Microsatellite Loci Research Articles

Molecular Characterization of Pongamia pinnata (L.) Pierre Genotypes in Respect to Seed Oil Using Microsatellite Markers

Twenty-four genotypes selected from different agroclimatic zones of Jharkhand (India) were assessed for seed oil content that varied from 21.86% to 41.86% with 14 genotypes recording above average 32.11 % for the trait which indicates towards efficiency of selection processes. Several genotypes in ‘Central and Western Plateau’ agroclimatic zone of Jharkhand displayed a good potential for high oil content. The employed 23 polymorphic microsatellite loci exhibited three to twenty one alleles per locus with an average of 12 while total 270 alleles were detected. Two primer set PpSSR21 and PpSSR27 showed 100% polymorphism among the genotypes. The high oil yielding plant K19 showed different band pattern with the locus PpSSR04. From the Nei’s analysis it was found that maximum diversity exists between the full sib genotypes K10 and K2. Thus, the genotypes (K2 and K10) which are more diverse could be used further in improvement programme. Overall, the genotypes included in the study showed a correlation with their geographical origins such that genotypes from the same region tend to have higher genetic similarity as compared to those from different regions. However, in UPGMA based Nei’s analysis, some genotypes were found not to be grouped based on geographical origins possibly due to the exchange of germplasm over time between farmers across the regions. Both molecular and oil content (biochemical) markers appeared useful in analyzing the extent of genetic diversity in P. pinnata. The result of these analyses will help to better understand the genetic diversity and relationship among populations constituting a set of useful background information that can be used as a basis for future breeding strategy and improvement of the species.

Genomics Reveals Complex Population History and Unexpected Diversity of Eurasian Otters (Lutra lutra) in Britain Relative to Genetic Methods.

Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated "stronghold" populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s-1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

Genetic imprints of grafting in wild iron walnut populations in southwestern China

BackgroundAnthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations.ResultsWe sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations.ConclusionsOur results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ and ex situ conservation.

Differentiation of Bos grunniens and Bos taurus based on STR locus polymorphism.

Differentiation of closely related biological species using molecular genetic analysis is important for breeding farm animals, creating hybrid lines, maintaining the genetic purity of breeds, lines and layering. Bos grunniens and Bos taurus differentiation based on STR locus polymorphism will help maintain the genetic isolation of these species and identify hybrid individuals. The aim of this study is to assess the differentiating potential of 15 microsatellite loci to distinguish between domestic yak (B. grunniens) bred in the Kalmak-Ashuu highland region (Kochkor district, Naryn region, Kyrgyz Republic) and cattle (B. taurus) of three breeds (Aberdeen-Angus, Holstein and Alatau) using molecular genetic analysis. The samples were genotyped at 15 microsatellite loci (ETH3, INRA023, TGLA227, TGLA126, TGLA122, SPS115, ETH225, TGLA53, BM2113, BM1824, ETH10, BM1818, CSSM66, ILSTS006 and CSRM60). Twelve of the loci were from the standard markers panel recommended by ISAG. Statistical analysis was performed using GenAlEx v.6.503, Structure v.2.3.4, PAST v.4.03, and POPHELPER v1.0.10. The analysis of the samples' subpopulation structure using the Structure v.2.3.4 and 15 STR locus genotyping showed that the accuracy of assigning a sample to B. taurus was 99.6 ± 0.4 %, whereas the accuracy of assigning a sample to B. grunniens was 99.2 ± 2.6 %. Of the 15 STRs, the greatest potential to differentiate B. grunniens and B. taurus was found in those with the maximal calculated FST values, including BM1818 (0.056), BM1824 (0.041), BM2113 (0.030), CSSM66 (0.034) and ILSTS006 (0.063). The classification accuracy of B. grunniens using only these five microsatellite loci was 98.8 ± 3.4 %, similar for B. taurus, 99.1 ± 1.2 %. The proposed approach, based on the molecular genetic analysis of 5 STR loci, can be used as an express test in Kyrgyzstan breeding and reproduction programs for B. grunniens.

Genetic diversity of Aedes aegypti and Aedes albopictus from cohabiting fields in Hainan Island and the Leizhou Peninsula, China

BackgroundAedes aegypti and Ae. albopictus are important human arbovirus vectors that can spread arboviral diseases such as yellow fever, dengue, chikungunya and Zika. These two mosquito species coexist on Hainan Island and the Leizhou Peninsula in China. Over the past 40 years, the distribution of Ae. albopictus in these areas has gradually expanded, while Ae. aegypti has declined sharply. Monitoring their genetic diversity and diffusion could help to explain the genetic influence behind this phenomenon and became key to controlling the epidemic of arboviruses.MethodsTo better understand the genetic diversity and differentiation of these two mosquitoes, the possible cohabiting areas on Hainan Island and the Leizhou Peninsula were searched between July and October 2021, and five populations were collected. Respectively nine and 11 microsatellite loci were used for population genetic analysis of Ae. aegypti and Ae. albopictus. In addition, the mitochondrial coxI gene was also selected for analysis of both mosquito species.ResultsThe results showed that the mean diversity index (PIC and SI values) of Ae. albopictus (mean PIC = 0.754 and SI = 1.698) was higher than that of Ae. aegypti (mean PIC = 0.624 and SI = 1.264). The same results were also observed for the coxI gene: the genetic diversity of all populations of Ae. albopictus was higher than that of Ae. aegypti (total H = 45 and Hd = 0.89958 vs. total H = 23 and Hd = 0.76495, respectively). UPGMA dendrogram, DAPC and STRUCTURE analyses showed that Ae. aegypti populations were divided into three clusters and Ae. albopictus populations into two. The Mantel test indicated a significant positive correlation between genetic distance and geographic distance for the Ae. aegypti populations (R2 = 0.0611, P = 0.001), but the correlation was not significant for Ae. albopictus populations (R2 = 0.0011, P = 0.250).ConclusionsThe population genetic diversity of Ae. albopictus in Hainan Island and the Leizhou Peninsula was higher than that of Ae. aegypti. In terms of future vector control, the most important and effective measure was to control the spread of Ae. albopictus and monitor the population genetic dynamics of Ae. aegypti on Hainan Island and the Leizhou Peninsula, which could theoretically support the further elimination of Ae. aegypti in China.Graphical

Population genetics of recent natural colonization by the bull-headed shrike (Lanius bucephalus; Aves) suggests the importance of recurrent immigration on remote islands

Abstract Founder effects and recurrent immigration are two major factors that might potentially contribute to genetic differentiation and population persistence in the early stage of remote island colonization. However, their relative importance remains controversial. By conducting population genetic analyses of four remote island populations of the bull-headed shrike (Lanius bucephalus) established naturally within several decades in Japan, we examined the contributions of founder effects and recurrent immigration to these island populations. Based on the standard genetic indices and population structure analyses using 15 microsatellite loci, we suggested island-specific scenarios of colonization. Notably, the founder effect strongly influenced genetic differentiation in the population on the most remote oceanic island, Chichi-jima Island, which, however, became extinct 20 years after colonization, possibly owing to a lack of recurrent immigration. In contrast, another oceanic island, Minami-Daito Island, was probably subjected to multiple recurrent immigration events from the mainland, which obscured any genetic differentiation previously established by the founder effect. Temporal samples collected over 8 years on this island confidently supported this scenario. Underlying the island-specific scenarios of colonization, we provide evidence that recurrent immigration strongly affected the population persistence, overwhelming the initial founder effects. We argue for the importance of recurrent immigration even in colonization of highly remote islands.

Inconsistent estimates of hybridization frequency in newts revealed by SNPs and microsatellites

Hybridization between the European smooth and palmate newts has recurrently been mentioned in the literature. The only two studies that attempted to quantify the frequency of hybridization and gene admixture between these two species came to strikingly opposite conclusions. According to Arntzen et al. (1998, 42 allozymes), hybrids are rare in nature and introgression negligible, while according to Johanet et al. (2011, 6 microsatellites), introgressive hybridization is significant and widespread across the shared distribution range. To clarify this question, we implemented high-throughput SNP genotyping with diagnostic biallelic SNPs on 965 specimens sampled across Europe. Our results are in line with Arntzen et al., since only two F1 hybrids were identified in two distinct French localities, and no further hybrid generations or backcrosses. Moreover, reanalysis of 78 of the samples previously studied by Johanet et al. (2011) using our SNPs panel could not reproduce their results, suggesting that microsatellite-based inference overestimated the hybridization frequency between these two species. Since we did not detect methodological issues with the analyses of Johanet et al., our results suggest that SNP approaches outperform microsatellite-based assessments of hybridization frequency, and that conclusions previously published on this topic with a small number of microsatellite loci should be taken with caution, and ideally be repeated with an increased genomic coverage.

Species and hybrid composition and genetic diversity of water frogs (Pelophylax esculentus complex) in western Ukrainian hemiclonal population systems

Background. Two species of water frogs, Pelophylax ridibundus and Pelophylax lessonae, and their hybrid, Pelophylax kl. esculentus, are widespread in Ukraine. The purpose of this study was to investigate the population structure of various types of hemiclonal population systems (HPS) of water frogs formed due to the coexistence of frogs in the same territory. In Ukraine, a hybridization centre with the spread of triploid individuals of hybrid nature has been identified. Triploid hybrids are an intriguing research subject due to the diverse hypotheses about their origin and role in HPS. Outside the hybridization centre in Kharkiv Region, triploids are not commonly found. In our study, we describe the initial findings of triploid specimens in Lviv Region and analyze the genetic structure of the HPS where such individuals were detected. Methods. In total, 193 specimens of green frogs were collected between 2011 and 2015. Here we present population structure analysis which was conducted using two microsatellite loci, Rrid059A and RlCA1b5. A wide range of software programs were utilized for processing the genetic analysis data, including GenePop 4.7.5, Micro-Checker and NewHybrids 1.1. Results. Three types of hemiclonal population systems were identified: R-E type in Perekalky and Lake Pisochne, L-E type in Lake Luky, and R-E-L type in Nyzhankovychi, Velykyi Lyubin, Zhovtantsi and Cholgyni. Additionally, population systems with hybrids of mixed ploidy (diploids and triploids) were found in Perekalky, Velykyi Lyubin and Zhovtantsi. Conclusions. Genetic diversity analysis revealed variations in the number of alleles per population. P. ridibundus individuals exhibited higher genetic diversity compared to P. lessonae individuals, whereas hybrids showed intermediate genetic diversity. Further investigations of the localities where potential triploids were detected are necessary to assess the survival and reproductive potential of hybrid individuals and determine all types of hybrids and individuals of both marsh and pool frogs.

Genetic Structure of Russian Riding Horse Breed

166 horses of the russian riding breed were studied using 17 microsatellite loci recommended by ISAG. Microsatellite polymorphism data of thirty European breeds (7874 individuals) was used for comparative analysis of genetic diversity. According to the level of allelic richness, the Russian riding horse has high values – 5. 4 alleles per microsatellite locus. In total, 122 alleles were found in the breed, including private 14 allele at the HTG7 locus. The population genetic structure was revealed for the Russian riding, Arabian, Akhal-Teke, German riding and thoroughbred breeds. Clustering of horse breeds on a circular UPGMA dendrogram revealed six main clusters. the Russian ridinghorse forms a single group with German and thoroughbred horses in PCA. Breeds were divided into three populations in STRUCTURE program: 1) Arabian, 2) Russian Riding, Akhal-Teke, thoroughbred riding, 3) German half-breed. Analysis of differentiation between breeds using the F-st method showed the greatest values (differences) between all breeds with the Akhal-Tekehorses. The smallest value (Fst = 0.22) between the Russian riding and German riding breed. Differences for each pair of breeds were significant (p-value 0.001).

Microsatellites and the Major Histocompatibility Complex of the Atlantic Walrus <i>Odobenus rosmarus rosmarus</i> (Linnaeus, 1758) in Haulouts of the Barents Sea

The allelic diversity of the DQB gene of the major histocompatibility complex (MHC) in Atlantic walrus from the European part of the Arctic was analyzed for the first time. We studied animals from the haulouts located on the Franz Josef Land archipelago and near the northern tip of Novaya Zemlya. We demonstrated that there are significant differences for this locus between the two studied haulouts. Simultaneous analysis of microsatellite loci reveals a lower level of differentiation, and also suggest that animals from more than one spatial-geographical stock (population) gather at the summer haulout on Novaya Zemlya. It has been suggested that individuals with different MHC genotypes may prefer different feeding areas, regardless of the areas of residence during the breeding season.

Phenotypic plasticity and genetic diversity shed light on endemism of rare Boechera perstellata and its potential vulnerability to climate warming.

The rapid pace of contemporary environmental change puts many species at risk, especially rare species constrained by limited capacity to adapt or migrate due to low genetic diversity and/or fitness. But the ability to acclimate can provide another way to persist through change. We compared the capacity of rare Boechera perstellata (Braun's rockcress) and widespread B. laevigata to acclimate to change. We investigated the phenotypic plasticity of growth, biomass allocation, and leaf morphology of individuals of B. perstellata and B. laevigata propagated from seed collected from several populations throughout their ranges in a growth chamber experiment to assess their capacity to acclimate. Concurrently, we assessed the genetic diversity of sampled populations using 17 microsatellite loci to assess evolutionary potential. Plasticity was limited in both rare B. perstellata and widespread B. laevigata, but differences in the plasticity of root traits between species suggest that B. perstellata may have less capacity to acclimate to change. In contrast to its widespread congener, B. perstellata exhibited no plasticity in response to temperature and weaker plastic responses to water availability. As expected, B. perstellata also had lower levels of observed heterozygosity than B. laevigata at the species level, but population-level trends in diversity measures were inconsistent due to high heterogeneity among B. laevigata populations. Overall, the ability of phenotypic plasticity to broadly explain the rarity of B. perstellata versus commonness of B. laevigata is limited. However, some contextual aspects of our plasticity findings compared with its relatively low genetic variability may shed light on the narrow range and habitat associations of B. perstellata and suggest its vulnerability to climate warming due to acclimatory and evolutionary constraints.

Host-associated genetic differentiation and origin of a recent host shift in the generalist parasitic weed Phelipanche ramosa.

Branched broomrape, Phelipanche ramosa (L.) Pomel, is a globally distributed parasitic weed of economic importance. In Europe, where it is native, it can infest several crops, notably tomato, tobacco, and hemp. In western France, it has recently adapted to a new host crop, oilseed rape, causing substantial damage. The aim of this study was to investigate the evolutionary relationships and genetic differentiation among P. ramosa populations infesting different hosts. We collected 1611 P. ramosa samples from 109 fields cultivated with six different crops (oilseed rape, tobacco, hemp, tomato, lentil, and celery) and distributed among six European countries. All samples were genotyped for ten microsatellite loci and a subset of samples was sequenced for two nuclear genes and two chloroplast genes. Genetic differentiation among populations was high (F ST = 0.807) and mainly driven by differentiation among different host crops, with no significant geographic structure. Genetic structure analysis identified up to seven biologically meaningful clusters that matched with host crops of origin. Reconstructed networks of sequence haplotypes and multilocus SSR genotypes showed a large genetic divergence between samples collected on oilseed rape and samples collected on other crops. The phylogeny inferred from DNA sequences placed samples collected from oilseed rape as a basal lineage. Approximate Bayesian Computations were used to compare different evolutionary scenarios of divergence among the three main genetic clusters, associated, respectively, with oilseed rape, tobacco, and hemp as host crops. The best-supported scenario indicated that P. ramosa infesting oilseed rape derived recently from an ancient, unknown lineage. Our results suggest that a more complete description of the genetic diversity of P. ramosa is still needed to uncover the likely source of the recent adaptation to oilseed rape and to anticipate future new host shifts.

Siberian Larch Reproduction Using the Somatic Embryogenesis Biotechnology

The biotechnology of somatic embryogenesis in vitro, combined with genomic selection and cryopreservation is used to create varietal genetically tested fast-growing plantations (Multi-Varietal Forestry program (MVF), Park, 2014, 2016, 2018). In 2008, the Sukachev Forest Institute of the Siberian Branch of the RAS has developed for the first time the biotechnology of somatic embryogenesis for Siberian larch (Larix sibirica Ledeb.) and obtained 42 proliferating cell lines consisting of embryonal-suspensor mass (ESM). The age of cell lines reaches 13 years. Significant variability was observed between cell lines in the number and size of globular embryos in proliferating embryogenic cultures, and in the ability of somatic embryos to mature and germinate. In different cell lines, the number of globular somatic embryos per 1 g of ESMs fresh weight ranges from 2040 to 11103, with 10 to 1220 embryos maturing. The regenerants germinate in a growth chamber, and plantlets of individual cell lines grow successfully in a greenhouse and then in the soil of the forest nursery at the Forest Institute’s Pogorelsky Bor station. Genotyping of clones at microsatellite loci showed their complete genetic identity to the cell line from which they were obtained. In cloned Siberian larch trees at the age of seven, the initiation of generative organs forming occurred. Thus, at present, it is possible to quickly implement the MVF program for plantation forestry in Russia.

Genetic resources of macroalgae: Development of an efficient method using microsatellite markers in non-model organisms

Red and brown seaweeds are species with high ecological and economic importance. Here we report the feasibility of cost-effective molecular marker development in 6 species from different phyla. Microsatellite markers of two brown seaweed species Alaria esculenta and Pylaiella littoralis, and of four red seaweed species Calliblepharis jubata, Gracilaria gracilis, Gracilaria dura and Palmaria palmata were identified and characterized in genomic sequences obtained using Double-Digest Restriction site Associated DNA (ddRAD). A total of 64,623,186 reads were generated from two runs of multiplexed Illumina Miseq sequencing, from which 30,636 reads containing microsatellite motifs including 15,443 where primer pairs could be designed. Five hundred seventy-six primers pairs were selected for amplification trials to determine levels of polymorphism. Of these 576 loci, 338 could be amplified and 142 of the 338 loci were polymorphic. A total of 28 usable polymorphic markers were developed in A. esculenta, 18 in P. littoralis, 11 in C. jubata, 14 in G. gracilis, 21 in G. dura and 13 in P. palmata. The overall number of alleles per locus ranged from 2 to 22. These 105 new microsatellite markers will be useful for further studies of population genetics, breeding programs and conservation genetics of these species. Compared with traditional approaches, our study yielded thousands of microsatellite loci for six different species in a short time and with affordable costs. This study, based on the use of ddRADseq, provided preliminary data about the genetic structure and reproduction mode of these six non-model species based on a small number of individuals from two geographically-distant populations and on the genetic structure and reproduction mode of two non-model species, i.e. detection of clonality for two red algae, C. jubata and G. dura and detection of highly genetically divergent populations corresponding probably to different cryptic species under the name P. littoralis.

GENETIC DIVERSITY OF THE AMUR TIGER (<i>PANTHERA TIGRIS ALTAICA</i>): ARE THERE DIFFERENCES BETWEEN THE 20 AND THE BEGINNING OF THE 21 CENTURIES?

The polymorphism of 9 microsatellite loci of the Sikhote-Alin tiger population in the modern and recent historical period was analyzed using blood, excrement, and museum bone samples. Tests for a population going through a period of low abundance in the analysis of excess heterozygotes and a low value of the Garza-Williamson coefficient indicate a high probability of such events in the recent and historical past. The mean effective population size Ne for the current sample was 34.4 (95% range 17–106.8). This fact is of great interest for the conservation and restoration of endangered large cat species.

Genomic Characteristics of Okamejei kenojei and the Implications to Its Evolutionary Biology Study.

Okamejei kenojei is an important economic species widely distributed in shallow coastal waters of the western North Pacific. In this study, the whole-genome survey analysis of O. kenojei was conducted to reveal its genomic characteristics. The genome size was estimated to be 2027.44Mb, the repeat sequence content was 44.90%, and the heterozygous ratio was 1.04%. The mitochondrial genome excavated from the sequencing data was 16,974bp, and it can form the closed circular molecule. The phylogenetic tree based on 13 protein-coding gene sequences supported the validity of Okamejei and assisted the conclusion that Raja porosa was the junior synonym of O. kenojei. Plenty of potential microsatellite loci were identified, and the distribution frequency was estimated to be approximately 236.3 SSRs per Mb. Among all motif types of microsatellites, the dinucleotide repeats were dominant (82.59%), followed by the trinucleotide repeats (8.05%), tetranucleotide repeats (5.80%), pentanucleotide repeats (2.83%), and hexanucleotide repeats (0.72%). The results of the present study could not only provide useful information for understanding the genome structure and functional characteristics of O. kenojei, but also lay the foundation for the subsequent mapping of the whole genome.

Genetic diversity and structure of mongolian gazelle (Procapra gutturosa) populations in fragmented habitats

BackgroundThe Mongolian gazelle (Procapra gutturosa) population has shown a considerable range of contractions and local extinctions over the last century, owing to habitat fragmentation and poaching. A thorough understanding of the genetic diversity and structure of Mongolian gazelle populations in fragmented habitats is critical for planning effective conservation strategies.ResultIn this study, we used eight microsatellite loci and mitochondrial cytochrome b (Cytb) to compare the levels of genetic diversity and genetic structure of Mongolian gazelle populations in the Hulun Lake National Nature Reserve (HLH) with those in the China-Mongolia border area (BJ). The results showed that the nucleotide diversity and observed heterozygosity of the HLH population were lower than those of the BJ population. Moreover, the HLH and BJ populations showed genetic differentiation. We concluded that the HLH population had lower genetic diversity and a distinct genetic structure compared with the BJ population.ConclusionThe genetic diversity of fragmented Mongolian gazelle populations, can be improved by protecting these populations while reinforcing their gene exchange with other populations. For example, attempts can be made to introduce new individuals with higher genetic diversity from other populations to reduce inbreeding.

A study of genetic profiles of grape plants preserved under the name of Dagestan variety ‘Khatmi’

Background. Traditionally, the description of grape varieties is a task of ampelographic studies. However, several different grape cultivars have similar phenotypic traits. Molecular genetic characterization is the most accurate tool for cultivar identification. The development of DNA fingerprinting of varieties is the first step in this direction. An extensive database of DNA profiles of grape genotypes makes it possible to determine the varietal affiliation of unknown forms, confirm or refute the varietal correspondence of planting material. ‘Khatmi’ is an autochthonous grape variety from Dagestan. The profile of ‘Khatmi’ is presented in the VIVC international database of DNA fingerprints for grape varieties. However, an application of DNA analysis methods in grape variety studies has determined that several ancient varieties were cultivated under one name, while for others a certain variability of genotypes was observed. The objectives of the work were to study samples of ‘Khatmi’ plants from different places of growth in Dagestan by standard microsatellite loci used for grape genotyping, to assess the level of genetic similarity of the samples, and to refine the DNA profile of ‘Khatmi’. Materials and methods. Molecular genetic study was carried out on 10 samples from different ‘Khatmi’ populations. The material was picked from the collection sites of Dagestan breeding experimental station of viticulture and vegetable growing and the Dagestan Experiment Station of VIR, as well as from production plantations. DNA was extracted from herbarium specimens of young grape shoot tips by the CTAB method. The samples were genotyped by polymerase chain reaction using a standard set of primers for 9 microsatellite markers VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79 recommended by the International Organization of Grapes and Wine (OIV) for grapevine DNA fingerprinting. Amplification products were separated, and their sizes were assessed using capillary electrophoresis on an ABI Prism 3130 genetic analyzer. Results. Genotyping was done for 10 samples of grapes growing in Dagestan under the name ‘Khatmi’, including samples from different collections and places of industrial cultivation, as well as clonal variations of this variety and putative clonal variations. The two base pair differences within one of the loci distinguished the DNA profiles of the analyzed samples from that of ‘Khatmi’ presented in the international grape varieties database VIVC. It was determined that the sample under the name ‘Khatmi krupnoyagodnyi’ is closely related to ‘Khatmi’ variety by its genotype, but probably represents a clonal variation of ‘Koz uzyum’, another local variety of Dagestan. Conclusion. The DNA profile of the local Dagestan grape variety ‘Khatmi’ has been refined.

Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000–2016)

Emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACT) is a major challenge for Greater Mekong Subregion countries in their goal to eliminate malaria by 2030. Tools to efficiently monitor drug resistance beyond resource-demanding therapeutic efficacy studies are necessary. A custom multiplex amplicon sequencing assay based on Illumina technology was designed to target the marker of partial resistance to artemisinin (K13), five candidate modulators of artemisinin resistance, the marker of resistance to chloroquine (crt), and four neutral microsatellite loci. The assay was used to genotype 635 P. falciparum-positive blood samples collected across seven provinces of Vietnam and one of Cambodia between 2000 and 2016. Markers of resistance to artemisinin partner-drugs piperaquine (copy number of plasmepsin-2) and mefloquine (copy number of multidrug-resistance 1) were determined by qPCR. Parasite population structure was further assessed using a 101-SNP barcode. Validated mutations of artemisinin partial resistance in K13 were found in 48.1% of samples, first detection was in 2000, and by 2015 prevalence overcame > 50% in Central Highlands and Binh Phuoc province. K13-C580Y variant became predominant country-wide, quickly replacing an outbreak of K13-I543T in Central Highlands. Mutations in candidate artemisinin resistance modulator genes paralleled the trends of K13 mutants, whereas resistance to piperaquine and mefloquine remained low (≈ 10%) by 2015–2016. Genomic tools applied to malaria surveillance generate comprehensive information on dynamics of drug resistance and population structure and reflect drug efficacy profiles from in vivo studies.

The influence of landscape features on the population connectivity and genetic structure of the Edible Dormouse Glis glis in Poland

Abstract In this study, we analyze and compare the genetic structure of edible dormice representing five populations, inhabiting forest complexes in southwestern Poland that differ in their degree of fragmentation and isolation. Total genomic DNA was isolated from hairs with bulbs of 145 individuals captured between 2018 and 2020. All dormice sampled were genotyped at 14 microsatellite loci. Population genetic analyses showed that genetic variability in the studied dormice, assessed based on observed and expected heterozygosity as well as allelic richness, was on average very low (0.39, 0.43, and 2.83, respectively). In the populations studied, genetic structure was detected indicating two or five distinct genetic clusters, the existence of which can be attributed to either historical factors or modern human activity. Genetic differentiation between dormice living in the studied localities, determined by FST, ranged from 0.07 to 0.40 (all FST coefficients were significant at P < 0.05). A significant positive correlation was detected between genetic differentiation and geographic distance (r = 0.645, P < 0.017), indicating that genetic differentiation increases with distance, but no correlation between genetic differentiation and habitat barriers (r = 0.359, P < 0.132). The results of this research are compared to those of other European populations of edible dormice, and implications for the future of this endangered species are discussed.