Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 71-80, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1523 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Surachest Aiumsumang, P a t c h a r a p o r n C h a i y a s a n , K a n Khoomsab, Weerayuth Supiwong, Alongklod Tanomtong Sumalee Phim- phan (2022) Comparative chromosome mapping of repetitive DNA in four min- now fishes (Cyprinidae, Cypriniformes). Caryologia 75(2): 71-80. doi: 10.36253/ caryologia-1523 Received: December 02, 2021 Accepted: May 20, 2022 Published: September 21, 2022 Copyright: © 2022 Surachest Aium- sumang, Patcharaporn Chaiyasan, Kan Khoomsab, Weerayuth Supi- wong, Alongklod Tanomtong Sumalee Phimphan. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) Surachest Aiumsumang1, Patcharaporn Chaiyasan2, Kan Khoomsab3, Weerayuth Supiwong4, Alongklod Tanomtong2 Sumalee Phimphan1,* 1 Biology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 2 Program of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 3 Education Science Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 4 Applied Science Program, Faculty of Interdisciplinary Studies, Khon Kaen University, Nong Khai Campus, Muang, Nong Khai 43000, Thailand *Corresponding author. E-mail: sumalee.phi@pcru.ac.th Abstract. The present study focused on the repetitive DNA of the chromosome in four minnow fishes from the genera Danio Hamilton, 1822, Devario Heckel, 1843 and Ras- bora Bleeker, 1859. Chromosomes were analysed using fluorescence  in situ  hybridiza- tion (FISH) with microsatellite probes including (CA)15, (CAC)10, (CGG)10, (GC)15 and (TA)15 staining. All species retained the diploid chromosome number 2n = 50 in male and female. The microsatellite sequences were mapped in the chromosomes of Danio albolineatus (Blyth, 1860), Devario regina (Fowler, 1934), Rasbora aurotaenia Tirant, 1885 and R. paviana Tirant, 1885. In most cases, the microsatellite was dispersed in the chromosome with conspicuous markings in the telomeric region and the whole genome, which suggests that sequences contribute to chromosome structure and may have played a role in the relationship of this fish group. The comparative genome map- ping data presented here provide novel information on the structure and organisa- tion of the repetitive DNA region of the minnow’s genome and contribute to a better understanding of the genomes of these minnows. Keywords: Cyprinidae, Danioninae, FISH, microsatellite. INTRODUCTION The level of molecular cytogenetics plays an important role in precise characterisation of the structure of fish genomes (Cioffi and Bertollo 2012). The family Cyprinidae is the most abundant and globally widespread fam- ily of freshwater fish, comprising 3,000 species are one of the largest groups (Eschmeyer and Fong 2015). Thailand is one of the important areas for fish fauna in terms of both diversity and endemicity. The total number of fish spe- 72 Surachest Aiumsumang et al. cies in Thai waters is over 2,700 with about 2,000 marine and 720 freshwater species (Vidthayanon 2005). Danio albolineatus (Blyth, 1860), Devario regina (Fowler, 1934), Rasbora aurotaenia Tirant, 1885 and R. paviana Tirant, 1885 are four of the species of minnows, belonging to the family Cyprinidae (subfamily Danioninae-Danionini). They are tropical freshwater fish of minor commercial importance, which are native in Thailand. Their distri- butions include the Mekong, Chao Phraya, and Meklong Basins (Froese and Pauly 2012) and they can be eas- ily found in large and small rivers, ponds, ditches, lakes, paddy field, and swamps. It rarely occurs in low oxygen waters (Brittan 1954; 1971; 1998). They could be used to assess if they were sensitive to change in environmental problems and aquatic pollution (Blazer 2002; Frame and Dickerson 2006; Raskovic et al. 2010; Yenchum 2010; Reddy and Rawat 2013). However, cytogenetic studies in minnows are quite scarce, in which only conventional technique reported to determine chromosome number and karyotype composition has been performed. Up to the present time, cytogenetic studies on sub- family Danioninae (Cyprinidae, Osteichthyes) with 20 genera and about 335 species have been undertaken for only 21 species from three genera (Danio, Devario and Rasbora) as yet, only conventional cytogenetics have been applied to determine chromosome numbers and karyotype complements. Diploid chromosomes number (2n) varies between 48-50. Cytogenetic studies of the genera Danio, Devario and Rasbora have been reported in Table 1. From the previous reports, in most cases, more descriptions of karyotypes seem to be inconclu- sive when they are not used in combination with other methods to produce more accurate chromosome mark- ers. More recently, molecular cytogenetics begun to be implemented in a finer-scale characterization of karyo- type structures in certain Cyprinid taxa (Spoz  et al. 2014; Saenjundaeng et al. 2018; Phimphan et al. 2020). More specifically, FISH-based repetitive DNA map- ping, multiple DNA copies of 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 sat- ellite DNAs; also, there are dispersed elements, such as transposons and retrotransposons, known as trans- posable elements (TEs) (Jurka et al. 2005). Repetitive DNA sequences display a high degree of polymorphism because of the variation in the number of repetitive units, which results from specific evolutionary dynam- ics. The taxonomic status by DNA genome as well as the phylogenetic relationships of Danioninae species is confirmed and well established based on previous study (Rüber et al., 2007; Tang et al., 2010). Recently, molecular cytogenetic studies, using fluo- rescence in situ hybridization (FISH) for mapping of repetitive DNA sequences, have provided important contributions to the characterisation of biodiversity and evolution of divergent fish groups (Cioffi and Ber- tollo 2012). Moreover, some microsatellite repeats are species-specific characters amongst some fish groups (Cioffi et al. 2015). An important role of repetitive DNAs in genome evolution has been reported for different fish groups (Cioffi and Bertollo 2012; Cioffi et al. 2010, 2015; Moraes et al. 2017; 2019; Sassi et al. 2019; Terencio et al. 2013; Yano et al. 2014). Thus, the present study is the report on chromo- somal characteristics of FISH mapping of the microsat- ellites repeats in Da. albolineatus, De. regina, R. aurotae- nia and R. paviana by using molecular cytogenetic pro- tocols. The knowledge gained can provide cytogenetic information potentially useful for further study in this family. MATHERIAL AND METHODS Individuals from both sexes of the four minnows were collected for analyses from river basins in, Thai- land (Table 2 and Fig. 1). The fishes were transferred to laboratory aquaria and kept under standard conditions for three days before the experiments. The procedures followed ethical protocols, as approved by the Ethics of Animal Experimentation of the National Research Council of Thailand U1-04498-2559. Preparation of fish chromosomes was from kidney cells (Phimphan et al. 2020). The chromosomes were stained with Giemsa’s Solution for 10 min. Metaphase figures were analysed according to the chromosome classification of Levan et al. (1964). Chromosomes were classified as metacentric (m), submetacentric (sm), subte- locentric (st) or acrocentric (a). The Fundamental num- ber, NF (number of chromosome arms) is obtained by assigning a value of two (2) to metacentric and submeta- centric chromosomes and one (1) to subtelocentric and acrocentric chromosomes. FISH was performed under stringent conditions on metaphase chromosome spreads with microsatel- lite (CA)15, (CAC)10, (CGG)10, (GC)15 and (TA)15 probes (Kubat et al. 2008; Liehr 2009) which were directly labelled with Cy3 at 5´terminal during synthesis by Sigma (St. Louis MO, USA). FISH, under stringent con- ditions on mitotic chromosome spreads (Pinkel et al. 1986), was carried out by previous protocols as report- ed by Supiwong et al. (2019) and Yano et al. (2017). The hybridzation signals were checked and analysed on an 73Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) Table 1. Reviews of cytogenetic reports in the genera Danio, Devario and Rasbora. (2n = diploid number, m = metacentric, sm = submeta- centric, st = subtelocentric, a = acrocentric, NORs = nucleolar organizer regions, NF = fundamental number, + = positive - = not available). Genus/Species 2n NF Karyotype formula FISH Reference Danio rerio 48 - - - Post (1965) 50 100 50m - Endo and Ingalls (1968) 50 100 10m + 40sm - Fontana et al. (1970) 50 100 10m + 12sm +28a - Rishi (1976) 50 100 16m + 32sm + 2a - Schreeb et al. (1993) 50 100 12m + 26sm + 12a - Pijnacker and Ferwerda (1995) 50 100 4m + 16sm + 30a - Daga et al. (1996) 50 100 4m + 16sm + 30a - Gornung et al. (1997) 50 100 F: 7m + 7sm + 36a M: 6m + 8sm + 36a - Sharma et al. (1998) 50 100 12m + 26sm + 12a - Ueda and Naoi (1999) 50 100 100 4m + 30sm + 16a or* 4m + 20sm + 26a - Amores and Postlethwait (1999) 50 100 4m + 16sm + 30a + Phillips and Reed (2000) 50 100 4m + 16sm + 30a + Sola and Gornung (2001) Da. Roseus 50 100 8m+34sm+8a - Kaewtip et al. (2021) Da. albolineatus 50 99 10m+39sm+1a - Arai (2011) 50 100 8m + 14sm + 28a + Present study Devario laoensis 50 50 100 96 6m+10sm+34a 6m+20sm+20a+4t - - Aiumsumang et al. (2021) Kaewtip et al. (2021) De. aequipinnatus 50 96 6m+34sm+6st+4t Sukham et al. (2013) De. regina 50 100 6m+12sm+32a - Aiumsumang et al. (2021) 50 100 6m+12sm+32a + Present study Rasbora agilis 50 100 24m+26sm - Donsakul et al. (2009) R. aurotaenia 50 92 14m+26sm+2a+8t - Seetapan and Moeikum (2004) 50 98 8m+16sm+24a+2t - Aiumsumang et al. (2012) 50 98 8m+16sm+24a+2t + Present study R. borapetensis 50 88 24m+14sm+12t - Donsakul et al. (2005) R. buchanani 50 100 30m+18sm+2a - Manna and Khuda-Bukhsh (1977) R. caudimaculata 50 98 20m+26sm+2a+2t - Donsakul and Magtoon (2002) R. daniconius 50 80 18m+6sm+6a+20t - Khuda-Bukhsh et al. (1979) 50 92 32m+8sm+2a+8t - Donsakul et al. (2005) R. dorsiocellata 50 92 18m+24sm+8t - Donsakul et al. (2009) R. einthovenii 50 94 6m+30sm+8a+6t - Donsakul et al. (2005) 50 100 16m+18sm+16a - Yeesaem et al. (2019) R. heteromorpha 48 - - - Post (1965) 48 74 14m+10sm+2a+22t - Donsakul et al. (2005) R. myersi 50 90 20m+14sm+6a+10t - Donsakul and Magtoon (2002) R. paviei 50 100 10m+24sm+16a - Donsakul and Magtoon (2002) R. paviana 50 98 8m+16sm+24a+2t - Aiumsumang et al. (2021) 50 98 8m+16sm+24a+2t + Present study R. retrodorsalis 50 88 26m+10sm+2a+12t - Donsakul and Magtoon (2002) R. rubrodorsalis 50 82 16m+16sm+18t - Donsakul et al. (2009) R. sumatrana 50 94 26m+16sm+2a+6t - Donsakul and Magtoon (1995) R. trilineata 48 - - - Post (1965) 50 94 26m+16sm+2a+6t - Donsakul et al. (2005) 74 Surachest Aiumsumang et al. epifluorescence microscope Olympus BX50 (Olympus Corporation, Ishikawa, Japan). RESULTS Diploid number and fundamental number of Da.  albolin- eatus, De. regina, R. aurotaenia and R. paviana The four minnow fishes have the same diploid num- ber of 2n = 50. Although the minnow fishes analysed share the same 2n, there are differences in the funda- mental number (NF) i.e. Da. albolineatus and De. regi- na NF = 100, while for the two Rasbora, NF = 98, The karyotype complements of D. albolineatus composed of 8m + 14sm + 28a, D. regina was m6+sm10+a34, while R. aurotaenia and R. paviana were 8m+16sm+24a+2t (Figs. 2A-D). Patterns of the microsatellite repeat in the genomes of Da. albolineatus, De. regina, R. aurotaenia and R. paviana The result of the mapping of the microsatellite repeats (CA)15 show that hybridization signals are abun- dantly distributed on telomeric regions in all species (Figs. 3E-H), (CAC)10 showing moderate abundance in Da. albolineatus and R. paviana, while De. regina and R. aurotaenia were not detected (Figs. 3I-L). The region hybridized (CGG)10 of Da. albolineatus and R. paviana identified a partial genome, De. regina has a hybridiza- tion pattern throughout the genomes and R. paviana was not detected (Figs. 3M-P). (GC)15 hybridized in Da. albolineatus and R. paviana as whole genomes, Da. albo- lineatus as telomeric regions and R. paviana was not detected (Figs. 3Q-T). The microsatellite (TA)15 probe showed hybridization on the whole genomes of Da. albolineatus and De. regina, while two Rabora were not detected (Figs. 3U-X) (Table 3). DISCUSSION Diploid number of and fundamental number Da. albolin- eatus, De. regina, R. aurotaenia and R. paviana Da. albolineatus had 2n = 50 which is in accordance with one single previous report (Arai 2011) and the same in other species in genus Danio (Post 1965; Endo and Ingalls 1968; Fontana et al. 1970; Rishi 1976; Schreeb et al. 1993; Pijnacker and Ferwerda 1995; Daga et al. 1996; Gornung et al. 1997; Sharma et al. 1998; Ueda and Naoi 1999; Amores and Postlethwait 1999; Phillips Table 2. Collection sites of the analyzed species show the sample number. Species Number of specimens in site sampling Mae Khong Basin Sirindhorn Peat Swamp Forest Pasak Basin Chi Basin Chao Phaya Basin Songkhram Basin Danio albolineatus - - 10 ♀ 10 ♂ 04 ♀ 05 ♂ 05 ♀ 05 ♂ - Devario regina 05 ♀ 06 ♂ 06 ♀ 08 ♂ - - - - Rasbora aurotaenia - - - - 08 ♀ 07 ♂ 05 ♀ 08 ♂ Rasbora paviana 05 ♀ 08 ♂ 03 ♀ 04 ♂ 05 ♀ 07 ♂ 04 ♀ 05 ♂ - - Figure 1. Map showing the collection sites of Danio albolineatus [red circles], Devario regina [blue circles], Rasbora aurotaenia [green circles] and Rasbora paviana [yellow circles] for studied herein. 75Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) and Reed 2000; Sola and Gornung 2001), Da. Roseus: 2n=50 (Kaewtip et al. 2021) and Da. albolineatus: 2n=50 (Arai 2011). The 2n of De. regina is the same as that of De. laoensis Pellegrin & Fang, 1940 (Aiumsumang et al. 2021; Kaewtip et al. 2021) and De. aequipinnatus report- ed by Sukham et al. (2013). R. aurotaenia has 2n=50 according to Seetapan and Moeikum (2004) and Aium- sumang et al. (2012). The diploid chromosome number of R. paviana was 50, which is the same as that from the previous study by Aiumsumang et al. (2021). This 2n is considered the same as for the other species of subfamily Danioninae (Danio, Devario and Rasbora). Patterns of microsatellite repeats in the genomes of Da. albolineatus, De. regina, R. aurotaenia and R. paviana The chromosomal distribution of repetitive DNA elements revealed remarkable differences amongst the analysed species. In this study, the number and dis- tribution of microsatellite sequence (CA)15, (CAC)10, (CGG)10, (GC)15 and (TA)15 were not conserved among four analyzed species. The microsatellite sequence (CA)15 was mapped on chromosomes of Da. albolineatus, De. regina, R. aurotaenia and R. paviana, and abundantly located and distributed in all chromosomes, usually in telomeric regions and a few chromosome pairs showed the strongest signal intensities, these not really being suited to serve as chromosomal markers. Microsatel- lites are usually located in the heterochromatic regions (telomeres/centromeres) of fish genomes, where a sig- nificant fraction of repetitive DNA is localized (Cioffi and Bertollo, 2012). Indeed, this distribution pattern is found in Epalzeorhynchos frenatum (Fowler, 1934), Pun- tigrus partipentazona (Fowler, 1934), Scaphognathops bandanensis Boonyaratpalin & Srirungroj, 1971 (Phim- phan et al, 2020); Catlocarpio siamensis Boulenger, 1898 and Probarbus jullieni Sauvage, 1880 (Saenjundaeng et Figure 2. Karyotype of Danio albolineatus [A], Devario regina [B], Rasbora aurotaenia [C], Rasbora paviana [D], m = metacentric, sm = submetacentric, a = acrocentric and t = telocentric chromosomes. Table 3. Molecular cytogenetic studies on four minnow fishes. [2n = diploid chromosome number, NF fundamental number (number of chromosome arm)]. Species 2n NF (CA)15 (CAC)10 (CGG)10 (GC)15 (TA)15 Da. albolineatus 50 100 telomere partial genome partial genome whole genome whole genome De. regina 50 100 telomere not detected whole genome telomere whole genome R. aurotaenia 50 98 telomere not detected not detected partial genome not detected R. paviana 50 98 telomere partial genome partial genome whole genome not detected 76 Surachest Aiumsumang et al. Figure 3. FISH using DAPI (A-D), [CA]15 (E-H), [CAC]10 (I-L), [CGG]10 (M-P), [GC]15 (Q-T), [TA]15 (U-Z) of Danio albolineatus, Devario regina, Rasbora aurotaenia and Rasbora paviana, respectively. Scale bar 5 µm. 77Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes) al. 2018); Clarias species (Maneechot et al. 2016) and Channa micropeltes (Cuvier, 1831) (Cioffi et al. 2015).This distribution pattern are differences known for Channa gachua (Hamilton, 1822), C. lucius (Cuvier, 1831), C. stri- ata (Bloch, 1793) (Cioffi et al. 2015), Hemibagrus wyckii (Bleeker, 1858) (Supiwong et al. 2017) and Monopterus albus (Zuiew, 1793) (Supiwong et al. 2019). For the map- ping of (CAC)10 repeats in Da. albolineatus and R. pavi- ana, partial genomes were identified, while De. regina and R. aurotaenia were not detected. In Da. albolineatus, De. regina, R. aurotaenia and R. paviana, most micros- atellites (CGG)10 and (CG)15 were abundantly distributed in all chromosomes with the exception of (CGG)10 in R. aurotaenia, this not being a signal probe. Nevertheless, comparative analyses among the species indicate that the microsatellites have also preferential zones of accu- mulation at telomeric heterochromatin. For De. regina the accumulation of microsatellites (GC)15 in all chro- mosomal pair could point out a species–specific type of heterochromatin is similar to previous reports in Chan- na micropeltes (Cioffi et al. 2015). In Da. albolineatus R. aurotaenia and R. paviana the same microsatellites are not only located at the telomeric region of some chromo- somes, but they are also found at the centromeres of the chromosome pair. (TA)15 repeats in Da. albolineatus and De. Regina, displaying high accumulations at the whole genome, while not detected in the Rasbora group. Repetitive DNA sequences display a high degree of polymorphism because of the variation in the number of repetitive units, which results from a specific evolu- tionary dynamic. Amongst these elements, microsatel- lites (or short tandem repeats) are the most polymorphic and consist of short sequences of one to six nucleotides repeated in tandem throughout the DNA (Tautz and Renz 1984). Due to their supposed neutral evolution, these molecular markers have been widely used in pop- ulation genetics, to identify taxonomic limits and in hybridization and forensic studies (Goldstein and Pol- lock 1997; Filcek et al. 2005; Racey et al. 2007; McCusker et al. 2008) and can be used to spot genomic evolution as previously been reported for different fish groups (Cioffi et al. 2010; Cioffi and Bertollo 2012; Terencio et al. 2013; Yano et al. 2014; Cioffi et al. 2015; Moraes et al. 2017; 2019; Sassi et al. 2019). The comparative study on four species showed that the diploid chromosome is the same, but the patterns of microsatellite repeat on chromosomes have differ- ences amongst them. Thus, the molecular cytogenetic data may be a tool for classification of fish species where there is similar morphology, such as the stripe. Overall, it is believed that the microsatellites have specific zones of accumulation in genomes, preferentially in hetero- chromatic regions (Supiwong et al. 2014). In fact, micro- satellites are located in the heterochromatic regions (telomeres, centromeres and in the sex chromosomes) of fish genomes (Cioffi and Bertollo 2012, including the present study). However, the distribution of microsatel- lites was not only restricted to heterochromatin, but also dispersed in euchromatic regions of the chromosomes (Getlekha et al. 2016). Additionally, such variability is also reinforced by the dynamism of repetitive elements in the genome, especially by the differential distribution and accumulation of rDNA sequences amongst chromo- somes (Cioffi et al. 2015). Although not yet completely understood, this marked diversity is likely linked to the lifestyle of these fishes and to population fragmentation, as already identified for other fish species. CONCLUSIONS Our study is the first one to offer reliable chromo- somal data for Da. albolineatus, De. regina, R. aurotae- nia and R. paviana by molecular cytogenetic protocols, this study were useful tools in highlighting the remarka- ble chromosomal diversification which was characterised in the four minnow fishes. Besides, data from compara- tive genomic hybridization experiments also highlighted an advanced stage of repetitive DNA divergence, evi- dencing their evolutionary diversification. ACKNOWLEDGMENTS This study was supported by the National Research Council of Thailand under the Phetchabun Rajabhat University (Grant No. FRB640052/04) and the Post- Doctoral Training Program from Research Affairs and Graduate School, Khon Kaen University, Thailand (Grant no. 59255). 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