Mitochondrial Sequence Variation, Haplotype Diversity, and Relationships Among Dromedary Camel-Types

Dromedary camels are outstanding livestock that developed efficient abilities to tolerate desert conditions. Many dromedary camel-types (i.e., named populations) exist but lack defined specific breed standards, registries, and breeders’ governing organizations. The breed status of dromedary camel-types can partly be assessed by exploring mitochondrial DNA (mtDNA) variation. Accordingly, this study aimed to examine the breed status and the inter-population relationships of dromedary camel-types by analyzing sequence variation in the mtDNA control region and in three coding genes [cytochrome b, threonine, and proline tRNA, and part of the displacement loop (D-loop)] (867 bp region). Tail hair samples (n = 119) that represent six camel-types from Kuwait were collected, extracted, sequenced, and compared to other publicly available sequences (n = 853). Within the sequenced mitochondrial region, 48 polymorphic sites were identified that contributed to 82 unique haplotypes across 37 camel-types. Haplotype names and identities were updated to avoid previous discrepancies. When all sequences were combined (n = 972), a nucleotide diversity of 0.0026 and a haplotype diversity of 0.725 was observed across the dromedary-types. Two major haplogroups (A and B) were identified and the B1 haplotype was predominant and found in almost all dromedary-types whereas the A haplotypes were more abundant in African regions. Non-metric multidimensional scaling revealed an increased similarity among Arabian Peninsula “Mezayen” camel-types, despite their defining coat colors. The relationships among dromedary camel-types can partly be explained by mtDNA. Future work aimed at a deeper understanding of camel-type breed status should focus on a high number of nuclear markers.

Early detection of the Root-knot Nematode Meloidogyne hapla through developing a robust qPCR approach compliant with the MIQE guidelines

Root-knot nematodes (RKNs) are major threats to crops through attacking the roots, which induces an abnormal development of the plant. Meloidogyne hapla Chitwood is of particular concern as it is currently expanding its distribution area and displays a wide host range. Effective plant protection against this RKN requires early detection as even a single individual can cause severe economic loses on susceptible crops. Molecular tools are of particular value for this purpose and among them, qPCR presents all the advantages, i.e. sensitivity, specificity, rapidity of diagnosis at a reduced cost. Although few studies already proposed detecting M. hapla through this technique, they lack experimental details and performance testing, and suffered from low taxonomic resolution and/or required expensive hydrolysis probes. Here we propose a qPCR detection method that uses SybrGreen with developed primers amplifying a fragment of COI mitochondrial region. The method is developed and evaluated following the MIQE guidelines to ensure its quality, i.e. sensitivity, specificity, repeatability, reproducibility, robustness. The results demonstrate that the newly developed method fulfills its goals as it proved specific to M. hapla and allowed for a reproductible detection level as low as 1.25 equivalent of a juvenile individual. All criteria associated with the MIQE guidelines were also met, what makes it of general use for the reliable early detection of M. hapla.

Ichtyological diversity in a tropical nature reserve (Etang de Saint-Paul, Réunion Island) from environmental DNA (eDNA) metabarcoding

The aim of this study was to test whether the ichthyological diversity of one natural reserve in Reunion Island (Réserve de l'Etang de Saint-Paul) could be established with a molecular tool, environmental DNA (eDNA). We hence filtrated the water (2L) at 10 different areas around the reserve. For each sampling area, 12 PCR replicas were performed and the identification of fish species was carried out by metabarcoding through a primer over a mitochondrial region (12S). This study showed the importance of reference sequencing databases as well as improvements through phylogenetic analyses. This first fish study by eDNA in La Réunion also revealed the coherence of the distribution of species and their habitat. See the Poster in Suppl. material 1. Collection : 1st DNAQUA International Conference - poster session.

Barcoding of ToLCV Resistant Tomato Germplasm in Bangladesh

The current study was carried out to confirm the existence of the ToLCV resistant genes (Ty-1 to Ty-5) in the germplasm using molecular markers and to identify at the genomic level following phylogenetic relationships analysis among some local tomato germplasm using DNA barcoding. Most of the tomato germplasm (Ten out of 14) contain the dominant Ty-genes as revealed by PCR analysis. “Barcoding” of the non-coding plastid trnH-psbA intergenic spacer region, three plastidal regions: rbcL, rpoB, rpoC1, spacer region of nuclear genome ITS and a mitochondrial region matK were employed following PCR and sequence analysis of the germplasm. Among all the barcode genes, rpoB, rbcL, trnH-psbA and ITS were leading candidates for successful amplification and used for the identification of the germplasm as S. lycopersicum in multi-locus identification based on their sequences. Neighbor-Joining phylogenetic tree was constructed in which the germplasm were clustered into five main clades. The current study was successful to establish an efficient barcoding protocol for the correct identification of tomatoes and was capable of establishing elite gene source(s) for biotic stress resistance tomato varieties which would serve as potential donor plants in modern breeding programs. Plant Tissue Cult. & Biotech. 30(1): 107-117, 2020 (June)

Detection of pork in meatballs using probe TaqMan Real-time Polymerase Chain Reaction

This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detection of pork adulteration in meatballs. The study is important as it described a TaqMan method for product adulteration analysis. TaqMan is known to have a more specific result compared to SYBR green analytical method. Assay in the study combined species-specific primers and TaqMan probes to targeting 153 bp fragment of D-loop mitochondrial region of pork. A specificity test was conducted on fresh tissues of pork, beef, chicken, wild boar, dog, and mouse. Meatballs as samples were prepared from a mixture of pork-beef and wild boar-beef with concentrations as follows: 5%, 10%, 25%, 75%, 90%, and 100%. The linearity and sensitivity of the method were performed by measuring the amplification curve from the dilution series, namely 1000, 200, 100, 10, 5, 1, and 0.5 pg/μL of DNA, extracted from 100% pork meatballs. A repeatability test was conducted as many as six repetitions on 100% pork and 100% wild boar meatballs. This study showed that mitochondrial D-loop species-specific primers and TaqMan probes could identify the DNA of pork and wild boar on the fresh tissues. Additionally, it also resulted in a threshold cycle (Ct) of 17.02 and 17.95 for pork, 22.22 for wild boar, while the negative result for others. The detection limit has shown 5 pg in the meatball formulation. The Relative Standard Deviation (RSD) of repeatability was 1.936% for pork, while 2.140% for wild boar. The developed method was also applied to analyzing commercial meatballs. A TaqMan real-time PCR analytical method using specific primer targeting on 153 bp fragment of the D-loop mitochondrial region could be applied as a standard method for identifying pork and wild boar in food samples intended for halal authentication studies

DNA barcoding in some Belarusian insects

Due to a number of factors in the anthropogenic load on ecosystems, environmental changes, and competition between native and invasive species, the loss of species diversity has currently risen to an alarming scale. One of the prospective approaches to remedy the current state and halt the loss is the international project “Barcode of Life”. The report describes the results of molecular genetic research on the insect representatives of such orders as Coleoptera (beetles), Lepidoptera (butterflies), and Trichoptera (caddisflies) using the cytochrome c oxidase subunit 1 of the mitochondrial region (COI). We proposed a technique for the sampling of biological material to isolate DNA from the hind legs of individuals. The technique prevents removal of the individuals of the most valuable species collected on the territory of the Republic of Belarus from their habitats. The DNA sample collection of order representatives was investigated. For the subfamily Cetoniinae (flower chafer), significant differences in haplotypes among the representatives of the Belarusian and European parts of their areal were found. For Trichoptera, it was shown that the COI gene has high variability to differentiate species. It was also revealed that some species of caddisflies, which Belarusian researchers believe to be synonymous ones, have sequences with big differences according to the BoldSystem database. This fact should be explored in the future. So, our analysis allows considering the COI gene as a satisfactory marker for the species identification in the Belarusian insects’ taxa studied.

Polymorphisms and distribution of South American manatees (Trichechus spp.)

Traditionally, the morphological attributes and the range of Trichechus species have been clearly established. However, we herein show that morphological traits, like belly and pectoral flipper coloration in South American manatees may be polymorphic. Karyotypic analysis of T. manatus allowed the precise identification of this species and confirmed the variability of the observed morphological findings. Molecular analysis based on cytochrome b DNA and the D-loop mitochondrial region showed shared haplotypes between T. inunguis and T. manatus, suggesting the presence of an ancestral polymorphism. These findings showed the need of improving the identification of these species before implementing conservation strategies. Finally, we present a complete report on the extant distribution of these species in South America.

Polymorphisms and distribution of South American manatees (Trichechus spp.)

Traditionally, the morphological attributes and the range of Trichechus species have been clearly established. However, we herein show that morphological traits, like belly and pectoral flipper coloration in South American manatees may be polymorphic. Karyotypic analysis of T. manatus allowed the precise identification of this species and confirmed the variability of the observed morphological findings. Molecular analysis based on cytochrome b DNA and the D-loop mitochondrial region showed shared haplotypes between T. inunguis and T. manatus, suggesting the presence of an ancestral polymorphism. These findings showed the need of improving the identification of these species before implementing conservation strategies. Finally, we present a complete report on the extant distribution of these species in South America.

Genetic diversity and population structure of Brycon nattereri (Characiformes: Bryconidae): a Neotropical fish under threat of extinction

ABSTRACT Brycon nattereri is an endangered Neotropical fish reported along conserved stretches of the upper Paraná, Tocantins and São Francisco rivers. Populations of this species have been very rare in some Paraná River sub basins. This study analyzes the genetic diversity and population structure of B. nattereri in a restricted area of occurrence recently identified in upper Paraná River basin. Seven microsatellite loci and 497 bp of D-Loop mitochondrial region were examined in 92 individuals from four points along the area of occurrence. Both molecular markers indicated a single population distributed along a stretch of the river approximately 80 km long. Although some of the data suggest an ancient bottleneck, current levels of genetic diversity (H E = 0.574 and h = 0.616) were similar to those of other species of the genus Brycon. The results suggest that the population of B. nattereri has been able to maintain satisfactory levels of genetic diversity, in spite of the small area of occurrence. These data have highlighted an important conservation area and action may prove essential to improve the quality of the environment, and especially the water and riparian plant life, if the area is to be managed and conserved efficiently.