Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497 bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905 single copy orthologous genes. The tetraploid and diploid M. anguillicaudatus grouped into a clade, and M. bipartitus showed a closer relationship with the M. anguillicaudatus. The overall evolutionary rates of tetraploid M. anguillicaudatus were significantly higher than those of other Misgurnus species. Meanwhile, 28 positively selected genes were identified in M. anguillicaudatus clade. These positively selected genes may play critical roles in the adaptation to various habitat environments for M. anguillicaudatus. This study could facilitate further exploration of the genomic signatures of M. anguillicaudatus and provide potential insights into unveiling the evolutionary history of tetraploid loach.

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.

Identifying the dispersal pathways of an invasive species is useful for adopting the appropriate strategies to prevent and control its spread. However, these processes are exceedingly complex. So, it is necessary to apply new technology and collect representative samples for analysis. This study used Approximate Bayesian Computation (ABC) in combination with traditional genetic tools to examine extensive sample data and historical records to infer the invasion history of the red swamp crayfish, Procambarus clarkii, in China. The sequences of the mitochondrial control region and the proPOx intron in the nuclear genome of samples from 37 sites (35 in China and one each in Japan and the USA) were analyzed. The results of combined scenarios testing and historical records revealed a much more complex invasion history in China than previously believed. P. clarkii was most likely originally introduced into China from Japan from an unsampled source, and the species then expanded its range primarily into the middle and lower reaches and, to a lesser extent, into the upper reaches of the Changjiang River in China. No transfer was observed from the upper reaches to the middle and lower reaches of the Changjiang River. Human-mediated jump dispersal was an important dispersal pathway for P. clarkii. The results provide a better understanding of the evolutionary scenarios involved in the rapid invasion of P. clarkii in China.

Misgurnus anguillicaudatus has several natural ploidy types. To investigate whether nuclear polyploidy have an impact on mitochondrial DNA (mtDNA), the complete mitochondrial genomes (mitogenomes) of five distinct ploidy M. anguillicaudatus (natural diploid, triploid, tetraploid, pentaploid and hexaploid), which were collected in central China, were sequenced and analyzed. The five mitogenomes share the same gene arrangement and have similar gene size, base composition and codon usage pattern. The most variable regions of the mitogenome were the protein-coding genes, especially the ND4L (5.39% mutation rate). Most variations occurred in tetraploids. The phylogenetic tree showed that the tetraploid M. anguillicaudatus separated early from other ploidy loaches. Meanwhile, the mitogenomes from pentaploids, and hexaploids have the closest phylogenetic relations, but far from that of tetraploids, implying that pentaploids and hexaploids could not be formed from tetraploids, possibly from the diploids and triploids. The genus Misgurnus species were divided into two divergent inter-genus clades, and the five ploidy M. anguillicaudatus were monophyletic, which support the hypotheses about the mitochondrial introgression in loach species.

The distribution of Triplophysa yarkandensis is restricted to Xinjiang’s Tarim River basin. We collected 119 T. yarkandensis samples from nine geographic populations in the Tarim River basin and utilized the RAD-seq method for SNP genotyping. In this study, a total of 164.81 Gb bases were generated with the Illumina platform, and 129,873 candidate SNPs were obtained with the Stacks pipeline for population genetic analyses. High levels of genetic diversity were detected among nine populations. The AMOVA results showed that the majority of genetic variations originated from among populations (F_ST = 0.67), and the pairwise F_ST values ranged from 0.4579 to 0.8736, indicating high levels of genetic differentiation among these populations. The discriminate analysis of principal components (DAPCs) and neighbor joining (NJ) tree revealed that the nine populations could be separated into two clusters (i.e., south and north populations), and modest genetic differentiation between south and north populations was observed, while the individuals from several populations were not clustered together by geographical location. The evidence of two genetic boundaries between south and north populations (except TTM) was supported by barrier analysis. The Bayesian skyline plotting indicated that T. yarkandensis populations in the Tarim River basin had not experienced genetic bottlenecks, and the effective population size remained stable. This study first clarified the genetic diversity and differentiation of T. yarkandensis populations in the Tarim River basin, and it provided valuable molecular data for conservation and management of natural populations.