A rapid and simple procedure for the whole-mount bone staining of small fish.

The cartilage and bone structure has provided insightful knowledge about evolution and ecology of fish, which is an important component of biological oceanography. However, the whole-body bone staining is a lengthy and complicated process that typically takes five days to several months, and the improvement of the conventional method has been one of the important issues in this field. Here we report a quick and easy whole-mount bone staining method for small fish, in which a newly designed fixative is applied. Compared to conventional methods, this novel protocol is a straightforward process that could be adopted for small estuarine fish and other small vertebrates.

Histological study of seventeen organs from dugong (Dugong dugon).

Background: Dugongs are marine mammals with a crescent-shaped tail fluke and a concave trailing margin that belong to the family Dugongidae., They are distributed widely in the warm coastal waters of the Indo-Pacific region. Importantly, the population of dugongs has decreased over the past decades as they have been classified as rare marine mammals. Previous studies have investigated the habitat and genetic diversity of dugongs. However, a comprehensive histological investigation of their tissue has not yet been conducted. This study provides unique insight into the organs of dugongs and compares them with other mammal species. Methods: Tissue sections were stained with Harris's hematoxylin and eosin Y. The histological structure of 17 organ tissues obtained from eight systems was included in this study. Tissue sections were obtained from the urinary system (kidney), muscular system (striated skeletal muscle and smooth muscle), cardiovascular system (cardiac muscle (ventricle), coronary artery, and coronary vein), respiratory system (trachea and lung), gastrointestinal system (esophagus, stomach, small intestine, liver, and pancreas), reproductive system (testis), lymphatic system (spleen and thymus), and endocrine system (pancreas). Results: While most structures were similar to those of other mammal species, there were some differences in the tissue sections of dugongs when compared with other mammalian species and manatees. These include the kidneys of dugongs, which were non-lobular and had a smooth, elongated exterior resulting in a long medullary crest, whereas the dugong pyloric epithelium did not have overlying stratified squamous cells and was noticably different from the Florida manatee. Discussion: Histological information obtained from various organs of the dugong can serve as an essential foundation of basal data for future microanatomical studies. This information can also be used as high-value data in the diagnosis and pathogenesis of sick dugongs or those with an unknown cause of death.

An Overview of Pulses Production in India: Retrospect and Prospects of the Future Food with an Application of Hybrid Models.

Forecasts are valuable to countries to make informed business decisions and develop data-driven strategies. The production of pulses is an integral part of agricultural diversification initiatives because it offers promising economic opportunities to reduce rural poverty and unemployment in developing countries. Pulses are the cheapest source of protein needed for human health. India's pulses production guidelines must be based on accurate and best forecast models. Comparing classical statistical and machine learning models based on different scientific data series is the subject of high-level research today. This study focused on the forecasting behaviour of pulses production for India, Karnataka, Madhya Pradesh, Maharashtra, Rajasthan and Uttar Pradesh. The data series was split into a training dataset (1950-2014) and a testing dataset (2015-2019) for model building and validation purposes, respectively. ARIMA, NNAR and hybrid models were used and compared on training and validation datasets based on goodness of fit (RMSE, MAE and MASE). This research demonstrates that due to the diverse agricultural conditions across different provinces in India, there is no single model that can accurately predict pulse production in all regions. This study's highest accuracy model is ARIMA. ARIMA outperforms NNAR, a machine learning model. Pulse production in India, Rajasthan, and Madhya Pradesh will expand by 26.11%, 12.62%, and 0.51% from 2020 to 2030, whereas it would decline by - 6.5%, - 6.21%, and - 6.76 per cent in Karnataka, Maharashtra, and Uttar Pradesh, respectively. The current forecast results could allow policymakers to develop more aggressive food security and sustainability plans and better Indian pulses production policies in the future.

RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.

Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.

Herbal species authentication by melting fingerprint coupled with high resolution melting analysis (MF-HRM).

BACKGROUND: Herbal medicines have recently attracted increasing attention for use as food supplements with health benefits; however, species authentication can be difficult due to incomplete morphological characters. Here, a molecular tool was developed for the identification of species in the National List of Essential Medicinal Plants in Thailand. METHODS: The identification process used DNA fingerprints including start codon targeted (SCoT) and inter simple sequence repeat (ISSR) polymorphisms, coupled with high resolution melting (HRM), to produce melting fingerprint (MF)-HRM. RESULTS: Results indicated that MF-HRM, SCoT-HRM and ISSR-HRM could be used for DNA fingerprints as S34, S36, S9 and S8 of SCoT and UBC873, S25 and UBC841 of ISSR. The melting fingerprints obtained from S34 of SCoT exhibited the best primers for identification of herbal species with 87.5% accuracy and relatively high repeatability. The presence of intraspecific variation in a few species affected the shift of melting fingerprints within species. MF-HRM using S34 showed improved species prediction compared to DNA fingerprints. The concentration of DNA with 10 ng/µl was recommended to perform MF-HRM. MF-HRM enabled species authentication of herbal commercialized products at only 20% resulting from the low quality of DNA isolated, while admixture of multiple product species interfered with the MF process. CONCLUSION: Findings suggested that MF-HRM showed promise as a molecular tool for the authentication of species in commercial herbal products with high specificity, moderate repeatability and rapidity without prior sequence information. This information will greatly improve quality control and traceability during the manufacturing process.

Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/µl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

The first study of genetic diversity and population structure of Indo-Pacific bottlenose dolphin (Tursiops aduncus) and pantropical spotted dolphin (Stenella attenuata) in the Thai Andaman Sea based on ISSR.

Background and Aim: The Indo-Pacific bottlenose dolphin, Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, are protected marine mammals in Thailand; however, knowledge regarding the populations of both species in Thai seas is minimal. We aimed to reveal the genetic diversity and population structure of two species, T. aduncus, and S. attenuata, based on inter-simple sequence repeats (ISSRs). Materials and Methods: Samples of stranded T. aduncus (n = 30) and S. attenuata (n = 23) found along Thai Andaman Sea coasts from 1998 to 2018 were used in this study. A total of 17 and 16 ISSR primers that produced clear and polymorphic bands were selected for T. aduncus and S. attenuata, respectively. Results: The highest percentages of polymorphic bands for T. aduncus and S. attenuata were 93.750% and 92.857%, respectively. Phylogenetic dendrograms indicated that the population of each species was clustered into three groups. This outcome was consistent with the genetic population structure, as both suggested three genetic clusters (DK = 3). Genetic diversity analysis revealed that the average Shannon's information index (I) was 1.926 ± 0.066 for T. aduncus and 1.714 ± 0.090 for S. attenuata, which indicate a high level of genetic variation. Further, low fixation index (F) values were observed for T. aduncus and S. attenuata at -0.231 ± 0.024 and -0.312 ± 0.042, respectively, suggesting that inbreeding is unlikely to have occurred for both species over the past decades. Conclusion: At least three genetic clusters of both species were found in the Thai Andaman Sea, and the diversity indices of each species indicated that these species are not at a critical level for extinction. However, monitoring their population status should be prioritized to observe any future changes in the level of diversity.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Microsatellite Polymorphism and the Population Structure of Dugongs (Dugong dugon) in Thailand.

The dugong (Dugong dugon) is an endangered species of marine mammals, so knowledge of genetic diversity of these populations is important for conservation planning within different habitats. In this study, six microsatellite markers were used to assess the genetic diversity and population structure of 77 dugongs from skin samples of stranded animals collected from 1994-2019 (69 from Andaman Sea and 8 from the Gulf of Thailand). Our results found that dugongs in the Andaman Sea had higher genetic variation than those in the Gulf of Thailand. Populations in Trang, Satun, and some areas of Krabi had highest diversity compared to other regions of Thailand. Bayesian genetic clustering analysis revealed that dugongs in Thailand consist of five genetic groups. Moreover, dugongs in the middle and lower Andaman Sea presented the greatest gene flow compared to other regions. However, based on calculation of inbreeding coefficients (Fis value = 0.239), dugong populations in the Sea of Thailand are experiencing some levels of inbreeding, and so may warrant special protections. These results provide important information for understanding the genetic status of dugongs that can lead to improved management and conservation of this endangered species.

Life Expectancy in Marine Mammals Is Unrelated to Telomere Length but Is Associated With Body Size.

Marine mammals vary greatly in size and lifespan across species. This study determined whether measures of adult body weight, length and relative telomere length were related to lifespan. Skin tissue samples (n = 338) were obtained from 23 marine mammal species, including four Mysticeti, 19 Odontoceti and one dugong species, and the DNA extracted to measure relative telomere length using real-time PCR. Life span, adult body weight, and adult body length of each species were retrieved from existing databases. The phylogenetic signal analysis revealed that body length might be a significant factor for shaping evolutionary processes of cetacean species through time, especially for genus Balaenoptera that have an enormous size. Further, our study found correlations between lifespan and adult body weight (R 2 = 0.6465, p < 0.001) and adult body length (R 2 = 0.6142, p ≤0.001), but no correlations with relative telomere length (R 2 = -0.0476, p = 0.9826). While data support our hypothesis that larger marine mammals live longer, relative telomere length is not a good predictor of species longevity.

Morphometric analysis of cervical vertebrae in some marine and land mammals.

Bones or skeletal remains can be used to answer a number of questions related to species, sex, age or cause of death. However, studies involving vertebrae have been limited as most were performed on skulls or long bones. Here, we have stated the hypothesis that the morphometry of cervical vertebrae can be used for species identification and body size estimation among marine and land mammals. The cervical vertebrae from eight and 14 species of marine and land mammals were used to collect morphometric data. Cluster dendrogram, principal component analysis, discriminant analysis and linear regression were used to analyse the data. The results indicate that, based on an index of C4 to C7, there were 13 out of 22 species for which identity could be correctly predicted in 100% of the cases. The correlations between cervical vertebrae parameters (height, width and length of centrum) in marine (average R2 =  0.87, p < .01) and land (average R2 =  0.51, p < .01) mammals were observed. These results indicate that vertebral morphometrics could be used for species prediction and verification of body weight in both marine and land mammals.

Feasibility of melting fingerprint obtained from ISSR-HRM curves for marine mammal species identification.

Currently, species identification of stranded marine mammals mostly relies on morphological features, which has inherent challenges. The use of genetic information for marine mammal species identification remains limited, therefore, new approaches that can contribute to a better monitoring of stranded species are needed. In that context, the ISSR-HRM method we have proposed offers a new approach for marine mammal species identification. Consequently, new approaches need to be developed to identify individuals at the species level. Eight primers of the ISSR markers were chosen for HRM analysis resulting in ranges of accuracy of 56.78-75.50% and 52.14-75.93% in terms of precision, while a degree of sensitivity of more than 80% was recorded when each single primer was used. The ISSR-HRM primer combinations revealed a success rate of 100% in terms of discrimination for all marine mammals included in this study. Furthermore, ISSR-HRM analysis was successfully employed in determining marine mammal discrimination among varying marine mammal species. Thus, ISSR-HRM analysis could serve as an effective alternative tool in the species identification process. This option would offer researchers a heightened level of convenience in terms of its performance and success rate. It would also offer field practice to veterinarians, biologists and other field-related people a greater degree of ease with which they could interpret results when effectively classifying stranded marine mammals. However, further studies with more samples and with a broader geographical scope will be required involving distinct populations to account for the high degree of intraspecific variability in cetaceans and to demonstrate the range of applications of this approach.

Mammalian species identification using ISSR-HRM technique.

Wildlife trading and the illegal hunting of wildlife are contributing factors to the biodiversity crisis that is presently unfolding across the world. The inability to control the trade of animal body parts or available biological materials is a major challenge for those who investigate wildlife crime. The effective management of this illegal trade is an important facet of wildlife forensic sciences and can be a key factor in the enforcement of effective legislation surrounding the illegal trade of protected and endangered species. However, the science of wildlife forensics is limited by the absence of a comprehensive database for wildlife investigations. Inter-simple sequence repeat markers (ISSR) coupled with high resolution melting analysis (HRM) have been effectively used for species identification of 38 mammalian species. Six primers of the ISSR markers were chosen for species identification analysis. From six ISSR primers resulting in a range of accuracy of 33.3%-100% and 100% in terms of precision in every primer. Furthermore, 161 mammalian samples were 100% distinguished to the correct species using these six ISSR primers. ISSR-HRM analysis was successfully employed in determining mammal identification among varying mammalian species, and thus could serve as an effective alternative tool or technique in the species identification process. This option would offer researchers a heightened level of convenience in terms of its performance and the ease with which researchers or field practice veterinarians would be able to interpret results in effectively identifying animal parts at wildlife investigation crime scenes.

Genetic diversity in a unique population of dugong (Dugong dugon) along the sea coasts of Thailand.

Dugong (Dugong dugon) populations have been shrinking globally, due in large part to habitat fragmentation, degradation and ocean pollution, and today are listed as Vulnerable by the IUCN. Thus, determining genetic diversity in the remaining populations is essential for conservation planning and protection. In this study, measures of inter-simple sequence repeat (ISSR) markers and mtDNA D-loop typing were used to evaluate the genetic diversity of 118 dugongs from skin samples of deceased dugongs collected in Thai waters over a 29-year period. Thirteen ISSR primers revealed that dugongs from the Andaman Sea and Gulf of Thailand exhibited more genetic variation in the first 12 years of the study (1990-2002) compared to the last decade (2009-2019). Dugongs from the Andaman Sea, Trang, Satun and some areas of Krabi province exhibited greater diversity compared to other coastal regions of Thailand. Eleven haplotypes were identified, and when compared to other parts of the world (235 sequences obtained from NCBI), five clades were apparent from a total 353 sequences. Moreover, dugongs from the Andaman Sea were genetically distinct, with a separate haplotype belonging to two clades found only in Thai waters that separated from other groups around 1.2 million years ago. Genetic diversity of dugongs in present times was less than that of past decades, likely due to increased population fragmentation. Because dugongs are difficult to keep and breed in captivity, improved in situ conservation actions are needed to sustain genetically healthy wild populations, and in particular, the specific genetic group found only in the Andaman Sea.

Association of IL-4 and IL-4R Polymorphisms with Litter Size Traits in Pigs.

The interleukin-4 (IL-4) and interleukin-4 receptor (IL-4R) are cytokines that are involved in the immune and reproductive systems. This study aimed to verify the polymorphisms in the porcine IL-4 and IL-4R genes and to assess their effects on litter size traits in commercial pigs. Single nucleotide polymorphisms (SNPs) in the porcine IL-4 and IL-4R genes were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A non-coding SNP of IL-4 g.134993898T > C and a non-synonymous SNP of IL-4R c.1577A > T (amino acid change at position 526, Q526L) were found to be segregating in Landrace sows. The IL-4 g.134993898T > C polymorphism was significantly associated with the number of piglets weaned alive (NWA) trait. The IL-4R c.1577A > T polymorphism was significantly associated with the number born alive (NBA) and NWA traits. Moreover, the accumulation of favorable alleles of these two SNP markers revealed significant associations with the NBA, NWA, and mean weight of piglets at weaning (MWW) traits. These findings indicate that the porcine IL-4 and IL-4R genes may contribute to the reproductive traits of pigs and could be used as candidate genes to improve litter size traits in the pig breeding industry.

Consistency of dark skeletal muscles in Thai native black-bone chickens (Gallus gallus domesticus).

Black-bone chickens (Gallus gallus domesticus) have become economically valuable, particularly in Southeast Asia as a consequence of popular traditional Chinese medical practices. Chickens with whole body organ darkness are considered to have higher value and are, therefore, more often requested. This research study aimed to investigate the darkness in 34 skeletal muscles of 10 Thai black-bone chickens (five males and five females). The evaluation of muscle darkness was done on two levels: (i) a color chart was employed at the macroanatomical level and (ii) by using melanin pigment to evaluate the structure at the microanatomy level. The results revealed that the accumulation of melanin pigment in the muscle tissue was observed in the endomysium, perimysium and epimysium. With respect to the results of the color chart test, iliotibialis lateralis pars preacetabularis, gastrocnemius, fibularis longus and puboischiofemoralis pars medialis showed the highest degree of darkness, while serratus profundus, pectoralis, iliotibialis cranialis, flexor cruris lateralis, and flexor cruris medialis appeared to be the least dark. In addition, we found that the highest and lowest amounts of melanin pigment was noted in the flexor carpi ulnaris and pectoralis (p < 0.05), respectively; however, there was no significant difference (p > 0.05) observed between the sexes. These results reveal that the 34 specified muscles of black-bone chickens showed uneven distribution of darkness due to the differing accumulations of melanin pigments of each muscle.This information may provide background knowledge for a better understanding of melanin accumulation and lead to breeding improvements in Thai black-bone chickens.

Phylogenetic analyses of distantly related clades of bent-toed geckos (genus Cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western Thailand.

Cyrtodactylus species are the most diverse of the geckos and are widely distributed in Southeast Asia, including Thailand. However, their patterns of distribution, especially in northern and western parts of Thailand, remain unknown because few Cyrtodactylus species in these regions have been described. Thus, a data set of mitochondrial NADH dehydrogenase 2 (ND2) gene and flanking tRNAs from Cyrtodactylus found in northern and western Thailand, including contiguous areas, was assembled to elucidate phylogenetic relationships and identify the distribution patterns of these geckos. The results showed four well-supported clades, a northwestern clade (A), a northern clade (B), a western clade (C), and a special clade characterized by specific morphological features (D). Clades A-C were grouped with strong support by the geography of their localities from northern Thailand (Mae Hong Son and Chiang Mai Provinces) along the Tenasserim mountain ranges to Phang-Nga Province, Thailand. Clade D is a distinct clade of Cyrtodactylus species characterized by a tuberculate and prehensile tail and distributed widely in mainland Southeast Asia. Overall, the results suggest a pattern of geographic separation and distribution of Cyrtodactylus in northern and western Thailand. Additionally, this study provides evidence of a hidden biodiversity of Cyrtodactylus in these regions.

Elemental Distribution in Animal Carpal and Tarsal Bones Using Differences in X-ray Fluorescence Energy.

Little is known as to whether different operating voltages of X-ray fluorescence (XRF) can affect the accuracy rate for species identification. Here, we have addressed this question by comparing the rate of correct species identification using the elemental composition of either the carpal or tarsal bone obtained from a determination of the different energy values of XRF at 15 and 50 kV using energy-dispersive XRF (ED-XRF). Carpal bones were taken from 16 species and tarsal bones from 11 of these species. The data on the elemental profiles were analyzed by stepwise discriminant analysis for species discrimination. The classification results indicated that 94.1% and 63.7% of the originally grouped cases were correctly classified as carpal bones using 15 kV and 50 kV, respectively. Additionally, 69.4% and 77.3% of the originally grouped cases were correctly classified as tarsal bones using 15 kV and 50 kV, respectively. When the datasets of the elemental profiles obtained using two operating voltages were gathered, the classification results of the prediction rate appeared to be more accurate at 89.7% and 90.7% in the carpal and tarsal bones, respectively. In conclusion, our findings suggest that the elemental profiles of bones obtained using two operating voltages could effectively facilitate accurate species discrimination.

Pelvic bone morphometric analysis in the dugong (Dugong dugon).

The dugong (Dugong dugon Müller) is recognized as an endangered marine mammal. There is limited available anatomical data on the dugong's skeletal system, while what is available has not been well established due to the limited number of archived samples and limited access to them. Importantly, there are certain key questions that should be answered when examining the bones and/or remains of animals such as; what kind of bone is it?; what species does it belong to?; what sex was the animal?; how old was the animal? or how big was it?, etc. In this study, we have focused on the pelvic bone of the dugong by asserting the hypothesis that pelvic bone morphology is related to age, sex, and body size. Here, we have established certain morphometric data encompassing 8 parameters and 5 indexes to analyze the morphology of the pelvic bones obtained from 88 specimens (45 dugongs). We will present three main findings: (1) the pelvic bone in mature male subjects is larger than it is in female subjects, (2) a high rate of accuracy can be established for sex identification using morphometric data obtained from the pelvic bone, and (3) the pelvic bone has the highest degree of correlation with body length, followed by body weight and age. Notably, the important data on the pelvic bone of the dugong acquired in this study can be reliable and extremely useful in sex identification and body size estimation.

Genetic variations and dog breed identification using inter-simple sequence repeat markers coupled with high resolution melting analysis.

The identification of differing physical characteristics of dogs is an uncomplicated and straightforward way to categorize dog breeds. However, many dog owners and veterinarians still struggle to distinguish between pure breed and mixed variations in certain breeds of dogs. Presently, the absence of the tools and methods needed to confirm a pure breed dog is a significant problem since the only method available to validate pure or mongrel breeds is the official pedigree system. Inter-simple sequence repeat markers have been successfully used to assess genetic variations and differentiations. Notably, inter-simple sequence repeat markers coupled with high resolution melting analysis were effectively used for the breed identification of 43 breeds of dogs (total 463 dogs). The 10 primers chosen for analysis resulted in a range of 31-78.6% of breed discrimination when using one primer, while a combination of two primers was able to successfully discriminate between all of the 43 dog breeds (100%). Shannon's index information (I = 2.586 ± 0.034) and expected heterozygosity (H e  = 0.908 ± 0.003) indicated a high level of genetic diversity among breeds. The fixation index (F st ) revealed a value of 10.4%, demonstrating that there was a high level of genetic subdivision between populations. This study showed that inter-simple sequence repeat marker analysis was effective in demonstrating high genetic diversity among varying breeds of dogs, while a combination of Inter-simple sequence repeat marker analysis and high resolution melting analysis could provide an optional technique for researchers to effectively identify breeds through genetic variations.