Research report: Microsatellite repeat mapping shows inner chromosomal diversification in highly conserved karyotypes of Asian cyprinid fishes

The research focused on the subfamilies Poropuntiinae and Smiliogastrinae within the family Cyprinidae, the most species-rich group of freshwater fishes on Earth. Thailand itself hosts roughly ten percent of the world's freshwater fish species, with at least 858 species identified across 81 families, making it a worldwide hub for aquatic diversity. Despite this abundance, the cytogenetic data for many Thai cyprinids remain sparse, and the new study set out to close that gap by combining conventional chromosome staining with modern molecular cytogenetic techniques.

Live individuals were transported to laboratory aquaria and kept at temperatures between twenty and twenty-eight degrees Celsius until analysis. All procedures were approved by the Institutional Animal Care and Use Committee of Khon Kaen University under the Ethics of Animal Experimentation of the National Research Council of Thailand, and voucher specimens were deposited in the Vertebrate Cytogenetics Laboratory at the university. Conventional cytogenetic analysis using Giemsa staining confirmed that every species possessed a diploid chromosome number of 2n = 50, the predominant and plesiomorphic pattern for the cyprinoid lineage.

However, the fundamental number, which counts the total number of chromosome arms per karyotype, proved unique to each species, ranging from 88 in Sikukia stejnegeri to 100 in Desmopuntius hexazona and Pethia stoliczkana. Chromosomes were classified as metacentric, submetacentric, subtelocentric, or acrocentric according to the centromere position, and the karyotypic composition varied considerably among species even though the overall chromosome count remained fixed. Notably, none of the fifteen species examined showed heteromorphic sex chromosomes in either males or females, consistent with the rarity of differentiated sex chromosomes across the entire family.

The molecular cytogenetic component of the study employed fluorescent in situ hybridization, or FISH, to physically map four distinct microsatellite motifs directly labeled with the fluorescent dye Cy3: (CA)15, (TA)15, (CAC)10, and (CGG)10. Microsatellites are short repetitive DNA sequences of one to six nucleotides found in the genomes of all eukaryotic species, and their chromosomal distribution can range from dispersed patterns across entire chromosomes to discrete banding patterns concentrated in specific regions. Because microsatellites are generally considered to evolve neutrally, their frequency and distribution are thought to reflect the underlying mutation process, making them valuable markers for studying chromosomal organization and evolution.

Variations in their patterns can further indicate chromosomal rearrangements such as inversions or translocations, which are important in understanding speciation and evolutionary processes. The FISH results revealed striking species-specific distribution patterns. Fourteen of the fifteen species exhibited a very similar distribution of the (CA)15 motif, which hybridized robustly and selectively in the telomeric regions of all chromosomes.

The single exception was Desmopuntius hexazona, in which the (CA)15 signal was evenly spread across the telomeric regions of all chromosomes but only one chromosome pair displayed a prominent signal. The (TA)15 probe produced no positive hybridization signals at all in Amblyrhynchichthys micracanthus, Poropuntius laoensis, Hampala dispar, and Hampala macrolepidota, while both the (CAC)10 and (CGG)10 probes failed to produce signals in Barbonymus schwanenfeldii. In other species, the same motifs were detected in telomeric regions, spread widely across entire chromosomes, or restricted to a few specific chromosome pairs.

These variations in microsatellite distribution reflect the evolutionary divergence and chromosomal diversity within the cyprinids. The comparative patterns suggest that the (CAC)10 and (CGG)10 repeats could serve as markers specific to the genus Barbonymus, while the (TA)15 repeats could serve as a marker distinct to the genus Cyclocheilichthys, and the (TA)15 and (CAC)10 repeats are appropriate as species-specific identifiers for the genus Hampala. The findings carry broader implications for understanding genome evolution in fishes.

The buildup of repetitive DNA is recognized as a main driver of karyotype diversification associated with speciation, and repetitive DNA mapping, particularly of microsatellites and ribosomal DNA classes, can be used to study and estimate evolutionary karyotype changes in fish species. Recent research has also identified functional microsatellites that influence an individual's physical attributes, and microsatellite sequences are known to be involved in chromosome organization, DNA recombination and replication, and gene expression. The distribution of microsatellite motifs in fish genomes may exhibit a bias toward specific noncoding regions but may also correlate with the distribution of ribosomal DNAs across chromosomes, adding another layer of complexity to the interpretation of these patterns.

While the diploid chromosome number of 2n = 50 remains remarkably stable across the cyprinoid lineages, the fundamental number and karyotypic structure vary widely, indicating that intrachromosomal evolutionary diversity has proceeded independently of gross chromosome count. The evolution of karyotypes in Cyprinidae is marked by multiple independent polyploidization events across various species, which have contributed significantly to the observed variation in chromosome numbers, ranging from 42 in Acheilognathus gracilis to 446 in Diptychus dipogon. The research applied both conventional and molecular cytogenetics to examine the karyotypic organization and microsatellite distribution in fifteen fish species from the two subfamilies, and the results indicate that microsatellites can be valuable genetic markers that differentiate within genera with similar morphology.

The comparative cytogenetic mapping results not only enhance comprehension of the genome of this fish family but also provide novel insights into the structure and organization of the repetitive DNA region in Systomini genomes.

Subject of Research: Microsatellite repeat mapping and karyotype analysis of Asian cyprinid fishes

Article Title: Microsatellite repeat mapping shows inner chromosomal diversification in highly conserved karyotypes of Asian cyprinid fishes

Article References: Buasriyot, P., Supiwong, W., Muanglen, N., Donbundit, N., Ditcharoen, S., Chumpol, P., Saenjundaeng, P., Tongnunui, S., Arunsang, S., Thongnetr, W., Juntharat, S., Seetapan, K., Liehr, T., Cioffi, M. B., Rab, P., & Tanomtong, A. (2025). Microsatellite repeat mapping shows inner chromosomal diversification in highly conserved karyotypes of Asian cyprinid fishes. Comparative Cytogenetics, 19, 29-50. https://doi.org/10.3897/compcytogen.19.141557

Image Credits: AI Generated

DOI: 10.3897/compcytogen.19.141557

Keywords: cyprinid fishes, microsatellites, karyotype, fluorescent in situ hybridization, chromosomal evolution, repetitive DNA, telomeres, Poropuntiinae, Smiliogastrinae, Thailand freshwater fishes, Comparative Cytogenetics, genome evolution