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

<div><p>The co-existence of several ploidy types in natural populations makes the cyprinid loach <i>Misgurnus anguillicaudatus</i> 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 <i>M</i>. <i>anguillicaudatus</i> and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple <i>M</i>. <i>anguillicaudatus</i> 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 <i>M</i>. <i>anguillicaudatus</i>.</p>