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

Abstract

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.

Development and Transferability of EST-SSR Markers for Pinus koraiensis from Cold-Stressed Transcriptome through Illumina Sequencing

“…Recently, EST-SSR markers developed using expressed sequence tags have been widely used in the fields of genetic diversity, phylogenetic development, and molecular marker-assisted breeding. At present, the development of universal EST-SSR primers is a cheap and simple approach for species without whole genome and developed microsatellite loci [40]. However, for some species with complex genetic background, such as non-model species, genome-free species, and endangered species, traditional methods for SSR marker development are complex and expensive.…”

A Target Capture-Based Method to Estimate Ploidy From Herbarium Specimens

“…For each of these studies a tetraploid upper limit was assumed in the bioinformatic pipelines. For example, EST-SSR nuclear markers have been used to distinguish between diploid and tetraploid individuals of the fish Misgurnus anguillicaudatus (Feng et al, 2018) and RAD-seq data were used to explore ploidy levels in Betula species (Zohren et al, 2016).…”

Development and Characterization of EST-SSR Markers From RNA-Seq Data in Phyllostachys violascens

“…Simple sequence repeat markers have been widely used in fingerprint construction, genetic diversity analysis and molecular marker-assisted breeding because of their high polymorphism, repeatability and codominant inheritance (Ran et al, 2010; Feng et al, 2018; Shi et al, 2018; Wang et al, 2018). However, traditional methods for developing SSR markers are not very efficient (Tang et al, 2010).…”