Tilapia skin mucus swabs for ddRAD and the major sex determining locus in GIFT tilapia. Tilapia BSA-ddRAD

Skin mucus swabs have been used as a source for fish DNA in many targeted locus PCR-based studies, but their suitability for assaying genome wide variability through RADseq and related next generation sequencing methodologies has not been examined. We extracted genomic DNA from mucus (collected from skin swabs using filter paper), muscle and fin samples of two Nile tilapia and performed double-digest restriction-site associated DNA (ddRAD) sequencing. The results indicate that DNA derived from skin mucus swabs can be reliably used for genotyping by sequencing projects for tilapia and is likely to be much more widely applicable. Different major sex-determining loci have been detected in tilapia. The ‘genetically improved farmed tilapia’ (GIFT) stock, founded from multiple Nile tilapia (Oreochromis niloticus) populations, is a key resource for tilapia aquaculture. The main sex-determining loci present in GIFT fish are unknown. A bulk segregant analysis (BSA) version of double-digest restriction-site associated DNA sequencing (BSA-ddRADseq) was used to detect and position sex-linked single nucleotide polymorphism (SNP) markers in 19 GIFT stock pedigrees and two Stirling stock pedigrees. Phenotypic sex in Stirling families showed strong association with LG1 (previously known), whereas SNPs located in LG23 showed clear association with sex in all 19 GIFT families. No other genomic regions linked to sex-determination were apparent.

在诸多基于靶向位点PCR的研究中，皮肤黏液拭子常被用作获取鱼类DNA的样本来源，但其通过限制性酶切位点相关DNA测序（RADseq）及相关下一代测序技术分析全基因组变异的适用性尚未得到验证。本研究从两尾尼罗罗非鱼的、采用滤纸片采集的皮肤拭子黏液、肌肉及鳍组织样本中提取基因组DNA，并进行双酶切限制性酶切位点相关DNA（ddRAD）测序。结果表明，皮肤黏液拭子来源的DNA可可靠应用于罗非鱼的测序分型研究，且其适用范围有望进一步拓展。罗非鱼中已发现多种主要的性别决定位点。由多个尼罗罗非鱼（Oreochromis niloticus）种群培育而来的遗传改良养殖罗非鱼（GIFT）品系，是罗非鱼养殖业的关键种质资源，但该品系鱼类中存在的主要性别决定位点尚未明确。本研究采用双酶切限制性酶切位点相关DNA测序结合混合分组分析（BSA-ddRADseq）技术，在19个GIFT品系家系与2个斯特林（Stirling）品系家系中检测并定位与性别连锁的单核苷酸多态性（SNP）标记。斯特林家系的表型性别与LG1（此前已有相关报道）存在显著关联，而位于LG23的SNP位点在全部19个GIFT家系中均与性别存在明确关联，未发现其他与性别决定相关的基因组区域。