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Research Article
Comparative karyotype study of three Cyprinids (Cyprinidae, Cyprininae) in Thailand by classical cytogenetic and FISH techniques
expand article infoSumalee Phimphan, Patcharaporn Chaiyasan§, Chatmongkon Suwannapoom|, Montri Reungsing, Sippakorn Juntaree#, Alongklod Tanomtong§, Weerayuth Supiwong#
‡ Phetchabun Rajabhat University, Phetchabun, Thailand
§ Khon Kaen University, Khon Kaen, Thailand
| University of Phayao, Phayao, Thailand
¶ Rajamangala University, Chon Buri, Thailand
# Khon Kaen University, Nong Khai, Thailand
Open Access

Abstract

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.

Keywords

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

Introduction

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).

Table 1.

Reviews of cytogenetic reports in the tribes Labeonini, Poropuntiini, and Smiliogastrini. (2n = diploid number, m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric and NORs = nucleolar organizer regions, NF = fundamental number, – = not available).

Tribe / Genus / Species 2n NF Formula NORs Reference
Tribe Labeonini
Barbichthys laevis (Valenciennes, 1842) 50 76 20m+6sm+4st+20a Donsakul et al. (2006)
Bangana devdevi (Hora, 1936) 50 86 20m+16sm+14a Donsakul et al. (2011)
Cirrhinus julleini 50 90 26m+14sm+4st+6a Magtoon and Arai (1993)
(Valenciennes, 1844) 50 92 36m+6sm+2st+6a Donsakul (1997)
C. microlepis Sauvage, 1878 50 88 22m+8sm+8st+12a Donsakul and Magtoon (1997)
50 72 12m+10sm+2st+26a Donsakul et al. (2007)
Epalzeorhynchos frenatum (Fowler, 1934) 48 72 14m+10sm+8st+16a Donsakul and Magtoon (1993)
50 78 18m+10sm+10st+12a 2 Present study
E. bicolor (Smith, 1931) 50 74 20m+4sm+2st+24a Donsakul and Magtoon (1993)
E. munensis (Smith, 1934) 50 84 22m+12sm+2st+14a Donsakul et al. (2012)
Garra cambodgiensis (Tirant, 1883) 50 82 20m+12sm+4st+14t Donsakul et al. (2016)
G. fasciacauda Fowler, 1937 50 84 18m+14sm+2st+16t Donsakul et al. (2016)
G. notata (Blyth, 1860) 50 80 20m+10sm+20t Donsakul et al. (2016)
Incisilabeo behri (Fowler, 1937) 50 78 12m+16sm+4st+18t Donsakul and Magtoon (2003)
Labeo chrysophekadian (Bleeker, 1850) 50 78 4m+10sm+14st+22a Seetapan (2007)
Labiobarbus lineatus (Sauvage, 1878) 50 80 20m+10sm+20a Magtoon and Arai (1990)
L. spiropleura (Sauvage, 1881) 50 90 34m+4sm+2st+10a Donsakul and Magtoon (1997)
Mekongina erythrospila Fowler, 1937 50 74 10m+14sm+26a(t) Donsakul and Magtoon (2003)
Osteochilus melanopleura (Bleeker, 1852) 50 96 36m+10sm+2st+2a Donsakul and Magtoon (1995)
O. microcephalus (Valenciennes, 1842) 50 86 26m+10sm+14st Donsakul et al. (2001)
O. vittatus (Valenciennes, 1842) 50 96 16m+30sm+4st Magtoon and Arai (1990)
50 86 26m+10sm+14st Donsakul (1997)
O. waandersi (Bleeker, 1853) 50 92 18m+24sm+4st+4a 2 Magtoon and Arai (1993)
Puntioplites falcifer Smith, 1929 50 80 14m+16sm+2st+18a Donsakul et al. (2007)
50 92 16m+10sm+16a+8t Sophawanus et al. (2017)
Tribe Smiliogastrini
Osteobrama alfrediana (Valenciennes, 1844) 50 96 24m+22sm+4a Donsakul et al. (2011)
Hampala disper Smith, 1934 50 70 5m+5sm+3st+12a Donsakul and Poopitayasathaporn (2002)
H. macrolepidota Kuhl & Van Hasselt, 1823 50 72 10m+12sm+8st+20a Donsakul and Poopitayasathaporn (2002)
Puntigrus partipentazona (Fowler, 1934) 50 90 6m+34sm+10a Taki et al. (1977)
50 80 6m+24sm+14st+6a 2 Present study
P. tetrazona (Bleeker, 1855) 50 84 34m+6st+10a Ohno et al. (1967)
50 84 6m+28sm+16a Hinegardner and Rosen (1972), Taki et al. (1977), Suzuki et al. (1995)
50 Krishnaja and Rege (1980) Vinogradov (1998)
P. tetrazona partipentazona (Fowler, 1937) 50 90 6m+34sm+10a Taki et al. (1977)
Puntius arulius (Jerdon, 1849) 50 82 6m+26sm+18a Taki and Suzuki (1977)
50 90 10m+18sm+12st+10t Arunachalan and Murugan (2007)
P. binotatus (Valenciennes, 1842) 50 92 8m+34sm+8a Taki et al. (1977)
P. brevis (Bleeker, 1850) 50 70 6m+14sm+8st+22a Khuda-Bukhsh (1975)
50 54 2m+2sm+2st+22a Donsakul and Poopitayasathaporn (2002)
48 56 2m+6st+40a Seetapan (2007)
50 62 4m+4sm+4a+38t 2 Nitikulworawong and Khrueanet (2014)
P. chola (Hamilton, 1822) 50 56 2m+4sm+44a Taki and Suzuki (1977)
50 54 2m+2sm+4st+42a Tripathi and Sharma (1987)
50 54 2m+2sm+46a Sahoo et al. (2007)
P. conchonius (Hamilton, 1822) 50 94 6m+38sm+6a Hinegardner and Rosen (1972),
Taki and Suzuki (1977)
48 78 10m+20sm+10st+8a Sharma and Agarwal (1981)
50 Vasiliev (1985)
50 90 16m+24sm+2st+8a Khuda et al. (1986), Ojima and Yamamoto (1990)
P. conchonius (Hamilton, 1822) 50 94 4m+40sm+6a Takai and Ojima (1988)
P. cumingi (Günther, 1868) 50 94 18m+26sm+6a Taki and Suzuki (1977)
P. daruphani Smith, 1934 50 70 12m+8sm+6st+24a Magtoon and Arai (1989)
P. denisonii (Day, 1865) 50 74 4m+20sm+18st+8a 8 Nagpure et al. (2004)
P. everetti (Boulenger, 1894) 50 86 6m+30sm+14a Hinegardner and Rosen (1972), Taki et al. (1977), Vinogradov (1998)
P. fasciatus (Jerdon, 1849) 50 80 30m+4st+16a Ohno et al. (1967)
50 82 6m+26sm+18a Taki et al. (1977)
P. filamentosus (Valenciennes, 1844) 50 84 8m+26sm+16a Taki and Suzuki (1977)
50 78 12m+16sm+12st+10a 8 Nagpure et al. (2003)
P. lateristriga (Valenciennes, 1842) 50 88 6m+32sm+12a Taki et al. (1977)
50 86 22m+14sm+6st+8a Sobita et al. (2004)
P. melanampyx Day, 1865 50 74 12m+12sm+14st+12a Khuda et al. (1986)
P. nigrofasciatus (Günther, 1868) 50 100 16m+34sm Taki and Suzuki (1977)
P. oligolepis (Bleeker, 1853) 50 88 8m+30sm+12a Taki et al. (1977)
50 80 14m+16sm+4st+16a Arai and Magtoon (1991)
50 92 6m+36sm+8a Taki et al. (1977)
P. pentazona (Boulenger, 1894) 50 98 22m+26sm+2a Taki et al. (1977)
P. sarana (Hamilton, 1822) 50 76 12m+14sm+12st+12a Rishi (1981)
P. sarana subnasutus (Valenciennes, 1842) 50 88 12m+26sm+8st+4a Nagpure et al. (2004)
P. semifasciolatus (Günther, 1868) 50 76 12m+14sm+14st+10a Gui et al. (1986), Yu et al. (1989)
50 76 12m+14sm+14st+10a 8 Nagpure et al. (2004)
50 76 8m+18sm+24a Suzuki (1991)
P. sophore (Hamilton, 1822) 48 52 2m+2sm+44a Rishi (1973)
48 54 2m+4sm+42a Rishi et al. (1977)
48 52 4m+2st+42a Rishi and Rishi (1981)
50 56 2m+4sm+44a Khuda et al. (1986)
48 52 4m+6st+38a Tripathi and Sharma (1987)
P. sophoroides (Günther, 1868) 50 54 2m+2sm+46a Magtoon and Arai (1989)
P. stoliczkanus (Day, 1871) 50 94 22m+22sm+4st+2a Magtoon and Arai (1989)
P. tambraparniei Silas, 1954 50 94 12m+16sm+16a+6t Arunachalan and Murugan (2007)
P. ticto (Hamilton, 1822) 50 82 20m+12sm+10st+8a Sharma et al. (1995), Vinogradov (1998)
50 100 28m+22sm Taki and Suzuki (1977)
50 94 28m+16sm+6st Sahoo et al. (2007)
P. titteya (Deraniyagala, 1929) 50 98 20m+28sm+2a Hinegardner and Rosen (1972), Taki and Suzuki (1977)
48 52 4m+2sm+42a Khuda-Bukhsh and Barat (1987)
Systomus sp.1 50 82 12m+20sm+6st+12a Donsakul et al. (2006)
S. binotatus (Valenciennes, 1842) 50 88 24m+14sm+12a Donsakul and Magtoon (2002)
S. orphoides (Valenciennes, 1842) 50 82 12m+20sm+4st+14a Piyapong (1999)
50 74 8m+16sm+10st+16a Donsakul and Poopitayasathaporn (2002)
S. stoliczkanus (Day, 1871) 50 94 24m+20sm+6a Donsakul et al. (2011)
Tribe Poropuntiini
Amblyrhynchichthys truncatus (Bleeker, 1851) 50 78 16m+12sm+22a Donsakul et al. (2006)
Balantiocheilos melanopterus (Bleeker, 1850) 50 72 10m+12sm+28a Ojima and Yamamoto (1990)
50 70 14m+6sm+10st+20a Donsakul and Poopitayasathaporn (2002)
Barbonymus gonionotus (Bleeker, 1850) 50 72 2m+20sm+4st+24a Magtoon and Arai (1989)
50 74 16m+8sm+26a Donsakul and Magtoon (1997)
50 72 6m+16sm+6st+22a Piyapong (1999)
50 66 2m+4sm+10st+34a Seetapan (2007)
50 74 6m+18sm+16st+10a 2 Khuda-Bukhsh and Das (2007)
Cosmochilus harmandi Sauvage, 1878 50 82 22m+10sm+10st+8a Donsakul et al. (2005)
Cyclocheilichthys apogon (Valenciennes, 1842) 50 70 12m+8sm+6st+24a Magtoon and Arai (1989)
50 76 18m+8sm+4st+20a Donsakul and Poopitayasathaporn (2002)
50 86 10m+16sm+10a+14t 6 Chantapan (2015)
C. lagleri Sontirat, 1989 50 86 12m+6sm+1st+6a Donsakul et al. (2006)
C. repasson (Bleeker, 1851) 50 78 12m+16sm+6st+16a Donsakul et al. (2005)
50 84 6m+6sm+22st+16a Seetapan (2007)
Cyclocheilos enoplos (Bleeker, 1849) 50 90 10m+30sm+4st+6a 4 Magtoon and Arai (1993)
50 72 14m+8sm+10st+18a Donsakul and Magtoon (1995a)
50 78 16m+12sm+6st+16a Donsakul and Poopitayasathaporn (2002)
Hypsibarbus lagleri Rainboth, 1996 50 74 4m+20sm+26a Donsakul and Magtoon (2001)
H. malcolmi (Smith, 1945) 50 64 10m+4sm+36a Donsakul et al. (2007)
H. vernayi (Norman, 1925) 50 58 6m+2sm+4st+38a Donsakul and Magtoon (2002)
H. wetmorei (Smith, 1931) 50 70 12m+8sm+6st+24a Magtoon and Arai (1989)
50 74 12m+12sm+4st+22a 2 Piyapong (1999)
50 74 12m+12sm+2st+24a Donsakul and Magtoon (2002)
50 82 10m+14sm+8a+18t 6 Chantapan (2015)
Mystacoleucus argenteus (Day, 1888) 50 76 6m+20sm+2st+22a Donsakul et al. (2006)
M. marginatus (Valenciennes, 1842) 50 76 16m+10sm+24a Arai and Magtoon (1991)
50 68 14m+4sm+2st+30a Donsakul and Poopitayasathaporn (2002)
Poropuntius deauratus (Valenciennes, 1842) 50 74 14m+10sm+26t Donsakul et al. (2005)
P. sinensis (Bleeker, 1871) 50 82 10m+22sm+18st Zen et al. (1984)
P. laoensis (Günther, 1868) 50 74 14m+10sm+10st+16a Donsakul and Magtoon (2008)
P. normani Smith, 1931 50 72 10m+12sm+28a Donsakul et al. (2007)
P. chonglingchungi (Tchang, 1938) 50 80 12m+18sm+20st Zen et al. (1986)
Scaphognathops bandanensis Boonyaratpalin & Srirungroj, 1971 50 66 10m+6sm+34a Donsakul et al. (2007)
50 66 10m+6sm+34a 2 Present study
Sikukia gudgeri (Smith, 1934) 50 68 10m+8sm+4st+28a Donsakul et al. (2005)

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.

Material and methods

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).

Results

Diploid number, fundamental number and karyotype of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops bandanensis

Results have shown that the three cyprinid species have the same diploid number of 2n = 50. Although the three species analyzed share the same 2n, there are differences in the fundamental number (NF) and karyotypes i.e. E. frenatum: NF = 78, 18 metacentric (m), 10 submetacentric (sm), 10 subtelocentric (st) and 12 acrocentric (a) chromosomes; P. partipentazona: NF = 80, 6m, 24sm, 14st, and 6a chromosomes; S. bandanensis: NF = 66, 4m, 12sm, and 34a chromosomes (Fig. 1).

Figure 1.

Specimens, metaphase chromosome plates and karyotypes of Epalzeorhynchos frenatum (A–C), Puntigrus partipentazona (D–F), Scaphognathops bandanensis (G–I) by conventional technique.

Chromosome marker of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops bandanensis

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 they were revealed at telomeric regions of the long arm of the chromosome pair 9 (acrocentric) in S. bandanensis (Fig. 2A, D, G and Table 2).

Figure 2.

Karyotypes of Epalzeorhynchos frenatum (A–C), Puntigrus partipentazona (D–F), Scaphognathops bandanensis (G–I) by NOR banding and FISH techniques. Arrows indicate NOR-bearing chromosomes. Scale bars: 5 µm.

Table 2.

Cytogenetic and FISH studies on three Cypinid fishes in Thailand. (2n = diploid chromosome number, NF = fundamental number (number of chromosome arm), m = metacentric, sm = submetacentric, a = acrocentric, st = subtelocentric chromosomes, NOR = nucleolar organizer region).

Species 2n NF Chromosome type Ag-NOR pair (type) CA15 pair CGG10 pair
m sm st a
E. frenatum 50 84 18 10 10 12 10(sm) 1–13,15–25 1–6,9–12,14–25
P. partipentazona 50 94 6 24 14 6 1(m) 1–16, 18–21, 23–25 1
S. bandanensis 50 66 4 12 - 34 9(a) 1–25 1, 3–5,9–11, 13, 15–16, 19–21

Patterns of microsatellite repeats on the genome of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops 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. In addition, interstitial signals of d(CA)15 and d(CGG)10 repeats can be observed at the short arm of the chromosome pairs 3 and 4, respectively in E. frenatum (Fig. 2 and Table 2). Figure 3 shows the idiograms representing the patterns of d(CA)15 and d(CGG)10 microsatellites distributions on the chromosomes of three studied species. Microsatellite d(CGG)10 sequences were detected disperse hybridization signals with high accumulation of them at telomeric regions of several chromosomes in E. frenatum and S. bandanensis. However, it is interesting that the microsatellite d(CGG)10 repeats coincide with the NOR positions in P. partipentazona.

Figure 3.

Idiograms represent the (CA)15 and (CGG)10 mapping on the chromosomes of Epalzeorhynchos frenatum A Puntigrus partipentazona B Scaphognathops bandanensis C.

Discussion

Diploid number, fundamental number and karyotype of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops bandanensis

The diploid numbers (2n) are same as found in P. partipentazona (Taki et al. 1977) and S. bandanensis (Donsakul et al. 2007) but there is difference in E. frenatum (2n = 48) reported by Magtoon and Donsakul (1993). The 2n in three cypinids studied have the same 2n = 50 as in several species in the subfamily Cyprininae (Arai 2011, Table 1). It seems to be that this subfamily is highly conserved for the 2n. To compare with the previous studies, the NF of S. bandanensis is same as the study of Donsakul et al. (2007) whereas ones of E. frenatum and P. partipentazona differ from the reports of Magtoon and Donsakul (1993) and Taki et al. (1977), respectively. The differences of NFs have cause to differences of karyotypes among these fishes. These differences may be causes from the species-specific variations among populations, and/or misidentification of species or different species due to complex species. Three studied species cannot be observed heteromorphic sex chromosomes between male and female specimens. This phenomenon is same as many species in this family (Arai 2011).

Chromosome marker of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops bandanensis

The determination of nucleolar organizer regions (NORs) for these species was firstly proposed. If these loci are active during the interphase before to mitosis, they can be detected by silver nitrate staining (Howell and Black 1980) since they specifically stain a set of acidic proteins related to ribosomal synthesis process. The single NOR-bearing chromosome pair in the present result is consistent with results from Barbonymus gonionotus (Bleeker, 1849) (Khuda-Bukhsh and Das 2007), Hypsibarbus wetmorei (Smith, 1931) (Piyapong 1999), Osteochilus waandersi (Bleeker, 1853) (Magtoon and Arai 1993) and Puntius brevis (Bleeker, 1849) (Nitikulworawong and Khrueanet 2014). This character is common characteristic found in many fish groups as well as vertebrates (Supiwong et al. 2012, 2013). However, some species had two pairs (Cyclocheilos enoplos (Bleeker, 1849): Magtoon and Arai 1993), three pairs (Cyclocheilichthys apogon (Valenciennes, 1842): Chantapan 2015) and four pairs (Puntius denisonii (Day, 1865), P. semifasciolatus (Günther, 1868): Nagpure et al. 2004; P. filamentosus (Valenciennes, 1844): Nagpure et al. 2003). NORs are chromosomal landmarks that consist of tandemly repeated sequences of ribosomal genes (rRNA). In eukaryotes, each unit is composed of three genes coding for 18S, 5.8S and 28S ribosomal RNA (Sharma et al. 2002). The number and position of the rDNA clusters have been widely used in systematics and phylogenetic reconstructions since these chromosomal characters are often species-specific (Britton-Davidian et al. 2012). Changes in chromosome number and structure can alter the number, and structure of NOR. Structure, number, and morphology of a NOR may be specific to populations, species, and subspecies. Robertsonian translocations (centric fusion) may cause losses of NOR. Studies on NOR variation in numerous organism groups have invariably described changes in the number and location of NORs even in closely related species, suggesting that rDNA clusters are highly mobile components of the genome (Britton-Davidian et al. 2012). Thus, species, which have limited gene exchange due to geographical isolation, have elevated karyotype varieties and NOR variations. The use of NORs in explaining phylogenetic relationships depends on a large extent on the uniformity of this characteristic and on the degree of variety within a taxon (Yüksel and Gaffaroğlu 2008). Normally, most fishes have only one pair of small NORs in a chromosome complement. If some fishes have more than two NORs, it may be caused by the translocation between NOR and another chromosome (Sharma et al. 2002).

Patterns of microsatellite repeats in the genome of Epalzeorhynchos frenatum, Puntigrus partipentazona and Scaphognathops bandanensis

The patterns of microsatellite d(CA)15 in three species in the present study except in S. bandanensis are different from the nine species of the Bagridae family including Hemibagrus filamentus (Fang & Chaux, 1949), H. spilopterus Ng & Rainboth, 1999, H. wyckii (Bleeker, 1858), H. wyckioides Fang & Chaux, 1949, Mystus atrifasciatus Fowler, 1937, M. multiradiatus Roberts, 1992, M. mysticetus Roberts, 1992, M. bocourti (Bleeker, 1864), and Pseudomystus siamensis (Regan, 1913) (Supiwong et al. 2013, 2014), Toxotes chatareus (Hamilton, 1822) (Supiwong et al. 2017). From the previous and current studies, it may seem that all heterochromatins in fish genomes consist of microsatellites (Cioffi and Bertollo 2012). However, microsatellites have also been found in noncentromeric regions, many of them were located either near or within genes (Rao et al. 2010). This is the same as in the pattern of microsatellite d(CGG)10 revealed in S. bandanensis.

Conclusions

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.

Acknowledgments

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.

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