Disclosing the hidden nucleotide sequences: a journey into DNA barcoding of raptor species in public repositories.

BACKGROUND: In nucleotide public repositories, studies discovered data errors which resulted in incorrect species identification of several accipitrid raptors considered for conservation. Mislabeling, particularly in cases of cryptic species complexes and closely related species, which were identified based on morphological characteristics, was discovered. Prioritizing accurate species labeling, morphological taxonomy, and voucher documentation is crucial to rectify spurious data. OBJECTIVE: Our study aimed to identify an effective DNA barcoding tool that accurately reflects the efficiency status of barcodes in raptor species (Accipitridae). METHODS: Barcode sequences, including 889 sequences from the mitochondrial cytochrome c oxidase I (COI) gene and 1052 sequences from cytochrome b (Cytb), from 150 raptor species within the Accipitridae family were analyzed. RESULTS: The highest percentage of intraspecific nearest neighbors from the nearest neighbor test was 88.05% for COI and 95.00% for Cytb, suggesting that the Cytb gene is a more suitable marker for accurately identifying raptor species and can serve as a standard region for DNA barcoding. In both datasets, a positive barcoding gap representing the difference between inter-and intra-specific sequence divergences was observed. For COI and Cytb, the cut-off score sequence divergences for species identification were 4.00% and 3.00%, respectively. CONCLUSION: Greater accuracy was demonstrated for the Cytb gene, making it the preferred primary DNA barcoding marker for raptors.

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.

Genetic admixture and diversity in Thai domestic chickens revealed through analysis of Lao Pa Koi fighting cocks.

Lao Pa Koi (LPK) chicken is a popular fighting breed in Thailand, prized for (its unique characteristics acquired by selective breeding), and a valuable model for exploring the genetic diversity and admixture of red junglefowls and domestic chickens. In this study, genetic structure and diversity of LPK chicken were assessed using 28 microsatellite markers and mitochondrial DNA (mtDNA) D-loop sequences, and the findings were compared to a gene pool library from "The Siam Chicken Bioresource Project". High genetic variability was observed in LPK chickens using mtDNA D-loop haplotype analysis, and six haplotypes were identified. Microsatellite data revealed 182 alleles, with an average of 6.5 alleles per locus. These results confirmed the occurrence of genetic admixture of red junglefowl and Thai domestic chickens in LPK chicken breed. A maximum entropy modeling approach was used to analyze the spatial suitability and to assess the adaptive evolution of LPK chickens in diverse local environments. The model identified 82.52% of the area studied as unsuitable, and 9.34%, 7.11%, and 2.02% of the area indicated moderate, low, and high suitability, respectively. The highest contribution rate to land suitability for LPK chickens was found at an elevation of 100-250 m, suggesting the importance of elevation for their potential distribution. The results of this study provide valuable insights into the genetic origin of LPK chicken breed and identify resources for future genetic improvement.

Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species.

DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.

Environmental and Socio-Cultural Factors Impacting the Unique Gene Pool Pattern of Mae Hong-Son Chicken.

Understanding the genetic diversity of domestic chicken breeds under the impact of socio-cultural and ecological dynamics is vital for the conservation of natural resources. Mae Hong Son chicken is a local breed of North Thai domestic chicken widely distributed in Mae Hong Son Province, Thailand; however, its genetic characterization, origin, and diversity remain poorly understood. Here, we studied the socio-cultural, environmental, and genetic aspects of the Mae Hong Son chicken breed and investigated its diversity and allelic gene pool. We genotyped 28 microsatellite markers and analyzed mitochondrial D-loop sequencing data to evaluate genetic diversity and assessed spatial habitat suitability using maximum entropy modeling. Sequence diversity analysis revealed a total of 188 genotyped alleles, with overall nucleotide diversity of 0.014 ± 0.007, indicating that the Mae Hong Son chicken population is genetically highly diverse, with 35 (M1-M35) haplotypes clustered into haplogroups A, B, E, and F, mostly in the North ecotype. Allelic gene pool patterns showed a unique DNA fingerprint of the Mae Hong Son chicken, as compared to other breeds and red junglefowl. A genetic introgression of some parts of the gene pool of red junglefowl and other indigenous breeds was identified in the Mae Hong Son chicken, supporting the hypothesis of the origin of the Mae Hong Son chicken. During domestication in the past 200-300 years after the crossing of indigenous chickens and red junglefowl, the Mae Hong Son chicken has adapted to the highland environment and played a significant socio-cultural role in the Northern Thai community. The unique genetic fingerprint of the Mae Hong Son chicken, retaining a high level of genetic variability that includes a dynamic demographic and domestication history, as well as a range of ecological factors, might reshape the adaptation of this breed under selective pressure.

Should the Identification Guidelines for Siamese Crocodiles Be Revised? Differing Post-Occipital Scute Scale Numbers Show Phenotypic Variation Does Not Result from Hybridization with Saltwater Crocodiles.

Populations of Siamese crocodiles (Crocodylus siamensis) have severely declined because of hunting and habitat fragmentation, necessitating a reintroduction plan involving commercial captive-bred populations. However, hybridization between Siamese and saltwater crocodiles (C. porosus) has occurred in captivity. Siamese crocodiles commonly have post-occipital scutes (P.O.) with 4-6 scales, but 2-6 P.O. scales were found in captives on Thai farms. Here, the genetic diversity and population structure of Siamese crocodiles with large P.O. variations and saltwater crocodiles were analyzed using mitochondrial DNA D-loop and microsatellite genotyping. Possible crocodile hybrids or phenotypic variations were ascertained by comparison with our previous library from the Siam Crocodile Bioresource Project. Siamese crocodiles with <4 P.O. scales in a row exhibit normal species-level phenotypic variation. This evidence encourages the revised description of Siamese crocodiles. Moreover, the STRUCTURE plot revealed large distinct gene pools, suggesting crocodiles in each farm were derived from distinct lineages. However, combining both genetic approaches provides evidence of introgression for several individual crocodiles, suggesting possible hybridization between Siamese and saltwater crocodiles. We proposed a schematic protocol with patterns observed in phenotypic and molecular data to screen hybrids. Identifying non-hybrid and hybrid individuals is important for long-term in situ/ex situ conservation.

Coincidence of low genetic diversity and increasing population size in wild gaur populations in the Khao Phaeng Ma Non-Hunting Area, Thailand: A challenge for conservation management under human-wildlife conflict.

The gaur (Bos gaurus) is found throughout mainland South and Southeast Asia but is listed as an endangered species in Thailand with a decreasing population size and a reduction in suitable habitat. While gaur have shown a population recovery from 35 to 300 individuals within 30 years in the Khao Phaeng Ma (KPM) Non-Hunting Area, this has caused conflict with villagers along the border of the protected area. At the same time, the ecotourism potential of watching gaurs has boosted the local economy. In this study, 13 mitochondrial displacement-loop sequence samples taken from gaur with GPS collars were analyzed. Three haplotypes identified in the population were defined by only two parsimony informative sites (from 9 mutational steps of nucleotide difference). One haplotype was shared among eleven individuals located in different subpopulations/herds, suggesting very low genetic diversity with few maternal lineages in the founder population. Based on the current small number of sequences, neutrality and demographic expansion test results also showed that the population was likely to contract in the near future. These findings provide insight into the genetic diversity and demography of the wild gaur population in the KPM protected area that can inform long-term sustainable management action plans.

Population Scale Analysis of Centromeric Satellite DNA Reveals Highly Dynamic Evolutionary Patterns and Genomic Organization in Long-Tailed and Rhesus Macaques.

Centromeric satellite DNA (cen-satDNA) consists of highly divergent repeat monomers, each approximately 171 base pairs in length. Here, we investigated the genetic diversity in the centromeric region of two primate species: long-tailed (Macaca fascicularis) and rhesus (Macaca mulatta) macaques. Fluorescence in situ hybridization and bioinformatic analysis showed the chromosome-specific organization and dynamic nature of cen-satDNAsequences, and their substantial diversity, with distinct subfamilies across macaque populations, suggesting increased turnovers. Comparative genomics identified high level polymorphisms spanning a 120 bp deletion region and a remarkable interspecific variability in cen-satDNA size and structure. Population structure analysis detected admixture patterns within populations, indicating their high divergence and rapid evolution. However, differences in cen-satDNA profiles appear to not be involved in hybrid incompatibility between the two species. Our study provides a genomic landscape of centromeric repeats in wild macaques and opens new avenues for exploring their impact on the adaptive evolution and speciation of primates.

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.

Reduced genetic variability in a captive-bred population of the endangered Hume's pheasant (Syrmaticus humiae, Hume 1881) revealed by microsatellite genotyping and D-loop sequencing.

Captive breeding programs are crucial to ensure the survival of endangered species and ultimately to reintroduce individuals into the wild. However, captive-bred populations can also deteriorate due to inbreeding depression and reduction of genetic variability. We genotyped a captive population of 82 individuals of the endangered Hume's pheasant (Syrmaticus humiae, Hume 1881) at the Doi Tung Wildlife Breeding Center to assess the genetic consequences associated with captive breeding. Analysis of microsatellite loci and mitochondrial D-loop sequences reveal significantly reduced genetic differentiation and a shallow population structure. Despite the low genetic variability, no bottleneck was observed but 12 microsatellite loci were informative in reflecting probable inbreeding. These findings provide a valuable source of knowledge to maximize genetic variability and enhance the success of future conservation plans for captive and wild populations of Hume's pheasant.

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.

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.

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.

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.