Genetic Diversity in the Wrinkled Frog, Glandirana rugosa, Evaluated Using Microsatellite Markers Identified by Nanopore Sequencing

Spread and performance of European earthworms invading North America as indicated by molecular markers and climate chamber experiments

Microsatellite Libraries Enriched for Several Microsatellite Sequences in Plants

Next generation biological control – an introduction

Abstract. The aim of the study was to describe the current state of genetic variability in the Haflinger breed in the Czech Republic using microsatellite markers, taking into consideration the numerous imports of breeding animals from abroad during the last 20 years and their impact on genetic diversity and population structure. A total of 443 horses from five countries of origin (Austria – AUT, Germany – GER, Czech Republic – CZE, Italy – ITA, and Slovakia – SVK) bred in the Czech Republic were included in the study. A set of 16 microsatellite markers for parentage control from the International Society for Animal Genetics (ISAG) was used for genotyping. The total number of alleles in individual subpopulations ranged from 53 (SVK) to 117 (CZE). The mean number of alleles per locus was 6.69. Observed heterozygosity (Ho) values ranged from 0.69 to 0.71 in all subpopulations. The most variable and informative locus (in terms of polymorphic information content – PIC) was VHL20, and the least variable was HTG6. The Fis index was mostly negative or close to 0 for all populations and was −0.033 for the whole population. The overall Fst was 0.010, indicating a low level of differentiation between subpopulations. Cavalli-Sforza and Edwards chord genetic distances were low between the CZE, AUT, and GER populations, while the ITA and SVK populations were more distinct. The results of the discriminant analysis of principal components (DAPC) and the STRUCTURE analysis indicated a high degree of admixture among subpopulations. However, three to four genetic groups were clustered. The most distant populations were ITA and SVK, which we attribute to the low number of representatives in these subpopulations. A higher level of admixture due to gene flow was observed between the populations of GER, CZE, and AUT. Higher admixtures and the discovery of more distinct genetic clusters suggest that there is more significant gene flow from the countries of origin in the population of the Haflinger breed in the Czech Republic and that there is sufficient genetic variability and diversity to suggest sufficient opportunities for more intensive breeding.

Советская тяжеловозная порода лошадей – уникальное достижение отечественных селекционеров. Цель исследования – провести мониторинг генетической структуры советской тяжеловозной породы лошадей по микросателлитным локусам ДНК, определить генетическое разнообразие в популяции, исследовать генетические особенности линий в породе с использованием микросателлитных маркеров. Объектом исследования являлись чистопородные лошади советской тяжеловозной породы с подтверждённым происхождением, материалом – ДНК сертификаты с результатами генотипирования образцов проб волоса лошадей по 17 микросателлитным локусам. При проведении генетико-популяционного анализа рассчитывали следующие показатели: общее число аллелей на локус (Na), частота встречаемости аллелей, уровень полиморфности (Ae), степень наблюдаемой (Ho) и ожидаемой (He) гетерозиготности. Коэффициенты внутрипопуляционного инбридинга (Fis), коэффициент фиксации (Fst), коэффициент инбридинга особей в популяции (Fit) оценивали с применением методов F-статистики и использованием программного обеспечения Microsoft Exсel 2010. Научная новизна исследования заключается в изучении генетических особенностей и внутрилинейной дифференциации, наличии внутренних ресурсов для успешного развития советской тяжеловозной породы без привлечения постороннего генетического потенциала. При тестировании лошадей советской тяжеловозной породы по 17 микросателлитным локусам ДНК определили 118 аллелей, из которых наиболее часто встречаются аллели: HTG4M (0,449), VHL20O (0,378), HMS6L (0,545), HTG10M (0,391), HTG7M (0,132), HMS2H (0,564). Выявлены редкие аллели: ANT4 M(0,002), HMS7Q (0,002), HTG6R(0,002), HMS3O (0,002). Результаты исследований позволяют заключить, что практически во всех линиях советской тяжеловозной породы отсутствует внутрипопуляционный инбридинг, а применяемые методы разведения способствуют поддержанию генетического разнообразия в популяции. The Soviet Heavy Draft horse breed is a unique achievement of domestic breeders. The goal of research is to monitor the genetic structure of the Soviet Heavy Draft horse breed by microsatellite DNA loci, determine the genetic diversity in the population, and study the genetic characteristics of lines in the breed using microsatellite markers. The object of the study was purebred horses of the Soviet Heavy Draft breed with confirmed origin, material – DNA certificates with the results of genotyping of horse hair samples at 17 microsatellite loci. The following parameters were calculated during genetic-population analysis: the total number of alleles per locus (Na), allele frequency, level of polymorphism (Ae), degree of observed (Ho) and expected (He) heterozygosity. The intrapopulation inbreeding coefficients (Fis), fixation coefficient (Fst), inbreeding coefficient of individuals in the population (Fit) were estimated using F-statistics methods and using Microsoft Excel 2010 software. The scientific novelty of the study lies in the study of genetic characteristics and intralinear differentiation, the availability of internal resources for the successful development of the Soviet Heavy Draft breed without attracting extraneous genetic potential. When testing horses of the Soviet Heavy Draft breed, 118 alleles were determined from 17 microsatellite DNA loci, of which the most common alleles are HTG4M (0.449), VHL20O (0.378), HMS6L (0.545), HTG10M (0.391), HTG7M (0.132), HMS2H (0.564). Rare alleles were identified: ANT4 M (0.002), HMS7Q (0.002), HTG6R (0.002), HMS3O (0.002). The research results allow us to conclude that almost all lines of the Soviet Heavy Draft breed do not have intrapopulation inbreeding, and the breeding methods used contribute to maintaining genetic diversity in the population.

Over the last 10 years plant pathologists have begun to realize that more knowledge about the genetic structure of populations of plant pathogens is needed to implement effective control strategies (48). Research on the genetic structure of fungal populations has mushroomed, and review papers that summarize these studies are numerous (7,27,33,34,38). Although the number of fungal studies has increased greatly, the most comprehensive work has focused on a small number of plant-pathogenic fungi. The majority of these fungi can be recognized easily by their fruiting bodies or disease symptoms on aboveground plant parts. It has proven more difficult to assess the genetic structure of fungal populations that exist mainly belowground, because the distribution of individuals cannot be visualized directly and appropriate sampling procedures are less obvious and more cumbersome. Nevertheless, substantial progress has been made in interpreting the population genetic structure of some soilborne fungi (1,17). The purpose of this paper is to provide an overview of the tools and techniques of fungal population genetics. I will try to emphasize approaches that may be applied to studies of soilborne fungi. Instead of providing detailed methods, I will cite recent references where appropriate. There are many opinions regarding which techniques and tools are best suited to studies of fungal populations. I will give a personal and biased viewpoint, which I believe will be most useful to those who are just entering the field.

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (HO = 0.312; HE = 0.436) and B. subterranea presented the highest values (HO = 0.687; HE = 0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q ≥ 0.976) and the low combined probability of identity (I ≤ 9.91 × 10-6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

It has been argued that genetic diversity in crop varieties has been on the decline in recent times due to plant breeding. This can have serious consequences for both the genetic vulnerability of crops and their plasticity when responding to changes in production environments. It is, therefore, vital for plant breeding programs to maintain sufficient diversity in the cultivars deployed for multi-period cultivation. In this study, to understand the temporal genetic diversity in durum wheat, 21 improved durum wheat cultivars released in Morocco, since 1956 and five exotic cultivars currently used in crossing programs were analyzed using 13 microsatellite markers. The analysis revealed a total of 44 alleles and average genetic diversity of 0.485 with genetic distances ranging from 0.077 to 0.846 at 13 microsatellite loci in Moroccan durum wheat cultivars. All the durum cultivars of Morocco could be distinguished using the 13 microsatellite markers. The total number of alleles and unique alleles were highest in cultivars developed before 1990, decreasing in cultivars developed during the 1990s and 2000s, indicating that recent durum breeding efforts have reduced allelic richness in recent cultivars. Thus, deployment of exotic durum wheat lines in breeding programs could enhance genetic diversity in durum wheat cultivars.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

Durum wheat (Triticum durum Desf.) is an important crop both in the world and in Kazakhstan. Durum wheat is used as a valuable raw material in bakery and pasta production. Effective breeding strategies require knowledge of the genetic diversity level of cultivars. Polymorphism of the twenty-nine durum cultivars was analyzed using 7 microsatellite markers. The total number of alleles was 20 and the effective allele number was an average of 2.8. The average polymorphic information content (PIC) value was 0.3658 and ranged from 0.1267 in Xgwm219 to 0.5457 in Xgwm247. The genetic diversity indices of Shannon and Nei were equal to 0.7174, 0.4243, respectively. The level of genetic diversity was relatively high. The genetic distance between cultivars was calculated. Also, with the help of microsatellite markers, a cluster analysis of the studied cultivars was conducted. The results of the study make it possible to assess the level of genetic polymorphism in the studied cultivars and indicate that the used markers are informative. Polymorphic markers were selected for the following studies on the durum genetic diversity. The obtained information will be used in breeding programs aimed at increasing yield and adaptability of durum wheat. Key words: Triticum durum, genetic resources, genetic diversity, microsatellites, SSR.

The implementation of a swift and economical molecular genetic approach, ensuring both efficacy and cost-effectiveness and facilitating population certification, is of utmost significance for breeders and the conservation of Turkiye's native pigeon biodiversity. In this study, we aimed to examine the genetic structure of racing pigeons (Columba livia domestica) raised in Turkiye using a genetic marker panel consisting of eight short tandem repeat (STR) loci. For this purpose, DNA was isolated from the shed feathers of 216 pigeons. Genomic DNA was amplified using the multiplex allele-specific PCR and subsequent capillary electrophoresis with ABI PRISM 3130XL Genetic Analyzer. Next, PCR products were analyzed in the GeneMapper Software program (Applied Biosystems). For parent testing, paternity index (PI), combined paternity index (CPI), and cumulative probability of paternity (CPP) were calculated. Furthermore, population genetic diversity was evaluated using heterozygosity (He), polymorphism information content (PIC), and Hardy–Weinberg equilibrium (HWE) testing. Results revealed that the total number of alleles is 81 and the number of alleles per locus varies between 4 and 19. The similarity rate between parent and offspring was calculated as 99.99% and above. Since no pedigree information was given when the samples were analyzed, obtaining this similarity ratio demonstrates the reliability of the panel. He values range from 0.362 to 0.919, and the PIC values range from 0.295 to 0.909. Loci PG-1, PG-2, and PG-3 show significant genetic diversity, with moderate to high PIC values reflecting varied allele frequencies in the population. Consequently, the set of seven STR markers (+ one sex marker) can be applied to identify and confirm parentage on a regular basis, thereby facilitating efficient breeding programs and ensuring genetic diversity conservation. This panel enables efficient pedigree analysis and gender determination, optimizing cost-effectiveness. The methodology presented in this study is ideal for pedigree analysis and breed certification in the Turkish pigeon breeding industry. Consequently, we affirm that the study data carries considerable national importance.

Wheat landraces could be unique sources of favorable genes for agronomic traits like yield and insect, pest, and disease resistances, as well as quality characteristics that are important for breeding programs. Molecular marker systems offer great opportunities to characterize wheat accessions, which in particular are not morphologically identifiable. In this study, 20 bread wheat landraces collected from different regions of Turkey were characterized by using microsatellite markers (SSRs) and morphological characters. Seventeen morphological characters were used. Fifteen SSR primers were prescreened and the 7 most polymorphic primers were employed in characterization. The most polymorphic SSR loci were Xgwm 95 and 295 with 11 alleles, followed by Xgwm 261 and 325 with 9 alleles. The total number of alleles was 63, with an average number of 9 alleles. The dendrogram showed that the bread wheat landraces can be divided into 2 major groups. Based on matrix values, the closest genotypes of landraces were TR 63445 and TR 63886 for molecular data, and TR 37179 and TR 46797 for morphological data. On the other hand, the most genetically different genotypes were TR 37179 and TR 3608 in the SSR analysis, and TR 14851 and TR 3608 in the morphological characterization. These results showed that SSR markers and morphological characters could be successfully used in genetic characterization and genetic diversity in bread wheat landraces that may be useful for wheat breeding programs as genetic resources.

Premise of the study:Microsatellite markers were developed for Garcinia paucinervis (Clusiaceae), an endangered and endemic tree species of karst habitats, to analyze its genetic diversity and genetic structure.Methods and Results:Using shotgun sequencing on an Illumina MiSeq platform, a total of 22 microsatellite primer sets were characterized, of which 17 were identified as polymorphic. For these polymorphic loci, the total number of alleles per locus ranged from two to 12 across 54 individuals from three populations. The observed and expected heterozygosities ranged from 0.000 to 1.000 and from 0.000 to 0.850, respectively. No pair of loci showed significant linkage disequilibrium. Three loci in one population deviated significantly from Hardy–Weinberg equilibrium (P < 0.05). Seven loci (JSL3, JSL5, JSL22, JSL29, JSL32, JSL39, and JSL43) were successfully amplified in G. bracteata.Conclusions:These markers will be useful in studies on genetic diversity and population structure of G. paucinervis.

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS) using 10 polymorphic microsatellite loci and cytochrome c oxidase subunit I (COI) sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data ( Fst =0.048 7, P <0.001) and mitochondrial DNA data (Fst =0.128, P <0.001) both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.

Genetic variability analysis of 26 sheep breeds in the Czech Republic