Genome-wide confirmation of isogenicity of clone founders (G1) in European seabass (Dicentrarchus labrax). Isogenicity seabass G1 (ddRAD)

Isogenic clonal lines of fish are a valuable tool for aquaculture-related research, as inbred animals have been in biomedical research, yet to date they are available in only a few species. Although the production of such lines can be achieved in two generations through induced parthenogenesis (either mitotic gynogenesis or androgenesis), challenges such as reduced survival of doubled haploid clone founders and spontaneous, partially heterozygous meiotic gynogenetics (due to non-targeted retention of second polar body in the mitotic gynogenesis process) hamper the successful establishment of such lines. Until recently only small numbers of genetic markers were available for the verification of such lines. Reliable marker technologies are needed for genome-wide screening during development of isogenic lines, and next generation sequencing offers this potential. In the present study, we analysed DNA from 17 putative mitotic gynogenetics (clone founders) of European seabass, using double-digestion restriction-associated DNA sequencing (ddRAD-seq). A total of over 31 million raw sequence reads were produced and assembled into an average of 6,830 unique ddRAD loci. Based on an average of 1,950 polymorphic single nucleotide polymorphism (SNP) loci, 17 out of 18 fish were identified as isogenic (mitotic gynogenetics), while one fish represented a clear case of spontaneous meiotic gynogenesis, with no sire contribution but heterozygous for 49% of informative maternal loci. Although these fish were genotyped first using 12 microsatellite loci which suggested isogenicity of all samples, the single meiotic gynogenetic was only detected with the higher power of genome-wide screening in one fish, proving the efficacy of NGS for this purpose. Provided that the clonal founders are fertile, they will be used for producing isogenic clonal lines in European seabass in the successive generation. Successful establishment of such lines in species of prime commercial interest in Europe is one of the objectives of the AQUAEXCEL2020 as a resource for aquaculture-related research. [Münevver Oral, PhD These: https://hdl.handle.net/1893/24909]

鱼类同源克隆系（isogenic clonal lines）是水产养殖相关研究的重要工具，正如近交动物在生物医学研究中的应用价值一般，但截至目前，这类克隆系仅在少数鱼类物种中得以建立。尽管通过诱导孤雌生殖（包括有丝分裂雌核发育（mitotic gynogenesis）与雄核发育（androgenesis））仅需两代即可获得此类克隆系，但双单倍体克隆创始个体存活率低下、以及有丝分裂雌核发育过程中因非靶向保留第二极体（second polar body）导致的自发部分杂合减数分裂雌核发育个体等问题，仍阻碍了这类克隆系的成功建立。直至近期，仅少量遗传标记可用于此类克隆系的验证。在同源克隆系的构建过程中，亟需可靠的标记技术用于全基因组筛选，而下一代测序（next generation sequencing, NGS）恰好具备这一应用潜力。本研究采用双酶切限制性位点关联DNA测序（double-digestion restriction-associated DNA sequencing, ddRAD-seq）技术，对17株疑似欧洲海鲈（European seabass）有丝分裂雌核发育个体（克隆创始个体）的DNA进行了分析。本次实验共获得超过3100万条原始序列读段，最终平均组装得到6830个独特的ddRAD位点。基于平均1950个多态性单核苷酸多态性（single nucleotide polymorphism, SNP）位点的基因分型结果，18尾受试个体中的17尾被鉴定为同源克隆系（有丝分裂雌核发育个体），剩余1尾则为典型的自发减数分裂雌核发育个体：该个体无父本遗传贡献，但在49%的有效母本位点上呈现杂合性。尽管研究团队最初采用12个微卫星位点（microsatellite loci）对这批个体进行基因分型，结果显示所有样本均为同源克隆系，但通过更高分辨率的全基因组筛选，才成功检测出这尾异常的减数分裂雌核发育个体，证实了NGS在该类鉴定中的应用效能。若这批克隆创始个体可正常繁育，后续将用于培育欧洲海鲈的同源克隆系。在欧洲具有重要商业价值的鱼类物种中成功建立此类克隆系，是作为水产养殖研究资源的AQUAEXCEL2020项目的目标之一。[Münevver Oral，博士学位论文：https://hdl.handle.net/1893/24909]