SNP markers developed here provide valuable tools to study hybridization over time, including the long-term impact of various environmental factors on hybridization and biodiversity changes. SNP-based genotyping will finally allow for revealing eco-evolutionary dynamics at different time scales.为解析杂交物种复合群中的种群群落动态与网状进化，亟需开展自然种群的长期比较研究，因此亟需开发适用于精细尺度遗传分析的工具。本研究针对杂交淡水甲壳类——多物种长棘溞（Daphnia longispina）复合群，开发了物种诊断性单核苷酸多态性(Single Nucleotide Polymorphism, SNP)标记。具体而言，本研究依托该杂交复合群关键物种的转录组数据、近缘溞类物种的注释基因组，以及来自3个亲本物种的明确参考基因型开展研究。通过对3个亲本物种及其杂交后代共22个个体的序列比对分析，共鉴定出11个核基因座，每个基因座均包含多个物种特异性SNP位点。为实现低成本高效的大规模种群SNP分型筛查，本研究开发了PCR-RFLP（聚合酶链反应-限制性片段长度多态性）检测方法。针对欧洲多个已筛查种群的分析显示，基于RFLP图谱的类群鉴定结果与微卫星分型结果几乎完全一致。最终，本研究成功从可追溯至1960年的甲醛固定保存样本中，扩增得到了上述基因座的两段短序列区域。本研究开发的物种特异性SNP标记，为长期杂交演化研究提供了宝贵工具，可用于解析各类环境因子对杂交过程的长期影响以及生物多样性变化。基于SNP的分型技术最终将助力揭示不同时间尺度下的生态-进化动态。