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

et al. 2021

Sci Rep

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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.

Section: Discussionmentioning

confidence: 87%

“…Only clearly observed and unambiguous fragments were scored in a binary manner for band presence (1) or absence (0), and a binary matrix was generated to determine the level of polymorphism for each primer represented by the percentage of polymorphic bands 19 , 20 .…”

Section: Methodsmentioning

confidence: 99%

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

Poommouang

Kriangwanich

et al. 2021

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“…This method has been confirmed in various studies involving genetic diversity, genetic structure and molecular identification ( Sarwat, 2012 ). A total of 34 ISSR primers were screened to obtain a melting profile of the DNA fingerprints based on the ISSR fingerprints established by HRM referred to as “ISSR-HRM” ( Kriangwanich et al, 2020 ). As a result, eight suitable ISSR primers that offered distinct melting curve patterns among all species were selected to create melting fingerprints using the ISSR-HRM technique ( Table 2 ).…”

Section: Methodsmentioning

confidence: 99%

“…High resolution melting analysis coupled with ISSR markers (ISSR-HRM) was performed on PCRmax Eco 48 (PCRmax Limited, Staffordshire, UK) following the protocol established by Kriangwanich et al, 2020 . This method was used with a final volume of 10 μl containing 1X using a SensiFast™ HRM kit (the EvaGreen ® dye, dNTPs and enhancers) (Bioline, Memphis, TN, USA), 0.5 μM ISSR primer and a 20 ng DNA template.…”

Section: Methodsmentioning

confidence: 99%

“…HRM can reveal results at different melting temperatures which can then be measured in real-time and reduce the subjective errors that are associated with human biases ( Erali & Wittwer, 2010 ; Power, 1996 ; Reed, Kent & Wittwer, 2007 ; Tulsiani et al, 2010 ; Vossen et al, 2009 ; Wittwer et al, 2003 ). For example, ISSR markers coupled with HRM analysis or ISSR-HRM have recently been reported for the evaluation of several dog breeds ( Canis lupus familiaris ) in terms of their identification and classification ( Kriangwanich et al, 2020 ). ISSR markers would be more effective in distinguishing between individuals and species due to the presence of a single primer that could produce multiple DNA bands from multiple loci ( Gupta et al, 1994 ; Ng & Tan, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

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

Kriangwanich

Poommouang

et al. 2021

PeerJ

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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.

Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn

Paenkaew

Sripairoj

et al. 2021

Sci Rep

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Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen’s DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, “Bar-cas12a” for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM’s gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.