Species-specific Markers Research Articles

Dietary effects on the reproductive performance of the sea urchin Tripneustes gratilla ll: Implications for offspring performance

Aquaculture practices can result in declines in the genetic diversity observed in progenitor natural populations, resulting in subsequent poor production output, particularly for broadcast spawning animals that frequently display differential parental contributions. This study aimed to assess the effect of different Tripneustes gratilla broodstock feeding regimes [formulated feed, kelp (Ecklonia maxima), green seaweed (Ulva lacinulata) and mixture of the three diets for four months] on larval growth, parental contributions and juvenile performance after a factorial breeding design was implemented. Larvae produced from broodstock fed kelp and the mixed diet survived for the full duration of larval rearing and displayed similar growth rates throughout (p > 0.05). Though larvae from Ulva-fed broodstock did not survive for the full duration of larval rearing, this could have been a consequence of larval rearing practices, as these larvae displayed a rapid increase in post-oral arm lengths prior to the mortality event. Furthermore, these larvae survived for the longest amount of time when left unfed, suggesting that the maternal reserves from animals fed Ulva as a broodstock conditioning diet could be beneficial to development in early larval stages. In contrast, larvae from animals fed a formulated feed did not survive the full duration of larval rearing and had developmental abnormalities. Three months post-metamorphosis, 10 species-specific microsatellite markers were PCR amplified across 16 broodstock and 364 offspring from kelp and mixed diet fed animals. Genetic diversity analyses showed that there were no statistically significant (p > 0.05) differences between the broodstock animals and their offspring, showing that factorial breeding designs are advantageous to preserve genetic diversity present in cultured cohorts. Parentage analyses revealed that a total of 26 out of 32 possible F1 parent pairs contributed to the F2 generation, with a low heritability estimate of approximately zero (0.050 ± 0.058) for offspring body diameter, likely as offspring phenotypic performance is not influenced by additive genetic effects during these early growth stages.

Species-Specific Nuclear Genetic Markers for the Detection of Hybridization between the Grey Smoothhound (Mustelus californicus) and the Brown Smoothhound (Mustelus henlei)

Many species retain the physiological capacity to hybridize over long periods of evolutionary time. Hybridization has been reported for sharks and may be the reason for uncertainty regarding phylogenetic relationships within the genus Mustelus. Because of similar life histories and morphologies, limited genetic divergence (both nuclear and mitochondrial DNA), and the degree of uncertainty regarding their phylogenetic relationship, 32 allozyme loci were used to determine the levels of genetic diversity for the Grey Smoothhound, Mustelus californicus, and the Brown Smoothhound, M. henlei, and to detect species-specific nuclear markers capable of determining the existence of hybridization within northeastern Pacific Mustelus. Tissue extracts from 26 adult M. henlei collected from Santa Catalina Island, California and 17 M. californicus collected from Marina Del Rey, California were subjected to starch gel electrophoresis. Gene products of the 32 loci were resolved for all specimens of both species. All 32 loci were monoallelic in M. californicus, whereas four polyallelic loci, three diallelic and one triallelic, were resolved in M. henlei. Nine of the 32 loci exhibited complete allelic divergence between the two species (i.e., no shared alleles) and can serve as diagnostic markers to reveal potential F1 and F2 hybrids.

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens.

Background Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment. Objective We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection. Methods N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations. Results The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for blaTEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates. Conclusions Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.

Utilization of an In Silico 16S Ribosomal RNA Gene-Based Restriction Enzyme Approach for Identification of Lactic Acid Bacteria

In silico 16S ribosomal RNA gene-based restriction enzyme approach is a computer-simulated polymerase chain reaction-restriction fragment length polymorphism analysis method, namely customizable in silico sequence evaluation for restriction sites. The purpose of this study was to identify lactic acid bacteria using restriction enzyme analysis of 16S gene (16S ribosomal RNA polymerase chain reaction-restriction fragment length polymorphism). An in silico investigation was conducted to detect species-specific molecular markers in different lactic acid bacteria strains. We selected six out of 20 16S ribosomal RNA gene sequences from probiotic-potential lactic acid bacteria strains. Initially, the corresponding 16S ribosomal RNA gene sequences were selected from the database aligned using the Clustal Omega program and their evolutionary relationships were determined. The results indicated that the studied lactic acid bacteria strains belonged to 6 distinct clades except for the genus of Lactobacillus. Furthermore, the in silico polymerase chain reactionrestriction fragment length polymorphism was conducted for further taxonomic classification. Different restriction enzymes were used to recognize sites present on the lactic acid bacteria 16S ribosomal RNA genes. Customizable in silico sequence evaluation for restriction sites output comprised a predicted agarose gel as virtual polymerase chain reaction-restriction fragment length polymorphism patterns, for proper selection of the appropriate enzyme (s) to be used for polymerase chain reaction-restriction fragment length polymorphism marker generation. Our study demonstrated that ApyPI, CchIII and NheI produced distinct restriction patterns as those of Ku324937_C species. The most abundant species-specific markers were seen for Ku324928_C (10 markers), followed by Ku324908_C (8 markers) of order Hemiptera, then Ku324909_C (5 markers), Ku324898_C (4 markers) and finally, Ku324937_C (3 markers). The results demonstrated that customizable in silico sequence evaluation for restriction sites is a powerful tool to detect species-specific markers to provide better understanding of organisms found in raw and/or fermented milk.

Species-Specific Flash Patterns Track the Nocturnal Behavior of Sympatric Taiwanese Fireflies.

Simple SummaryNocturnal fireflies are facing various challenges to their survival (e.g., artificial light pollution and extreme climate change). However, there are still no effective diagnostic tools to distinguish firefly species and monitor how their population size and nocturnal behavior change in the wild. Here, we demonstrated that flash interval (FI), a timing character of firefly flash signaling, can be a reliable species-specific luminescent marker for identifying three species (Abscondita cerata, Luciola kagiana, and Luciola curtithorax) in a wild sympatric population and evaluating their nightly behaviors in video-imagery analysis, as well as a traditional field specimen collection. Our studies provide key information and valuable references for future studies of (1) the behaviors of sympatric fireflies in the wild and (2) the development of automatic and rapid digital diagnostic tools for identifying firefly species and quantifying population size, especially for firefly conservation.It is highly challenging to evaluate the species’ content and behavior changes in wild fireflies, especially for a sympatric population. Here, the flash interval (FI) and flash duration (FD) of flying males from three sympatric species (Abscondita cerata, Luciola kagiana, and Luciola curtithorax) were investigated for their potentials in assessing species composition and nocturnal behaviors during the A. cerata mating season. Both FI and FD were quantified from the continuous flashes of adult fireflies (lasting 5–30 s) via spatiotemporal analyses of video recorded along the Genliao hiking trail in Taipei, Taiwan. Compared to FD patterns and flash colors, FI patterns exhibited the highest species specificity, making them a suitable reference for differentiating firefly species. Through the case study of a massive occurrence of A. cerata (21 April 2018), the species contents (~85% of the flying population) and active periods of a sympatric population comprising A. cerata and L. kagiana were successfully evaluated by FI pattern matching, as well as field specimen collections. Our study suggests that FI patterns may be a reliable species-specific luminous marker for monitoring the behavioral changes in a sympatric firefly population in the field, and has implication values for firefly conservation.

Novel microsatellites and investigation of faecal DNA as a non-invasive population monitoring tool for the banded hare-wallaby (

Monitoring programs for populations of small or medium-sized animals often use live-capture or photo-monitoring trapping methods to estimate population size. The banded hare-wallaby (Lagostrophus fasciatus), a small macropodiform marsupial, does not readily enter traps or have individually unique distinguishing physical features and is consequently difficult to monitor using these methods. Isolating DNA from faecal material to obtain individual genotypes is a promising monitoring technique and may present an alternative approach for this species. We developed novel species-specific microsatellite markers and undertook trials to assess faecal DNA degradation in ambient environmental conditions at two locations where this species has been translocated. The quality of DNA yielded from faecal pellets was evaluated through amplification failure and genotyping error rates of microsatellite markers. Error rates were compared for different treatments and exposure duration across multiple individuals. DNA was successfully obtained from all samples and error rates increased with exposure duration, peaking after 14–30 days depending on the site and treatment. The level of solar exposure was the most significant factor affecting degradation rate but both this and exposure duration had significant effects on amplification failure. Analysing DNA obtained from faecal pellets may represent a practical non-invasive method of deriving population estimates for this species and warrants further development.

Rapid Detection of Red Rot Disease Pathogens (Pythium chondricola and P. porphyrae) in Pyropia yezoensis (Rhodophyta) with PCR-RFLP.

Red rot disease is one of the best-known algal diseases infecting red algae Pyropia species. This disease decreases the quality and quantity of Pyropia aquaculture products in Korea, Japan, and China. Recently we found that Pythium chondricola (Oomycetes) infects blades of Pyropia yezoensis. Therefore, two Pythium species (P. chondricola and P. porphyrae) have been reported as red rot disease pathogens. In this study, we developed a species-specific molecular marker for distinguishing between the two red rot disease pathogens. Using a polymerase chain reaction restriction fragment length polymorphism method based on the mitochondrial cytochrome c oxidase subunit 2 (cox2) and nuclear ribosomal RNA large subunit regions, we classified these two Pythium species without a sequencing step. This new method had high specificity and efficiency for detecting red rot disease pathogens at the species level for both of the cultured and field samples. Therefore, the molecular markers developed in this study are effective for long-term monitoring of the infection and distribution pattern of each Pythium species in Pyropia aquaculture farms. Moreover, molecular monitoring can provide useful information for predicting infection and preventing mass mortality of Pyropia species by red rot disease.

ASSESSMENT OF ECOLOGICAL AND GENETIC DIVERSITY OF THE GENUS VIGNA SAVI USING BIOTECHNOLOGICAL METHODS - ISSR MARKERS

The world's genetic resources of plants are the main source of improving crops for decades to come. The gene pool of plants has a hidden resource of new genes, or their combinations, including - selection-important features. The study of the potential of the plant gene pool with the main biological and economic characteristics allows to expand the genetic base of crops for the successful implementation of breeding programs in various areas. The genus Vigna has about 200 species that are grown in warm regions of the planet. The centers of origin of the species are in Africa, but mung beans, urd, azuki and rice beans are of Asian origin. The collection includes 20 specimens belonging to 7 species of the genus Vigna: V. aconitifolia (Jacq.) Marechal (willow aconitolista, mott) – 3 specimens, V. angularis (Willd.) Ohwi et Ohashi (adzuki) – 4, V. radiata (L.) R. Wilczek (mash) – 4, V. umbellata (Thunb.) Ohwi et Ohashi (vigna rice) – 4, V. unguiculata (L.) Walp. (Chinese) – 5. These are mainly local varieties, about 10 % are breeding varieties and 1% are forms that grow in nature.
 The multifaceted use of cultivated species of the genus Vigna contributed to their spread throughout the tropical, subtropical and temperate zones of the globe. They are economically important crops in many developing countries. Geographical analysis showed that most of the samples came from the areas of world agriculture and crop formation. most samples of V. radiata, V. mungo, V. aconitifolia, V. trilobata, V. umbellata were obtained from India and Pakistan, V. angularis – from East Asia and China, V. unguiculata – East Africa (Ethiopia, Kenya), V unguiculata – from China. However, the exact place of domestication of this culture has not been established, and in numerous sources of literature can be found different opinions of scientists on the origin of cowpea and the centers of its diversity. Recently, using the methods of molecular genetics (RAPD, AFLP and others) it was confirmed that the northern part of Africa is the center of origin of the cultured, because the wild types of West Africa are closer to cultural forms than the wild types of East and South Africa. It should be noted that the species of cowpea have a significant intraspecific polymorphism. The samples showed strong variability of morphological and economically valuable features. Such a wide range of variable variability is due to the places of cultivation of samples, different environmental conditions (plains, mountains, climate).
 According to the results of the evaluation of the DNA polymorphism of cowpea using molecular genetic markers, it was found that the species of cowpea involved in the study are characterized by a high level of DNA polymorphism, which averaged 78.6 %. 145 loci were identified, including 31 unique, specific to a particular sample, and 31 monomorphic, characteristic of all samples. Monomorphic loci are conserved regions of DNA that indicate the common origin of the species of cowpea involved in the work, and can be used as genus and species-specific markers. Unique loci indicate genetic divergence of the studied material and can serve as markers of certain samples. The average level of intrapopulation polymorphism of cowpea DNA (37.2–93.8 %, depending on the genotype) was established, which indicates the existence of significant variability in the studied samples of cowpea. It shows a high level of genetic divergence of cowpea species and testifies in favor of the polyphyletic theory of their origin.

Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD).

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The blaCARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this blaCARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg2+, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63°C for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

Resprouters Versus Reseeders: Are Wild Rooibos Ecotypes Genetically Distinct?

Aspalathus linearis (Burm. F.) R. Dahlgren (Fabaceae) or rooibos, is a strict endemic species, limited to areas of the Cederberg (Western Cape) and the southern Bokkeveld plateau (Northern Cape) in the greater Cape Floristic Region (CFR) of South Africa. Wild rooibos, unlike the cultivated type, is variable in morphology, biochemistry, ecology and genetics, and these ecotypes are broadly distinguished into two main groups, namely, reseeders and resprouters, based on their fire-survival strategy. No previous assessment of genetic diversity or population structure using microsatellite markers has been conducted in A. linearis. This study aimed to test the hypothesis that wild rooibos ecotypes are distinct in genetic variability and that the ecotypes found in the Northern Cape are differentiated from those in the Cederberg that may be linked to a fire-survival strategy as well as distinct morphological and phytochemical differences. A phylogeographical and population genetic analyses of both chloroplast (trnLF intergenic region) and newly developed species-specific nuclear markers (microsatellites) was performed on six geographically representative wild rooibos populations. From the diversity indices, it was evident that the wild rooibos populations have low-to-moderate genetic diversity (He: 0.618–0.723; Ho: 0.528–0.704). The Jamaka population (Cederberg, Western Cape) had the lowest haplotype diversity (H = 0.286), and the lowest nucleotide diversity (π = 0.006) even though the data revealed large variations in haplotype diversity (h = 0.286–0.900) and nucleotide diversity (π = 0.006–0.025) between populations and amongst regions where wild rooibos populations are found. Our data suggests that populations of rooibos become less diverse from the Melkkraal population (Suid Bokkeveld, Northern Cape) down towards the Cederberg (Western Cape) populations, possibly indicative of clinal variation. The largest genetic differentiation was between Heuningvlei (Cederberg, Western Cape) and Jamaka (FST = 0.101) localities within the Cederberg mountainous region, and, Blomfontein (Northern Cape) and Jamaka (Cederberg) (FST = 0.101). There was also a significant isolation by distance (R2 = 0.296, p = 0.044). The presence of three main clusters is also clearly reflected in the discriminant analysis of principal components (DAPC) based on the microsatellite marker analyses. The correct and appropriate management of wild genetic resources of the species is urgently needed, considering that the wild Cederberg populations are genetically distinct from the wild Northern Cape plants and are delineated in accordance with ecological functional traits of reseeding or resprouting, respectively. The haplotype divergence of the ecotypes has also provided insights into the genetic history of these populations and highlighted the need for the establishment of appropriate conservation strategies for the protection of wild ecotypes.

The Development of New Species-Specific Molecular Markers Based on 5S rDNA in Elaeagnus L. Species.

The Elaeagnus L. species are trees and bushes that mainly grow in temperate zones of Western Europe; Minor, Central, and Southeast Asia; the Far East; and North America. Some species are used as fruit or ornamental plants and have economic value. Problems with the identification of species in the Elaeagnus genus by molecular genetical methods arise in the study of populations, systematics, breeding, and other areas of plant science and practice. Recently, the polymorphism of 5S ribosomal DNA non-transcribed spacers (5S rDNA NTSs) in Elaeagnaceae Adans. has been described. The results were used in our study as a basis for development of new species-specific molecular markers for some members of the Elaeagnus genus. The author’s method was applied for finding regions that were potentially applicable for species-specific primer design. As a result, some species-specific molecular markers were developed for Elaeagnus angustifolia L., E. commutata Bernh., E. pungens Thunb., and E. multiflora Thunb. These markers were tested in a range of samples and showed the presence of amplified fragments in lanes of the marked species only. Samples of other species showed no amplifications. Thus, the developed markers may be useful for the species identification of the studied Elaeagnus plants in botanical, dendrological, and genetic research (especially in a leafless period of year), as well as in breeding and hybridization experiments.

Occasional long-distance dispersal may not prevent inbreeding in a threatened butterfly

BackgroundTo set up successful conservation measures, detailed knowledge on the dispersal and colonization capacities of the focal species and connectivity between populations is of high relevance. We developed species-specific nuclear microsatellite molecular markers for the grayling (Hipparchia semele), a butterfly endemic to Europe and of growing conservation concern in North-West Europe, and report on its population genetics, in a fragmented, anthropogenic landscape in Belgium. Our study included samples from 23 different locations nested in two regions and additional historical samples from two locations. We assessed contemporary, long-distance dispersal based on genetic assignment tests and investigated the effect of habitat loss and fragmentation on the population genetic structure and genetic variation using data of nine microsatellite loci.ResultsDetected dispersal events covered remarkably long distances, which were up to ten times larger than previously reported colonisation distances, with the longest movement recorded in this study even exceeding 100 km. However, observed frequencies of long-distance dispersal were low. Our results point to the consequences of the strong population decline of the last decades, with evidence of inbreeding for several of the recently sampled populations and low estimates of effective population sizes (Ne) (ranging from 20 to 54 individuals).ConclusionsOur study shows low frequencies of long-distance dispersal, which is unable to prevent inbreeding in most of the local populations. We discuss the significance for species conservation including future translocation events and discuss appropriate conservation strategies to maintain viable grayling (meta) populations in highly fragmented, anthropogenic landscapes.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species.

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S. indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S. indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Complete Mitochondrial Genome of Great Frigatebird (Fregata minor): Phylogenetic Position and Gene Rearrangement.

The complete mitogenome sequence of the Great Frigatebird, Fregata minor was sequenced for the first time in this study. The mitogenome (16,899bp) comprises of 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes, and a control region (CR). The mitogenome was AT-rich (55.60%) with 11 overlapping and 18 intergenic spacer regions. Most of the PCGs were started by a typical ATG initiation codon except for cox1 and nad3. A maximum-likelihood phylogeny of concatenated PCGs resulted in a well-resolved phylogeny of all the species of Suliformes and illuminates the sister relationship of F. minor with F. magnificens. The present mitogenome-based phylogeny clearly enlightens the evolutionary position of Suliformes and Pelecaniformes species. Unique tandem repeats were identified in both F. minor and F. magnificens, which can be employed as a species-specific marker. To illuminate the population structure of this migratory seabirds, the present study advocate more sampling and the generation of additional molecular data to clarify their genetic diversity. The present study also rejects an earlier hypothesis on the mitochondrial gene order of Suliformes and corroborated the typical avian gene order in frigatebirds.

Site fidelity and shallow genetic structure in the common smooth-hound shark Mustelus mustelus confirmed by tag-recapture and genetic data.

The common smooth-hound shark, Mustelus mustelus, is a widely distributed demersal shark under heavy exploitation from various fisheries throughout its distribution range. To assist in the development of appropriate management strategies, the authors evaluate stock structure, site fidelity and movement patterns along the species' distribution in southern Africa based on a combination of molecular and long-term tag-recapture data. Eight species-specific microsatellite markers (N=73) and two mitochondrial genes, nicotinamide adenine dehydrogenase subunit 4 and control region (N=45), did not reveal any significant genetic structure among neighbouring sites. Nonetheless, tagging data demonstrate a remarkable degree of site fidelity with 76% of sharks recaptured within 50 km of the original tagging location. On a larger geographic scale, dispersal is governed by oceanographic features as demonstrated by the lack of movements across the Benguela-Agulhas transition zone separating the South-East Atlantic Ocean (SEAO) and South-West Indian Ocean (SWIO) populations. Microsatellite data supported very shallow ocean-based structure (SEAO and SWIO) and historical southward gene flow following the Agulhas Current, corroborating the influence of this dynamic oceanographic system on gene flow. Moreover, no movements between Namibia and South Africa were observed, indicating that the Lüderitz upwelling formation off the Namibian coast acts as another barrier to dispersal and gene flow. Overall, these results show that dispersal and stock structure of M. mustelus are governed by a combination of behavioural traits and oceanographic features such as steep temperature gradients, currents and upwelling systems.

A Comprehensive Study of the Genus Sanguisorba (Rosaceae) Based on the Floral Micromorphology, Palynology, and Plastome Analysis.

Sanguisorba, commonly known as burnet, is a genus in the family Rosaceae native to the temperate regions of the Northern hemisphere. Five of its thirty species are distributed in Korea: Sanguisorba officinalis, S. stipulata, S. hakusanensis, S. longifolia, and S. tenuifolia. S. officinalis has been designated as a medicinal remedy in the Chinese and Korean Herbal Pharmacopeias. Despite being a valuable medicinal resource, the morphological and genomic information, as well as the genetic characteristics of Sanguisorba, are still elusive. Therefore, we carried out the first comprehensive study on the floral micromorphology, palynology, and complete chloroplast (cp) genome of the Sanguisorba species. The outer sepal waxes and hypanthium characters showed diagnostic value, despite a similar floral micromorphology across different species. All the studied Sanguisorba pollen were small to medium, oblate to prolate-spheroidal, and their exine ornamentation was microechinate. The orbicules, which are possibly synapomorphic, were consistently absent in this genus. Additionally, the cp genomes of S. officinalis, S. stipulata, and S. hakusanensis have been completely sequenced. The comparative analysis of the reported Sanguisorba cp genomes revealed local divergence regions. The nucleotide diversity of trnH-psbA and rps2-rpoC2, referred to as hotspot regions, revealed the highest pi values in six Sanguisorba. The ndhG indicated positive selection pressures as a species-specific variation in S. filiformis. The S. stipulata and S. tenuifolia species had psbK genes at the selected pressures. We developed new DNA barcodes that distinguish the typical S. officinalis and S. officinalis var. longifolia, important herbal medicinal plants, from other similar Sanguisorba species with species-specific distinctive markers. The phylogenetic trees showed the positions of the reported Sanguisorba species; S. officinalis, S. tenuifolia, and S. stipulata showed the nearest genetic distance. The results of our comprehensive study on micromorphology, pollen chemistry, cp genome analysis, and the development of species identification markers can provide valuable information for future studies on S. officinalis, including those highlighting it as an important medicinal resource.

Molecular Phylogenetic Analysis of Vicia L. (Fabaceae) Taxa Growing in the Southeastern Anatolia Region of Turkey: Based on Internal Transcribed Spacer (ITS)

Leguminosae or Fabaceae is the third-largest flowering plant family and is important in terms of both food production and soil fertility. Wild Vicia species and the genetic diversity of the Southeastern Anatolia Region provide an invaluable resource for the improvement of cultivated temperate feed and legume crops. The rapid progress of technology in recent years has nowmade it possible to use modern techniques in phylogenetic studies and to examine plants in a greater detail using biochemical, cytological and molecular methods to supplement purely systematic studies. The use of molecular phylogenetic analysis is the most attractive alternative strategy for a more accurate identification of the species of the Vicia genus. In the current study, some Vicia L. taxa growing naturally in the Southeastern Anatolia Region were investigated using molecular phylogenetic analysis. Internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced in order to study the phylogenetic relationships of Vicia L. taxa. Lathyrus inconspicuous L. and Lathyrus cassius Boiss. were used as an outgroup. The ITS area was determined to be approximately 479- 672 bp. The ITS sequences were submitted to the NCBI database and accession numbers obtained. The resulting tree clearly groups and separates the sect. Narbonensis, Ervilia, Peregrinae, Lathyroides, Vicia and Cracca species but was less able to distinguish species from sect. Hypechusa and Lentopsis. The data acquired were observed to be reliable in terms of solving the taxonomical problems of the Vicia L. taxa. The morphological distinctions are greatly supported by DNA sequence studies. The species-specific markers developed in this study are useful for early detection of targeted Vicia taxa and can act as a guide to the basic data required for the evolution of systematic breeding and conservation strategies, as well as for germplasm resources.

Developing an efficient DNA barcoding system to differentiate between Lilium species

BackgroundLilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties.ResultsTo develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG).ConclusionsA rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Unrestricted gene flow between two subspecies of translocated brushtail possums (Trichosurus vulpecula) in Aotearoa New Zealand

Two lineages of brushtail possums (Trichosurus vulpecula) were historically introduced to Aotearoa New Zealand, and these two subspecies have different phenotypic forms. Despite over 100 years of potential interbreeding, they appear to retain morphological differences, which may indicate reproductive isolation. We examined this using population samples from a confined landscape and scored each specimen for phenotype using a number of fur colour traits. This resulted in a bimodal trait distribution expected for segregated grey and black lineages. We also sought evidence for genetic partitioning based on spatial and temporal effects. Genetic structure and rates of genetic mixing were determined using seven neutral, species-specific nuclear microsatellite markers and mitochondrial DNA control region sequence. Genotype analyses indicated high levels of variation and mtDNA sequences formed two major haplogroups. Pairwise tests for population differentiation of these markers found no evidence of subdivision, indicating that these brushtail possums behave as a single randomly mating unit. Despite maintenance of two main colour phenotypes with relatively few intermediates, previous inference of assortative mating and anecdotes of distinct races, our data indicate that New Zealand brushtail possums can freely interbreed, and that in some locations they have formed completely mixed populations where neutral genetic markers are unrelated to phenotype. This has implications for effective pest management towards eradication.

Mass spectrometry determination of potential species-specific peptide markers in commercial seasoning cubes

In recent years, issues related to food product authenticity have been alarming, particularly in meat-based products. Proteomic techniques in food authentication and their applications in determining quality and safety issues of ingredients have been progressively developed by researchers. The complexity of matrices in commercial seasoning cubes is one of the main concerns in developing a robust authentication method. Hence, the present study focuses on the development of a protein extraction method for commercial seasoning cubes and to determine potential species-specific peptide markers in commercial seasoning cubes. The efficacy of solvent extraction was determined using Bradford assays, while the electrophoretic profiles of commercial seasoning cubes were visualized via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The determination of potential species-specific peptide markers in commercial seasoning cubes was carried out via Liquid Chromatography (LC)-Orbitrap Fusion-Mass spectrometry (MS/MS) analysis (Thermo Fisher Scientific). In this study, the urea-thiourea solvent was ideal in extracting protein from commercial seasoning cubes as it produces a homogenous solution upon mixing, suggesting the diffusibility of solutes that is essential for continuing analysis. Moreover, a bovine-specific peptide marker ‘VGGHAAEYGAEALER’ and two porcine-specific peptide markers; ‘VGGQAGAHGAEALER’ and ‘EAVLGLWGK’ were discovered. The methodology developed in this study was successful in identifying species-specific peptide markers in seasoning cubes and can be used as a reference for validating the authenticity of food products, especially commercial seasonings.