Genome-wide SNP analysis of Siamese cobra (Naja kaouthia) reveals the molecular basis of transitions between Z and W sex chromosomes and supports the presence of an ancestral super-sex chromosome in amniotes.

Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to transposable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.

Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes.

Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion­deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.

Preserving Pure Siamese Crocodile Populations: A Comprehensive Approach Using Multi-Genetic Tools.

Hybrids between the critically endangered Siamese crocodile (Crocodylus siamensis) and least-concern saltwater crocodile (C. porosus) in captive populations represent a serious challenge for conservation and reintroduction programs due to the impact of anthropogenic activities. A previous study used microsatellite and mitochondrial DNA data to establish the criteria for identifying species and their hybrids; however, the results may have been influenced by biased allelic frequencies and genetic drift within the examined population. To overcome these limitations and identify the true signals of selection, alternative DNA markers and a diverse set of populations should be employed. Therefore, this study used DArT sequencing to identify genome-wide single nucleotide polymorphisms (SNPs) in both species and confirm the genetic scenario of the parental species and their hybrids. A population of saltwater crocodiles from Australia was used to compare the distribution of species-diagnostic SNPs. Different analytical approaches were compared to diagnose the level of hybridization when an admixture was present, wherein three individuals had potential backcrossing. Approximately 17.00-26.00% of loci were conserved between the Siamese and saltwater crocodile genomes. Species-diagnostic SNP loci for Siamese and saltwater crocodiles were identified as 8051 loci and 1288 loci, respectively. To validate the species-diagnostic SNP loci, a PCR-based approach was used by selecting 20 SNP loci for PCR primer design, among which 3 loci were successfully able to differentiate the actual species and different hybridization levels. Mitochondrial and nuclear genetic information, including microsatellite genotyping and species-diagnostic DNA markers, were combined as a novel method that can compensate for the limitations of each method. This method enables conservation prioritization before release into the wild, thereby ensuring sustainable genetic integrity for long-term species survival through reintroduction and management programs.

Concerted and Independent Evolution of Control Regions 1 and 2 of Water Monitor Lizards (Varanus salvator macromaculatus) and Different Phylogenetic Informative Markers.

Duplicate control regions (CRs) have been observed in the mitochondrial genomes (mitogenomes) of most varanids. Duplicate CRs have evolved in either concerted or independent evolution in vertebrates, but whether an evolutionary pattern exists in varanids remains unknown. Therefore, we conducted this study to analyze the evolutionary patterns and phylogenetic utilities of duplicate CRs in 72 individuals of Varanus salvator macromaculatus and other varanids. Sequence analyses and phylogenetic relationships revealed that divergence between orthologous copies from different individuals was lower than in paralogous copies from the same individual, suggesting an independent evolution of the two CRs. Distinct trees and recombination testing derived from CR1 and CR2 suggested that recombination events occurred between CRs during the evolutionary process. A comparison of substitution saturation showed the potential of CR2 as a phylogenetic marker. By contrast, duplicate CRs of the four examined varanids had similar sequences within species, suggesting typical characteristics of concerted evolution. The results provide a better understanding of the molecular evolutionary processes related to the mitogenomes of the varanid lineage.

Genome-Wide SNP Analysis of Hybrid Clariid Fish Reflects the Existence of Polygenic Sex-Determination in the Lineage.

The African catfish (Clarias gariepinus) may exhibit the co-existence of XX/XY and ZZ/ZW sex-determination systems (SDSs). However, the SDS of African catfish might be influenced by a polygenic sex-determination (PSD) system, comprising multiple independently segregating sex "switch" loci to determine sex within a species. Here, we aimed to detect the existence of PSD using hybrid. The hybrid produced by crossing male African catfish with female bighead catfish (C. macrocephalus, XX/XY) is a good animal model to study SDSs. Determining the SDS of hybrid catfish can help in understanding the interactions between these two complex SDS systems. Using the genotyping-by-sequencing "DART-seq" approach, we detected seven moderately male-linked loci and seventeen female-linked loci across all the examined hybrid specimens. Most of these loci were not sex-linked in the parental species, suggesting that the hybrid exhibits a combination of different alleles. Annotation of the identified sex-linked loci revealed the presence of one female-linked locus homologous with the B4GALNT1 gene, which is involved in the spermatogenesis pathway and hatchability. However, this locus was not sex-linked in the parental species, and the African catfish might also exhibit PSD.

Complete mitochondrial genome of Mekong fighting fish, Betta smaragdina (Teleostei: Osphronemidae).

Mekong fighting fish (Betta smaragdina) are found in Northeast Thailand. A complete mitochondrial genome (mitogenome) of B. smaragdina was assembled and annotated. Mitogenome sequences were 16,372 bp in length, with slight AT bias (59.8%), containing 37 genes with identical order to most teleost mitogenomes. Phylogenetic analysis of B. smaragdina showed closer relationship with B. splendens and B. mahachaiensis as the bubble-nesting group, compared to the mouthbrooder group (B. apollon, B. simplex, and B. pi). Results will allow the creation of a reference annotated genome that can be utilized to sustain biodiversity and eco-management of betta bioresources to improve conservation programs.

Mitochondrial genome of bronze-winged jacana (Metopidius indicus, Latham 1790).

We reported the mitochondrial genome (mitogenome) of bronze-winged jacana (Metopidius indicus, Latham 1790). The circular mitogenome was 17,208 base pairs (bp) in length, containing 13 protein-coding genes, two rRNAs, 22 tRNAs, and a non-coding control region. A DNA spacer 109 bp long was also detected between ND5 and Cytb. Phylogenetic analysis indicated that M. indicus was more closely related with the genera Himantopus, Jacana and Hydrophasianus. This annotated mitogenome reference can be utilized as a data resource for comparative mitogenomics of waders or shorebirds, with possible use in ecological and evolutionary studies.

High-Level Gene Flow Restricts Genetic Differentiation in Dairy Cattle Populations in Thailand: Insights from Large-Scale Mt D-Loop Sequencing.

Domestication and artificial selection lead to the development of genetically divergent cattle breeds or hybrids that exhibit specific patterns of genetic diversity and population structure. Recently developed mitochondrial markers have allowed investigation of cattle diversity worldwide; however, an extensive study on the population-level genetic diversity and demography of dairy cattle in Thailand is still needed. Mitochondrial D-loop sequences were obtained from 179 individuals (hybrids of Bos taurus and B. indicus) sampled from nine different provinces. Fifty-one haplotypes, of which most were classified in haplogroup "I", were found across all nine populations. All sampled populations showed severely reduced degrees of genetic differentiation, and low nucleotide diversity was observed in populations from central Thailand. Populations that originated from adjacent geographical areas tended to show high gene flow, as revealed by patterns of weak network structuring. Mismatch distribution analysis was suggestive of a stable population, with the recent occurrence of a slight expansion event. The results provide insights into the origins and the genetic relationships among local Thai cattle breeds and will be useful for guiding management of cattle breeding in Thailand.

Overview of the betta fish genome regarding species radiation, parental care, behavioral aggression, and pigmentation model relevant to humans.

BACKGROUND: The Siamese fighting fish (Betta splendens, also known as the betta) is well known in aquarium markets, and also presents an exciting new research model for studying parental care, aggressive behavior, and cryptically diverse pigmentation. However, concentrated efforts are required, both in the context of conservation biology and in its genetics, to address the problems of ongoing outbreeding depression, loss of biodiversity, and lack of scientific biological information. OBJECTIVE: The evolutionary dynamics of the betta must be better understood at the genomic scale in order to resolve the phylogenetic status of unrecognized species, develop molecular markers to study variation in traits, and identify interesting sets of genes encoding various bioresource functions. METHODS: The recent revolution in multi-omics approaches such as genomics, transcriptomics, epigenomics, and proteomics has uncovered genetic diversity and gained insights into many aspects of betta bioresources. RESULTS: Here, we present current research and future plans in an ongoing megaproject to characterize the betta genome as de novo assemblies, genes and repeat annotations, generating data to study diverse biological phenomena. We highlight key questions that require answers and propose new directions and recommendations to develop bioresource management to protect and enhance the betta genus. CONCLUSION: Successful accomplishment of these plans will allow the creation of a reference annotated genome and provide valuable information at the molecular level that can be utilized to sustain biodiversity and eco-management of the betta to improve breeding programs for future biomedical research.

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.

An Investigation of ZZ/ZW and XX/XY Sex Determination Systems in North African Catfish (Clarias gariepinus, ).

An investigation of sex-specific loci may provide important insights into fish sex determination strategies. This may be useful for biotechnological purposes, for example, to produce all-male or all-female fish for commercial breeding. The North African catfish species, Clarias gariepinus, has been widely adopted for aquaculture because its superior growth and disease resistance render the species suitable for hybridization with other catfish to improve the productivity and quality of fish meat. This species has either a ZZ/ZW or XX/XY sex determination system. Here, we investigate and characterize these systems using high-throughput genome complexity reduction sequencing as Diversity Arrays Technology. This approach was effective in identifying moderately sex-linked loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 30 perfectly sexed individuals of C. gariepinus. However, SNPs based markers were not found in this study. In total, 41 loci met the criteria for being moderately male-linked (with male vs. female ratios 80:20 and 70:30), while 25 loci were found to be moderately linked to female sex. No strictly male- or female-linked loci were detected. Seven moderately male-linked loci were partially homologous to some classes of transposable elements and three moderately male-linked loci were partially homologous to functional genes. Our data showed that the male heterogametic XX/XY sex determination system should co-exist with the ZZ/ZW system in C. gariepinus. Our finding of the co-existence of XX/XY and ZZ/ZW systems can be applied to benefit commercial breeding of this species in Thailand. This approach using moderately sex-linked loci provides a solid baseline for revealing sex determination mechanisms and identify potential sex determination regions in catfish, allowing further investigation of genetic improvements in breeding programs.

Complete mitochondrial genome of Mahachai betta, Betta mahachaiensis (Teleostei: Osphronemidae).

Mahachai bettas (Betta mahachaiensis) are distributed in areas of brackish water with Nipa Palms in Samut Sakhon, Thailand but urbanization is restricting their biodiversity. A complete mitochondrial genome (mitogenome) of B. mahachaiensis was determined to support conservation programs. Mitogenome sequences were 16,980 bp in length with slight AT bias (61.91%), containing 37 genes with identical order to most teleost mitogenomes. Phylogenetic analysis of B. mahachaiensis showed a closer relationship with B. splendens. Results will allow the creation of a reference annotated genome that can be utilized to sustain biodiversity and eco-management of the betta to improve conservation programs.

Next-generation sequencing yields complete mitochondrial genome assembly of peaceful betta fish, Betta imbellis (Teleostei: Osphronemidae).

The complete mitochondrial genome (mitogenome) of the peaceful betta (Betta imbellis) was obtained using next-generation sequencing. The sample of B. imbellis was collected from its native habitat in Southern Thailand. The mitogenome sequence was 16,897 bp in length, containing 37 genes with identical order to most teleost mitogenomes. Overall nucleotide base composition of the complete mitogenome was determined as AT bias. Phylogenetic analysis of B. imbellis showed a closer relationship with bubble-nesting fighting fish. This annotated mitogenome reference can be utilized as a bioresource for phylogenetic studies to support betta conservation programs.

Genome Complexity Reduction High-Throughput Genome Sequencing of Green Iguana (Iguana iguana) Reveal a Paradigm Shift in Understanding Sex-Chromosomal Linkages on Homomorphic X and Y Sex Chromosomes.

The majority of lizards classified in the superfamily Iguanoidea have an XX/XY sex-determination system in which sex-chromosomal linkage shows homology with chicken (Gallus gallus) chromosome 15 (GGA15). However, the genomics of sex chromosomes remain largely unexplored owing to the presence of homomorphic sex chromosomes in majority of the species. Recent advances in high-throughput genome complexity reduction sequencing provide an effective approach to the identification of sex-specific loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA), and a better understanding of sex chromosome dynamics in Iguanoidea. In this study, we applied Diversity Arrays Technology (DArTseqTM) in 29 phenotypic sex assignments (14 males and 15 females) of green iguana (Iguana iguana). We confirmed a male heterogametic (XX/XY) sex determination mode in this species, identifying 29 perfectly sex-linked SNP/PA loci and 164 moderately sex-linked SNP/PA loci, providing evidence probably indicative of XY recombination. Three loci from among the perfectly sex-linked SNP/PA loci showed partial homology with several amniote sex chromosomal linkages. The results support the hypothesis of an ancestral super-sex chromosome with overlaps of partial sex-chromosomal linkages. However, only one locus among the moderately sex-linked loci showed homology with GGA15, which suggests that the specific region homologous to GGA15 was located outside the non-recombination region but in close proximity to this region of the sex chromosome in green iguana. Therefore, the location of GGA15 might be further from the putative sex-determination locus in green iguana. This is a paradigm shift in understanding linkages on homomorphic X and Y sex chromosomes. The DArTseq platform provides an easy-to-use strategy for future research on the evolution of sex chromosomes in Iguanoidea, particularly for non-model species with homomorphic or highly cryptic sex chromosomes.

Existence of Bov-B LINE Retrotransposons in Snake Lineages Reveals Recent Multiple Horizontal Gene Transfers with Copy Number Variation.

Transposable elements (TEs) are dynamic elements present in all eukaryotic genomes. They can "jump" and amplify within the genome and promote segmental genome rearrangements on both autosomes and sex chromosomes by disruption of gene structures. The Bovine-B long interspersed nuclear element (Bov-B LINE) is among the most abundant TE-retrotransposon families in vertebrates due to horizontal transfer (HT) among vertebrate lineages. Recent studies have shown multiple HTs or the presence of diverse Bov-B LINE groups in the snake lineage. It is hypothesized that Bov-B LINEs are highly dynamic and that the diversity reflects multiple HTs in snake lineages. Partial sequences of Bov-B LINE from 23 snake species were characterized. Phylogenetic analysis resolved at least two Bov-B LINE groups that might correspond to henophidian and caenophidian snakes; however, the tree topology differed from that based on functional nuclear and mitochondrial gene sequences. Several Bov-B LINEs of snakes showed greater than 80% similarity to sequences obtained from insects, whereas the two Bov-B LINE groups as well as sequences from the same snake species classified in different Bov-B LINE groups showed sequence similarities of less than 80%. Calculation of estimated divergence time and pairwise divergence between all individual Bov-B LINE copies suggest invasion times ranging from 79.19 to 98.8 million years ago in snakes. Accumulation of elements in a lineage-specific fashion ranged from 9 × 10-6% to 5.63 × 10-2% per genome. The genomic proportion of Bov-B LINEs varied among snake species but was not directly associated with genome size or invasion time. No differentiation in Bov-B LINE copy number between males and females was observed in any of the snake species examined. Incongruence in tree topology between Bov-B LINEs and other snake phylogenies may reflect past HT events. Sequence divergence of Bov-B LINEs between copies suggests that recent multiple HTs occurred within the same evolutionary timeframe in the snake lineage. The proportion of Bov-B LINEs varies among species, reflecting species specificity in TE invasion. The rapid speciation of snakes, coinciding with Bov-B LINE invasion in snake genomes, leads us to better understand the effect of Bov-B LINEs on snake genome evolution.

Diversity of PBI-DdeI satellite DNA in snakes correlates with rapid independent evolution and different functional roles.

To better understand PBI-DdeI satellite DNA located in the centromeric region of python, molecular evolution analysis was conducted on 40 snake species. A ladder-like pattern of DNA bands with repetition of the 194-210 bp monomer was observed in 15 species using PCR. Molecular cloning was performed to obtain 97 AT-rich monomer sequences. Phylogenetic and network analyses showed three PBI-DdeI subfamilies with sequences grouped in species-specific clusters, suggesting rapid evolution. Slow evolution was found in eight species with shared PBI-DdeI sequences, suggesting recent species diversification, allowing PBI-DdeI no time to diverge, with limited homogenization and fixation processes. Quantitative real-time PCR showed large differences in copy number between Python bivittatus and other snakes, consistent with repeat scanning of whole genome sequences. Copy numbers were significantly higher in female Naja kaouthia than in males, concurring with chromosomal distribution of PBI-DdeI specifically localized to female W chromosomes. PBI-DdeI might act as an evolutionary driver with several repeats to promote W chromosome differentiation and heterochromatinization in N. kaouthia. Analysis revealed PBI-DdeI with a reduced copy number, compared to P. bivittatus, in most snakes studied, and it is possible that it subsequently dispersed and amplified on W chromosomes with different functional roles in N. kaouthia.

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes.

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female-specific 250-bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.

Assessment of snake DNA barcodes based on mitochondrial COI and Cytb genes revealed multiple putative cryptic species in Thailand.

DNA barcodes of mitochondrial cytochrome c oxidase I (COI), cytochrome b (Cytb) genes, and their combined data sets were constructed from 35 snake species in Thailand. No barcoding gap was detected in either of the two genes from the observed intra- and interspecific sequence divergences. Intra- and interspecific sequence divergences of the COI gene differed 14 times, with barcode cut-off scores ranging over 2%-4% for threshold values differentiated among most of the different species; the Cytb gene differed 6 times with cut-off scores ranging over 2%-6%. Thirty-five specific nucleotide mutations were also found at interspecific level in the COI gene, identifying 18 snake species, but no specific nucleotide mutation was observed for Cytb in any single species. This suggests that COI barcoding was a better marker than Cytb. Phylogenetic clustering analysis indicated that most species were represented by monophyletic clusters, suggesting that these snake species could be clearly differentiated using COI barcodes. However, the two-marker combination of both COI and Cytb was more effective, differentiating snake species by over 2%-4%, and reducing species numbers in the overlap value between intra- and interspecific divergences. Three species delimitation algorithms (general mixed Yule-coalescent, automatic barcoding gap detection, and statistical parsimony network analysis) were extensively applied to a wide range of snakes based on both barcodes. This revealed cryptic diversity for eleven snake species in Thailand. In addition, eleven accessions from the database previously grouped under the same species were represented at different species level, suggesting either high genetic diversity, or the misidentification of these sequences in the database as a consequence of cryptic species.