Distribution and amplification of interstitial telomeric sequences (ITSs) in Australian dragon lizards support frequent chromosome fusions in Iguania

Telomeric sequences are generally located at the ends of chromosomes; however, they can also be found in non-terminal chromosomal regions when they are known as interstitial telomeric sequences (ITSs). Distribution of ITSs across closely related and divergent species elucidates karyotype evolution and speciation as ITSs provide evolutionary evidence for chromosome fusion. In this study, we performed physical mapping of telomeric repeats by fluorescence in situ hybridisation (FISH) in seven Australian dragon lizards thought to represent derived karyotypes of squamate reptiles and a gecko lizard with considerably different karyotypic feature. Telomeric repeats were present at both ends of all chromosomes in all species, while varying numbers of ITSs were also found on microchromosomes and in pericentromeric or centromeric regions on macrochromosomes in five lizard species examined. This suggests that chromosomal rearrangements from ancestral squamate reptiles to Iguania occurred mainly by fusion between ancestral types of acrocentric chromosomes and/or between microchromosomes, leading to appearance of bi-armed macrochromosomes, and in the reduction of microchromosome numbers. These results support the previously proposed hypothesis of karyotype evolution in squamate reptiles. In addition, we observed the presence of telomeric sequences in the similar regions to heterochromatin of the W microchromosome in Pogona barbata and Doporiphora nobbi, while sex chromosomes for the two species contained part of the nucleolar organiser regions (NORs). This likely implies that these ITSs are a part of the satellite DNA and not relics of chromosome fusions. Amplification of telomeric repeats may have involved heterochromatinisation of sex-specific W chromosomes and play a role in the organisation of the nucleolus

ZW Sex Chromosomes in Australian Dragon Lizards (Agamidae) Originated from a Combination of Duplication and Translocation in the Nucleolar Organising Region

Sex chromosomes in some reptiles share synteny with distantly related amniotes in regions orthologous to squamate chromosome 2. The latter finding suggests that chromosome 2 was formerly part of a larger ancestral (amniote) super-sex chromosome and raises questions about how sex chromosomes are formed and modified in reptiles. Australian dragon lizards (Agamidae) are emerging as an excellent model for studying these processes. In particular, they exhibit both genotypic (GSD) and temperature-dependent (TSD) sex determination, show evidence of transitions between the two modes and have evolved non-homologous ZW sex microchromosomes even within the same evolutionary lineage. They therefore represent an excellent group to probe further the idea of a shared ancestral super-sex chromosome and to investigate mechanisms for transition between different sex chromosome forms. Here, we compare sex chromosome homology among eight dragon lizard species from five genera to identify key cytological differences and the mechanisms that may be driving sex chromosome evolution in this group. We performed fluorescence in situ hybridisation to physically map bacterial artificial chromosome (BAC) clones from the bearded dragon, Pogona vitticeps’ ZW sex chromosomes and a nucleolar organising region (NOR) probe in males and females of eight Agamid species exhibiting either GSD or TSD. We show that the sex chromosome derived BAC clone hybridises near the telomere of chromosome 2q in all eight species examined. This clone also hybridises to the sex microchromosomes of three species (P vitticeps, P. barbata and Diporiphora nobbi) and a pair of microchromosomes in three others (Ctenophorus pictus, Amphibolurus norrisi and Amphibolurus muricatus). No other chromosomes are marked by the probe in two species from the closely related genus Physignathus. A probe bearing nucleolar organising region (NOR) sequences maps close to the telomere of chromosome 2q in all eight species, and to the ZW pair in P. vitticeps and P. barbata, the W microchromosome in D. nobbi, and several microchromosomes in P. cocincinus. Our findings provide evidence of sequence homology between chromosome 2 and the sex chromosomes of multiple agamids. These data support the hypothesis that there was an ancestral sex chromosome in amniotes that gave rise to squamate chromosome 2 and raises the prospect that some particular property of this chromosome has favoured its role as a sex chromosome in amniotes. It is likely that the amplification of repetitive sequences associated with this region has driven the high level of heterochromatinisation of the sex-specific chromosomes in three species of agamid. Our data suggest a possible mechanism for chromosome rearrangement, including inversion and duplication near the telomeric regions of the ancestral chromosome 2 and subsequent translocation to the ZW sex microchromosomes in three agamid species. It is plausible that these chromosome rearrangements involving sex chromosomes also drove speciation in this group

Identification of native Dendrobium species in Thailand by PCR-RFLP of rDNA-ITS and chloroplast DNA

ABSTRACT: The PCR-restriction fragment length polymorphism (PCR-RFLP) approach was successfully developed to identify 25 native Dendrobium species in Thailand. PCR-RFLP of the rDNA-ITS with six restriction enzymes and three chloroplast (cp) DNA regions with five primer-enzyme combinations produced 24 types of DNA patterns altogether. Twenty-three out of the 25 species determined in this study were found to belong to unique classes and were successfully differentiated. Two species, D. crumenatum and D. formosum, possessing the same DNA pattern, however, were identified after cutting the chloroplast DNA fragment amplified by psbC-trnS primer with MboI enzyme. An effective procedure for identifying each Dendrobium species was developed. PCR-RFLP of the rDNA-ITS with TaqI, which is the most informative enzyme, was used for the early detection of 16 Dendrobium species. To identify the remaining Dendrobium species, PCR-RFLP analysis was performed using one more primer-enzyme combination. Our study provides a rapid, simple, and reliable identification method for these Dendrobium species

Chromosomal localization of the 18S-28S and 5S rRNA genes and (TTAGGG)n sequences of butterfly lizards (Leiolepis belliana belliana and Leiolepis boehmei, Agamidae, Squamata)

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution

Correction: Insights into avian molecular cytogenetics — with reptilian comparisons

Correction: Molecular Cytogenetics (2024) 17:24 https://doi.org/10.1186/s13039-024-00696-y The original article contained numerous minor typesetting mistakes which were mistakenly carried forward by the production team which handled the manuscript. Each error has since been amended

Insights into avian molecular cytogenetics—with reptilian comparisons

In last 100 years or so, much information has been accumulated on avian karyology, genetics, physiology, biochemistry and evolution. The chicken genome project generated genomic resources used in comparative studies, elucidating fundamental evolutionary processes, much of it funded by the economic importance of domestic fowl (which are also excellent model species in many areas). Studying karyotypes and whole genome sequences revealed population processes, evolutionary biology, and genome function, uncovering the role of repetitive sequences, transposable elements and gene family expansion. Knowledge of the function of many genes and non-expressed or identified regulatory components is however still lacking. Birds (Aves) are diverse, have striking adaptations for flight, migration and survival and inhabit all continents most islands. They also have a unique karyotype with ~ 10 macrochromosomes and ~ 30 microchromosomes that are smaller than other reptiles. Classified into Palaeognathae and Neognathae they are evolutionarily close, and a subset of reptiles. Here we overview avian molecular cytogenetics with reptilian comparisons, shedding light on their karyotypes and genome structure features. We consider avian evolution, then avian (followed by reptilian) karyotypes and genomic features. We consider synteny disruptions, centromere repositioning, and repetitive elements before turning to comparative avian and reptilian genomics. In this context, we review comparative cytogenetics and genome mapping in birds as well as Z- and W-chromosomes and sex determination. Finally, we give examples of pivotal research areas in avian and reptilian cytogenomics, particularly physical mapping and map integration of sex chromosomal genes, comparative genomics of chicken, turkey and zebra finch, California condor cytogenomics as well as some peculiar cytogenetic and evolutionary examples. We conclude that comparative molecular studies and improving resources continually contribute to new approaches in population biology, developmental biology, physiology, disease ecology, systematics, evolution and phylogenetic systematics orientation. This also produces genetic mapping information for chromosomes active in rearrangements during the course of evolution. Further insights into mutation, selection and adaptation of vertebrate genomes will benefit from these studies including physical and online resources for the further elaboration of comparative genomics approaches for many fundamental biological questions

Snake W Sex Chromosome: The Shadow of Ancestral Amniote Super-Sex Chromosome

Heteromorphic sex chromosomes, particularly the ZZ/ZW sex chromosome system of birds and some reptiles, undergo evolutionary dynamics distinct from those of autosomes. The W sex chromosome is a unique karyological member of this heteromorphic pair, which has been extensively studied in snakes to explore the origin, evolution, and genetic diversity of amniote sex chromosomes. The snake W sex chromosome offers a fascinating model system to elucidate ancestral trajectories that have resulted in genetic divergence of amniote sex chromosomes. Although the principal mechanism driving evolution of the amniote sex chromosome remains obscure, an emerging hypothesis, supported by studies of W sex chromosomes of squamate reptiles and snakes, suggests that sex chromosomes share varied genomic blocks across several amniote lineages. This implies the possible split of an ancestral super-sex chromosome via chromosomal rearrangements. We review the major findings pertaining to sex chromosomal profiles in amniotes and discuss the evolution of an ancestral super-sex chromosome by collating recent evidence sourced mainly from the snake W sex chromosome analysis. We highlight the role of repeat-mediated sex chromosome conformation and present a genomic landscape of snake Z and W chromosomes, which reveals the relative abundance of major repeats, and identifies the expansion of certain transposable elements. The latest revolution in chromosomics, i.e., complete telomere-to-telomere assembly, offers mechanistic insights into the evolutionary origin of sex chromosomes

The application of microsatellite markers as molecular tools for studying genomic variability in vertebrate populations

Vertebrate molecular genetic research methods typically employ single genetic loci (monolocus markers) and those involving a variable number of loci (multilocus markers). The former often employ microsatellites that ensure accuracy in establishing inbreeding, tracking pan-generational dynamics of genetic parameters, assessing genetic purity, and facilitating genotype/phenotype correlations. They also enable the determination and identification of unique alleles by studying and managing marker-assisted breeding regimes to control the artificial selection of agriculturally important traits. Microsatellites consist of 2–6 nucleotides that repeat numerous times and are widely distributed throughout genomes. Their main advantages lie in their ease of use for PCR amplification, their known genome localization, and their incredible polymorphism (variability) levels. Robust lab-based molecular technologies are supplemented by high-quality statistics and bioinformatics and have been widely employed, especially in those instances when more costly, high throughput techniques are not available. Here, we consider that human and livestock microsatellite studies have been a “roadmap” for the genetics, breeding, and conservation of wildlife and rare animal breeds. In this context, we examine humans and other primates, cattle and other artiodactyls, chickens and other birds, carnivores (cats and dogs), elephants, reptiles, amphibians, and fish. Studies originally designed for mass animal production have thus been adapted to save less abundant species, highlighting the need for molecular scientists to consider where research may be applied in different disciplines

Avian cytogenomics: Small chromosomes, long evolutionary history

This review considers fundamental issues related to the genomics of birds (Aves), including the special organization and evolution of their chromosomes. In particular, we address the capabilities of molecular genetic/genomic approaches to clarify aspects of their evolutionary history, including how they have adapted to multiple habitats. We contemplate general genomic organization, including the small size and typical number of micro/macrochromosomes. We discuss recent genome sequencing efforts and how this relates to cytogenomic studies. We consider the emergence of this unique organization ~245 million years ago, examining examples where the “norm” is not followed. We address the functional role of synteny disruptions, centromere repositioning, repetitive elements, evolutionary breakpoints, synteny blocks and the role of the unique ZW sex chromosome system. By analyzing the cytogenetic maps and chromosomal rearrangements of eight species, the possibility of successfully applying modern genomic methods/technologies to identify general and specific features of genomic organization and an in-depth understanding of the fundamental patterns of the evolution of avian genomes are demonstrated. An interpretation of the observed genomic “variadicity” and specific chromosomal rearrangements is subsequently proposed. We also present a mathematical assessment of cross-species bacterial artificial chromosome (BAC) hybridization during genomic mapping in the white-throated sparrow, a species considered a key model of avian behavior. Building on model species (e.g., chicken), avian cytogenomics now encompasses hundreds of genomes across nearly all families, revealing remarkable genomic conservation with many dynamic aspects. Combining classical cytogenetics, high-throughput sequencing and emerging technologies provides increasingly detailed insights into the structure, function and evolutionary organization of these remarkable genomes

Saving the Mahachai Betta: Genetic erosion and conservation priorities under urbanization pressure

Background/Objectives: Mahachai Betta (Betta mahachaiensis) is a bubble-nesting fighting fish endemic to brackish habitats in Bangkok, Samut Sakhon, and Samut Prakan, where rapid urbanization and industrial growth threaten persistence. We evaluated genetic structure and diversity across 10 populations (81 individuals) to inform conservation planning. Methods: This study combined microsatellite genotyping (13 loci) with ecological niche modeling to assess genetic variability, population connectivity, and landscape–environmental drivers of differentiation. Results: Habitat loss and fragmentation were associated with reduced gene flow and decreased genetic diversity. Mean allelic richness was 2.65 and expected heterozygosity ranged from 0.20 to 0.46, with FST values up to 0.400. Forward simulations predicted severe erosion of diversity within the next 12.5–37.5 years. Populations showed clear genetic subdivision, most pronounced in Samut Prakan and Samut Sakhon, with two Samut Sakhon populations (SKN3 and SKN7) reflecting strong environmental heterogeneity. Conclusions: Improving habitat connectivity and intensifying local community engagement are priority actions to enhance the resilience and long-term persistence of Mahachai Betta. This study provides the first integrated genetic and landscape-based assessment of the species, highlighting its rapid genetic erosion under urbanization and offering a foundation for targeted, evidence-based conservation strategies