Three species of ornamental fishes in the subfamily Cyprininae (family Cyprinidae) namely, Epalzeorhynchos frenatum (Fowler, 1934), Puntigrus partipentazona (Fowler, 1934), Scaphognathops bandanensis Boonyaratpalin et Srirungroj, 1971 were studied by classical cytogenetic and fluorescent in situ hybridization (FISH) techniques. Chromosomes were directly prepared from kidney tissues and stained by using conventional and Ag-NOR banding techniques. Microsatellite d(CA)₁₅ and d(CGG)₁₀ probes were hybridized to the chromosomes of three cyprinids. The results show that the three cyprinid species share the same diploid number as 2n=50 but there are differences in the fundamental number (NF) and karyotypes i.e. E. frenatum: NF = 78, 18m+10sm+10st+12a; P. partipentazona: NF = 80, 6m+24sm+14st+6a; S. bandanensis: NF = 66, 4m+12sm+34a. NOR positive masks were observed at the regions adjacent to the telomere of the short arm of the chromosome pairs 10 (submetacentric) and 1 (metacentric) in E. frenatum and P. partipentazona, respectively whereas those were revealed at telomeric regions of the long arm of the chromosome pair 9 (acrocentric) in S. bandanensis. The mapping of d(CA)₁₅ and d(CGG)₁₀ microsatellites shown that hybridization signals are abundantly distributed in telomeric regions of several pairs except d(CA)₁₅ repeats in S. bandanensis, which are distributed throughout all chromosomes and d(CGG)₁₀ repeats in P. partipentazona display the high accumulation only in the first chromosome pair.

Chromosome, Epalzeorhynchos frenatum, FISH, Puntigrus partipentazona, Scaphognathops bandanensis

There are about 200 species of freshwater fish used as ornamentals in Thailand. More than half of all ornamental fishes in Thailand belong to the family Cyprinidae. The most popular species include Betta splendens Regan, 1910, Gyrinocheilus aymonieri (Tirant, 1883), Epalzeorhynchos bicolor (Smith, 1931), E. frenatum (Fowler, 1934), Puntigrus tetrazona (Bleeker, 1855), Channa micropeltes (Cuvier, 1831), Barbonymus alter Bleeker, 1853, Bar. schwanenfeldii (Bleeker, 1854) and Balantiocheilos melanopterus (Bleeker, 1850) (Sermwatanakul 2005).

Family Cyprinidae is the most abundant and globally widespread family of freshwater fish, comprising 3,000 extant and extinct species in about 370 genera (Eschmeyer et al. 2015). The subfamily Cyprininae is one of the largest groups of this family. The essential large tribes such as Labeonini, Poropuntiini and Smiliogastrini have many species that are economically important ornamental fish of Thailand, namely Epalzeorhynchos frenatum (Fowler, 1934), Puntigrus partipentazona (Fowler, 1934), Scaphognathops bandanensis Boonyaratpalin et Srirungroj, 1971 (Fig. 1A, D, G). However, there are few studies of cytogenetics of these ornamental fishes. To date, most reports are of conventional technique studies to determine chromosome number and karyotype composition and only a few ionclude NOR banding analysis. The 2n ranges from 48–50 in the tribes Labeonini and Smiliogastrini while the tribe Poropuntiini is more conserved as 2n = 50 (Arai 2011) (Table 1). Understanding of the basic information on cytogenetics can be applied to the development of potentially commercial stains/species in the future. The studies on the karyotypes help to investigate the genetic structure of aquatic animal species in each habitat, thus it can determine what species are related to each other in an accurate manner. This may help to facilitate the hybridization between them in the future for strain improvement (Sofy et al. 2008), breeding practices of organisms by using chromosome set management (Na-Nakorn et al. 1980), brood stock selection (Mengampan et al. 2004).

For some species, the simple characterization of the karyotype may be sufficient to identify intra- and inter-specific variants. However, in most cases, just the karyotype description appears to be inconclusive when not coupled with other methods capable of generating more accurate chromosomal markers. In this sense, the use of molecular cytogenetic analyses has played an important role in the precise characterization of the structure of genomes (Cioffi and Bertollo 2012). Multiple DNA copies or repetitive DNAs are a large substantial portion of the genome of eukaryotes that can be generally classified into two main classes: tandem repeats, such as the multigene families and the satellite DNAs; and the dispersed elements, such as transposons and retrotransposons, known as Transposable elements (TEs) (Jurka et al. 2005). Among the tandem repeats we can find the highly-repeated satellite DNAs and “moderate repeats”, like mini- and microsatellite DNA (Charlesworth et al. 1994). These non-coding DNA sequences are organized as long arrays of head-to-tail linked repeats (Plohl et al. 2008).

Recently, the molecular cytogenetic studies using fluorescence in situ hybridization (FISH) for mapping repetitive DNA sequences have provided important contributions to the characterization of the biodiversity and the evolution of divergent fish groups (Cioffi and Bertollo 2012). Moreover, some microsatellite repeats are species-specific characters among some fish group (Cioffi et al. 2015). Most molecular cytogenetic studies in cypinid fishes were performed by FISH technique using rDNA probes (Inafuku et al. 2000; Kikuma et al. 2000; Ocalewicz et al. 2004; Zhu et al. 2006; Singh et al. 2009; Rossi et al. 2012; Nabais et al. 2013; Kirtiklis et al. 2014; Spoz et al. 2014; Han et al. 2015; Kumar et al. 2016; Han et al. 2017). However, NOR banding including fluorescence in situ hybridization (FISH) techniques to investigate chromosomal distribution of repetitive DNA sequences on the chromosomes of E. frenatum, P. partipentazona, S. bandanensis have not been performed.

In present study, we carried out an analysis of chromosomal structures and genetic markers on E. frenatum, P. partipentazona, and S. bandanensis using cytogenetics, and molecular cytogenetics techniques. The knowledge revealed will provide a powerful tool for the next generation of genome research in Thai freshwater fishes and discovering biodiversity, with useful applications in fish breeding for conservation and commercials of ornamental species. Moreover, it is useful applications in evolution, systematics, phylogenetics, fish fauna management and suitable conservation of river basin.

Ten males and ten females of each species including E. frenatum, P. partipentazona, S. bandanensis, were collected from the Song Khram, Chi and Mekong Basins, respectively. Preparation of fish chromosomes was from kidney cells (Pinthong et al. 2015; Supiwong et al. 2015). The chromosomes were stained with Giemsa’s solution for 10 min. Ag-NOR banding was performed by applying two drops of 2% gelatin on the slides, followed with four drops of 50% silver nitrate (Howell and Black 1980). Metaphase figures were analyzed according to the chromosome classification of Levan et al. (1964). Chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a). Fundamental number, NF (number of chromosome arm) is obtained by assigning a value of two to metacentric and submetacentric chromosomes and one to subtelocentric and acrocentric chromosomes.

The use of microsatellite d(CA)₁₅ and d(CGG)₁₀ probes described by Kubat et al. (2008) was followed here with slight modifications. These sequences were directly labeled with Cy3 at 5´ terminal during synthesis by Sigma (St. Louis, MO, USA). FISH was performed under high stringency conditions on mitotic chromosome spreads (Pinkel et al. 1986). After denaturation of chromosomal DNA in 70% formamide/ 2×SSC at 70 °C, spreads were incubated in 2×SSC for 4 min at 70 °C. The hybridization mixture (2.5 ng/µL probes, 2 µg/µL salmon sperm DNA, 50% deionized formamide, 10% dextran sulfate) was dropped on the slides, and the hybridization was performed overnight at 37 °C in a moist chamber containing 2×SSC. The post hybridization wash was carried out with 1×SSC for 5 min at 65 °C. A final wash was performed at room temperature in 4×SSCT for 5 min. Finally, the slides were counterstained with DAPI and mounted in an antifade solution (Vectashield from Vector laboratories) and analyzed in an epifluorescence microscope Olympus BX50 (Olympus Corporation, Ishikawa, Japan).

The present research is the first report on the NOR -banding and FISH techniques in E. frenatum, P. partipentazona, S. bandanensis. Although all studied species have the same diploid chromosome number (2n = 50) and two NOR-bearing chromosomes, there are differences in the fundamental numbers, numbers of chromosomes with equal sizes, pairs having NORs, and patterns of microsatellites distributions on chromosomes. The NORs can be observed at the regions adjacent to the telomeres of pairs 10, 1 and 9, respectively. The microsatellites are distributed throughout the chromosomes with high accumulations at some positions or all chromosomes which are species-specific characteristics. This result indicated that cytogenetic data can be used for classification in related fish species which have similar morphology.

This work was financially supported by the research grant of the Office of the Higher Education Commission and the Thailand Research Fund (MRG6080020), and the Post-Doctoral Training Program from Research Affairs and Graduate School (Grant no 59255), Khon Kaen University, and Unit of Excellence 2020 on Biodiversity and Natural Resources Management, University of Phayao (UoE63005), Thailand.