Sampled fish were treated according to the “Ethical justification for the use and treatment of fishes in research” (Anonymous 2006). This study was carried out in strict accordance with the recommendations in the Guide for the care and use of laboratory animals of the National Institutes of Health (Anonymous 2011b). The fish dissection was performed under MS-222 anesthesia, and all efforts were made to minimize the pain. The collection of I. longicorpa, C. hankugensis, and their hybrids was carried out in three areas with several localities (Fig. 2, Table 2), i.e., in the Seomjin River basin, Imsil, Jeollabuk-do, where I. longicorpa occurs, in the Nakdong River basin, Hapcheon, Gyeongsangnam-do, where C. hankugensis occurs and in the Nakdong River basin, Namwon, Jeollabuk-do.

Figure 2. 

Sampling localities of Cobitis hankugensis × Iksookimia longicorpa hybrid complex used in this study.

The identification of collected specimens was based on previously published methods. In particular, we examined each specimen by morphology, which is known to consistently distinguish both parental species from each other as well as their hybrids (albeit, we stress that morphological analysis may not reliably distinguish among different genomotypes of hybrids) (Kim and Park 2002). The ploidy was evaluated by erythrocyte size measurement (Ko 2009). To cross-validate our determination and to precisely evaluate the genomic composition, we employed karyotype analysis to a subset of specimens. This method provides reliable determination of sexual or hybrid genomotype given that both sexual species differ by chromosomal numbers (I. longicorpa 2n = 50; C. hankugensis 2n = 48), therefore allowing easy determination of both parental species (hereafter also labeled as LL and HH, respectively) from their diploid (HL, 2n = 49) and triploid (HHL 3n = 73 or HLL 3n = 74) hybrid forms (Fig. 3).

Figure 3. 

Metaphase plates and karyotypes of two triploid Cobitis hankugensis × Iksookimia longicorpa hybrids. A: genotype HLL; B: genotype HHL.

Finally, to obtain comparative material with known origin and genome composition, we have also performed 4 experimental crosses of parental species to obtain strict F1 HL hybrids, and we also crossed natural diploid HL hybrid females with either LL (3 families) or HH (3 families) males to obtain triploid HHL and HLL hybrids, altogether yielding a total of 133 experimental progeny of verified origin for microsatellite genotyping.

For the cross-species amplification analysis, the aforementioned fish samples were scrutinized for previously published microsatellite markers developed for related species of the Cobitidae family. The list of loci is shown in Table 1.

For DNA analysis a piece of pectoral fin was dissected from each specimen and stored in 100% ethyl alcohol. Total DNA was purified with the genomic DNA Prep Kit for blood and tissue (QIAGEN Co., USA). PCR reactions were completed in a total volume of 50 µL, consisting of 2 µL of genomic DNA, 1 µL of the 10 uM forward (fluorescently labeled) and reverse primer solutions, 24 µL of Premix Taq (Takara, Japan), and 22 µL of distilled water (Takara, Japan). Polymerase chain reactions for all specimens were executed in GeneAtlas G-02 thermocycler (Astec, Japan) with the initial denaturing step at 95°C for 5 min and 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C. A final extension step at 72°C for 5 min. The PCR amplicons were visualized on a 2% agarose gel stained with LoadingStar (Dyne, Korea) together with negative controls and Takara 1 KB molecular size ladder for preliminary size determination. The final PCR products were run on an ABI-3730XL sequencer (Applied Biosystems, USA) with the size standard at 350 ROX. The resulting electropherograms were analyzed in Peak Scanner v1.0 (Applied Biosystems, USA).

To evaluate whether particular markers bear consistent information about the allelic dosage in diploid and triploid hybrids, we used the Gene scan peak analysis with the Peak Scanner v1.0 (Applied Biosystems, USA) to analyze and compare the relative intensities of alleles in analyzed individuals.

Altogether, based on the classical determination methods including karyotype analysis we selected for marker validation 25 individuals of I. longicorpa, 25 of C. hankugensis, and also 5 HL, 5 HLL, and 5 HHL hybrid individuals. We further included into the analysis 59 natural hybrids (without karyotype analysis) sampled at five sites in the Nakdong River basin, and we also scrutinized 133 progenies generated by artificial crossing experiments with known origin and genomic composition.

The cross-amplification of 23 published markers showed that 19 loci were amplified in all genomotypes of the hybrid complex. Moreover, we further noticed that the IC875 marker did not amplify with I. longicorpa but it did amplify in the C. hankugensis and hybrids while the Iko_AAT08 marker did not amplify at C. hankugensis, but it did amplify in the I. longicorpa and in hybrids (Table 1). We observed that 50.0% of tested loci were monomorphic in I. longicorpa, 59.1% in C. hankugensis, and 43.5% in all three types of hybrids. The number of alleles per locus, except for monomorphic ones, ranged from 2–6 (mean 3.2) in I. longicorpa, 2–9 (mean 4.0) in C. hankugensis, 2–5 (mean 2.5) in hybrid (HL type), 2–4 (mean 2.9) in hybrid (HLL type), and 2–5 (mean 2.7) in hybrid (HHL type), respectively.

We note that in each locus, the numbers of detected alleles were always lower than those reported in the reference species for which given microsatellite marker has been developed and where 2–33 alleles per locus per species (mean 12.2) have been reported in the original publications. When compared to the reference species, analyzed hybrids had the highest numbers of alleles in markers taken from C. taenia (mean value 2.3 alleles per locus), the second-highest numbers of alleles in loci taken from C. choii (mean value 2.2), while the markers taken from different genera showed lowest numbers of alleles, i.e., mean value 1.5 in K. naktongensis markers and 1.4 in I. koreensis markers, respectively. This is in line with the general expectation that the efficiency of cross-species amplification tends to decrease with increasing phylogenetic distance between the reference species and the target species (Moore et al. 1991; Peakall et al. 1998). This study used the cross-amplification between species of the same genus or family and showed relatively high amplification efficiency. However, nearly half of essayed loci appeared as monomorphic and many of other loci shared the same alleles between species, making them of limited use for species diagnosis. Future studies of the genetic diversity of I. longicorpa and C. hankugensis and their hybrids would certainly profit from the direct development of microsatellite loci from their DNA.

Nevertheless, we discovered three loci, which seem very useful for fast and efficient identification of genomotypes from the studied hybrid complex because they possess non-overlapping allelic size ranges between species. Specifically, the loci IC654, IC783, and Iko_TTA01 always distinguished between the specimens identified as pure I. longicorpa and C. hankugensis, respectively, while they always provided amplification products of both species in the hybrid individuals. Furthermore, we found two additional loci with selective amplification (IC875, Iko_AAT08), where one sexual species was characterized by absence of amplification, while the other species and all hybrid individuals provided specific amplification product (Table 1).

This altogether suggests that tested cross-amplification identified three markers with species-specific allelic variants and two loci with species-selective amplification that may be used as haploid detection markers for C. hankugensis and I. longicorpa, respectively. In addition, some other loci also appear as useful for subsequent population genetic studies given they possess a moderate number of alleles per species, which may allow for frequency-based analyses.

Given that the scope of this paper was to find a fast and efficient method to discern parental species and hybrid genomotypes, we will describe in the following text the properties of three markers that we propose for such a purpose given their ability to diagnose both species as well as the ploidy of hybrid individuals. The IC654 and IC783 markers derived from C. choii and the Iko_TTA01 marker derived from I. koreensis were of particular interest for us as they were fixed for different alleles in both parental species and showed the consistent presence of both species-specific amplification products in hybrids with different relative peak intensities depending on the genotypes (Fig. 3).

The patterns were straightforward in IC654 and Iko_TTA01 markers (Fig. 4A–B) since in diploid hybrid HL genomotype with one genome of I. longicorpa and the other of C. hankugensis, the heights of the amplified peaks were similar to each other. In triploid hybrid HLL genomotype with two genomes of I. longicorpa and one of C. hankugensis, the allele peak intensity of I. longicorpa—specific allele was approximately twice as strong as that of C. hankugensis – specific allele, while in triploid hybrid HHL genomotype the situation was opposite with approximately double intensity of the C. hankugensis allele compared to I. longicorpa allele. The patterns in IC783 were more complicated by the presence of two alleles in I. longicorpa. Consequently, the diploid HL form always possessed one allele diagnostic for I. longicorpa and the other for C. hankugensis, HHL triploids also possessed two alleles with clear dosage pattern, but HLL triploids either possessed two alleles with the apparent dosage pattern or three alleles, each with similar intensity (Fig. 4C).

Figure 4. 

Demonstration of Microsatellite analysis of IC654 (A), Iko_TTA01 (B) and IC783 (C) loci in Cobitis hankugensis × Iksookimia longicorpa hybrid complex. Upper panels A–C show the electropherograms of the three loci in all biotypes. Boxplots in the lower panel depict for each locus the relative intensity (%) of the minor peak to the major one (D–F) and the mean relative values of the total of three markers for each genomotype (G).

To verify the possibility of applying the three selected markers (IC783, IC654, and Iko_TTA01) for the identification of unknown hybrid genomotype, we demonstrate the relative size ratio of the minor peak to the major one (Fig. 4 D–F). As a result, three hybrid genomotypes were clearly distinguished by the ratio of the peaks. Diploid HL genomotype had relatively similar intensities of the less intense allele, i.e., 92.7% (IC654), 84.4% (IC783), and 87.9% (Iko_TTA01), while in triploid HLL genomotype, the ratios were 54.3%, 62.3%, and 45.3%, respectively, and in HHL genomotype, we found 51.6%, 45.6%, and 54.1%, respectively (Fig. 4, D–F). Finally, we also plotted the mean relative values of the total of three markers for each genomotype, (Fig. 4, G), where the mean value in HL type was 89.8%, in HLL type was 55.3%, HHL type was 49.2%. This result strongly supported that the microsatellite marker can be used to the correct method of discrimination of known genomotypes of the C. hankugensis × I. longicorpa complex.

To date, the identification of C. hankugensis × I. longicorpa hybrid complex had a fatal disadvantage in that it requires complex processing and fish sacrifice. In this study, we provided a reliable identification method of the C. hankugensis × I. longicorpa hybrid complex using microsatellite markers through a single Genescan analysis using only a small piece of fin tissues. This has the great advantage that the fish are kept alive and can be used for additional hybridization experiments by reducing the stress.

Microsatellite markers have indeed been previously used to identify hybrid groups of fish, including the family Cobitidae, (e.g., You et al. 2007; de Gelas et al. 2008) and these markers have also been applied to polyploid hybrids, (e.g., Janko et al. 2012; Mishina et al. 2014), when triploids were typically inferred by the possession of three peaks in at least one locus. However, the identification method proposed in this study suggests that microsatellite markers can be used as a powerful method to determine triploids even in cases when hybrids possess no more than two alleles, relying on the relative amplification intensities of species-specific alleles.

In a summary, the cross-species amplification of microsatellite markers can be used as an easy and fast identification method in studies of reproductive modes of investigated hybrids.