Development of novel EST-SSR markers for ploidy identification based on <i>de novo</i> transcriptome assembly for <i>Misgurnus anguillicaudatus</i>

The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus.自然种群中多种倍性类型共存，使得鳅科泥鳅（Misgurnus anguillicaudatus）成为研究倍性变异的遗传与表型效应的极具吸引力的模式系统。开展此类研究的首要步骤，便是鉴定个体的特定倍性。目前主流的核型分析（通过细胞学样本制备）或流式细胞术方法，需要采集大量组织（如血液）样本，这可能对鱼类造成损伤甚至致死。本研究开发了来自泥鳅的新型微卫星标记（SSR标记，Simple Sequence Repeat），并证实其可通过微创方式采集的样本有效区分倍性。具体而言，我们利用Illumina双端测序技术对多尾泥鳅进行全转录组测序，获得约1.5亿条原始读段，经组装得到76544个非冗余单基因簇（unigene）。共鉴定出8194个潜在的SSR标记，我们从中筛选出98对包含5个以上串联重复序列的标记用于后续验证。在45个可成功扩增并在二倍体中呈现多态性的EST-SSR（表达序列标签-SSR，Expressed Sequence Tag-SSR）标记中，有11个标记在四倍体中表现出高变异性。我们进一步证实，从上述标记中筛选出的5个EST-SSR标记即可有效区分倍性水平：首先在69个已知倍性的参考标本上对这些标记进行验证，随后将其应用于新采集的96个倍性未知的标本。EST-SSR标记的检测结果与独立流式细胞术分析的结果高度一致。本研究开发的新型EST-SSR标记，将助力新兴模式系统泥鳅的多倍体遗传学研究。