Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228 bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships.

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.

Population Genetics of Polyploid Complex Psidium cattleyanum Sabine (Myrtaceae): Preliminary Analyses Based on New Species-Specific Microsatellite Loci and Extension to Other Species of the Genus.

Polyploidy is a phenomenon that alters the genetic diversity of populations and has been reported as one of the most important evolutionary forces for plant diversification. The Psidium cattleyanum complex comprises a group of wild populations with several ploidy levels reported in the literature. The multiple cytotypes, associated with its wide distribution area, make this species a potential key model for understanding evolutionary processes related to polyploidization. In this study, we isolated and characterized nuclear microsatellite markers of P. cattleyanum and tested their transferability to other nine species of the genus. We performed a preliminary analysis of genetic diversity and population structure in three populations of P. cattleyanum. The three populations analyzed had different chromosome numbers, being polyploid cytotypes (2n = 6x = 66, 2n = 7x = 77 and 2n = 8x = 88). We designed 46 primer pairs and successfully amplified 37 markers, from which the 10 best were selected for analysis. Considering both the PIC and DP values, most of markers were highly informative. The new SSR markers were used to assess the levels of genetic diversity of the populations and detected one population with predominance of sexual reproduction. DAPC analysis pointed the formation of three groups, which corresponded to the populations analyzed. The markers were successfully amplified in related species, with some species presenting 80% transferability. By producing this panel of polymorphic microsatellites, we contribute to the understanding evolution in groups of natural polyploids for future studies.

Development of microsatellite markers for Lippia alba and related Lippia species.

Microsatellite primers were developed in Lippia alba complex to better understanding the origins and evolution of the species. We sought to increase the numbers of available simple sequence repeat (SSR) markers. We performed low-coverage (~ twofold) genomic DNA sequencing of a diploid accession and generated a de novo assembly comprising 175,572 contigs. Sixteen SSR loci were selected and of these 13 SSR loci were successfully amplified in 20 L. alba tetraploid accessions and in 12 other Lippia species. Only one SSR locus was monomorphic, whereas 12 loci were polymorphic, yielding one to nine alleles. The heterozygosity was similar among markers, with values of 0.274-0.485; the polymorphism information content values varied from 0.237 to 0.367. These markers were successfully amplified in related species with 85% of transferability on average. Thus, we demonstrate the utility of including a de novo assembly step to obtain SSR markers from low-coverage genomic datasets.

Genetic diversity and karyotype of Pitcairnia azouryi: an endangered species of Bromeliaceae endemic to Atlantic Forest inselbergs.

Plant species of various families, such as those of Bromeliaceae, occur on inselbergs where they are subject to geographic isolation and environmental conditions that can lead to genetic erosion. This, in turn, can result in the loss of natural populations due to homozygosis, or changes in ploidy that may lead to reproductive isolation. The genetic diversity of five natural populations of Pitcairnia azouryi was measured using nine microsatellite markers transferred from P. albiflos and P. geyskesii. Chromosome numbers and nuclear DNA content were also evaluated. The results indicated moderate genetic differentiation among populations (FST = 0.188), and significant gene flow (Nm = 1.073) in four of the five populations. P. azouryi has, predominantly, 2n = 50 chromosomes and DNA content of 2C = 1.16 pg, but the tetraploid condition was found (2n = 100 and 2C = 2.32 pg) in seedlings of an individual of the most geographically isolated population. The moderate level of genetic structuring observed for P. azouryi seems to be related to its disjoint geographical distribution and the locally aggregated spatial structure of the populations, which are isolated from each other, hindering the inter and intrapopulational gene flow. This interpretation was also evidenced by the mantel test (r = 0.777, P < 0.05). The occurrence of diploid individuals with tetraploid seedlings is indicative of events of eupolyploidization, possibly due to the environmental conditions of this geographically isolated population.

Development and characterization of eleven microsatellite loci for the tropical understory tree Paypayrola blanchetiana Tul. (Violaceae).

Microsatellites markers were developed for Paypayrola blanchetiana (Violaceae), a near-dispersing forest tree forming aggregated populations, to investigate genetic diversity and gene flow among subpopulations in a fragmented environment. Next generation sequencing (Illumina platform) was used to develop ten nuclear microsatellite loci and one plastid microsatellite locus that amplify in P. blanchetiana. Polymorphism was tested in two subpopulations separated by a distance of approximately 11 km. The identified loci contained between two and five alleles per locus. Observed heterozygosity ranged between 0.063 and 0.563 in both subpopulations, while expected heterozygosity ranged from 0.063 to 0.567 in the first, and 0.063-0.627 in the second subpopulation. The microsatellites are among the first in the family Violaceae and will be useful for population genetic studies in this species. Amplification was successful in one further Paypayrola species from Amazonia, which suggest a wider usefulness of the present markers.

Detecting population structure of Paleosuchus trigonatus (Alligatoridae: Caimaninae) through microsatellites markers developed by next generation sequencing.

We isolated and characterized 10 new microsatellites loci for Paleosuchus trigonatus using ION TORRENT Sequencing Technology. We tested the transferability of these loci to three related species of the subfamily Caimaninae, and used these bi-parental markers to test population structure and genetic diversity of two populations of P. trigonatus impacted by hydroelectric dam construction on the Madeira (N = 16) and Xingu (N = 16) rivers. We also investigated the transferability of these markers to three related species: Paleosuchus palpebrosus (N = 5), Caiman crocodilus (N = 6) and Melanosuchus niger (N = 6). The genetic diversity of P. trigonatus was low in both the Madeira (He: 0.535 ± 0.148) and Xingu (He: 0.381 ± 0.222) populations, but the loci were sufficiently polymorphic to be used in system of mating and kinship studies in P. trigonatus. DAPC analysis with our set of microsatellites loci was able to separate the four species of Caimaninae studied and to detect a shallow genetic structure between Madeira and Xingu populations of P. trigonatus. AMOVA and STRUCTURE analyses using locprior model corroborate this shallow genetic structure. These novel molecular markers will be also useful in conservation genetics and phylogeographic studies of P. trigonatus, since they improve our ability to monitor the putative effects of dams on the loss of genetic diversity and allow us to investigate population dynamics and microevolutionary processes that occurred in the species.

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

Development of microsatellite markers for six Tetranychus species by transfer from Tetranychus urticae genome.

Microsatellite markers are frequently used to explore the population genetic structure of organisms. Spider mites (genus Tetranychus) are important agricultural pests. Several markers have been developed for T. urticae, but for other spider mites, few such markers are available, hampering studies of their population genetics. In this study, we developed and characterized microsatellite markers for six non-model spider mite species (T. truncatus, T. kanzawai, T. ludeni, T. piercei, T. phaselus and T. pueraricola) by cross-species amplification of markers in the T. urticae genome, in order to better understand the population structure of Tetranychus species. Among 228 screened loci, many were polymorphic, including 13 loci in T. urticae, 11 loci in T. truncatus, 15 loci in T. pueraricola, 23 loci in T. kanzawai, 19 loci in T. piercei, 11 loci in T. phaselus and 9 loci in T. ludeni. Sequence analysis determined that the fragment length variations of the transferred microsatellites were mainly due to the variations of the numbers of repeats. These new microsatellite markers should be useful for studying the population genetics of the seven Tetranychus species.

Isolation and characterization of cross-amplification microsatellite panels for species of Procapra (Bovidae; Antilopinae).

The three Procapra species, Tibetan gazelle (P. picticaudata), Mongolian gazelle (P. gutturosa) and Przewalski's gazelle (P. przewalskii) are endemic to Asia. Several intraspecific genetic issues have been studied with species-specific microsatellite loci in these Asian gazelles. However, cross-species microsatellite panels are absent, which inhibits comparative conservation and evolutionary studies of the Procapra. In this study, we isolated 20 cross-species microsatellite loci for Procapra from both related species and the genomic library of P. przewalskii. Fifty-three samples of the three gazelles were used to characterize the markers. Allele numbers ranged from three to 20, with a mean of 7.93 per locus. Observed heterozygosity (H(O)) averaged 0.680 and expected heterozygosity (H(E)) 0.767. The mean polymorphic information content (PIC) was 0.757 for P. picticaudata, 0.803 for P. gutturosa and 0.590 for P. przewalskii. Nine loci were significantly deviated from Hardy-Weinberg (H-W) equilibrium in the three species. Significant linkage disequilibrium was detected in four pairs of loci in P. przewalskii, five pairs in P. gutturosa and 51 pairs in P. picticaudata. Considering the abundance of published loci and their high success rates of cross-amplification, testing and utilization of loci from related species is efficient for wild species of Bovidae. The cross-species microsatellite loci we developed will facilitate further interspecies genetic studies in Procapra.

Microsatellite development for an endangered bream Megalobrama pellegrini (Teleostei, Cyprinidae) using 454 sequencing.

Megalobrama pellegrini is an endemic fish species found in the upper Yangtze River basin in China. This species has become endangered due to the construction of the Three Gorges Dam and overfishing. However, the available genetic data for this species is limited. Here, we developed 26 polymorphic microsatellite markers from the M. pellegrini genome using next-generation sequencing techniques. A total of 257,497 raw reads were obtained from a quarter-plate run on 454 GS-FLX titanium platforms and 49,811 unique sequences were generated with an average length of 404 bp; 24,522 (49.2%) sequences contained microsatellite repeats. Of the 53 loci screened, 33 were amplified successfully and 26 were polymorphic. The genetic diversity in M. pellegrini was moderate, with an average of 3.08 alleles per locus, and the mean observed and expected heterozygosity were 0.47 and 0.51, respectively. In addition, we tested cross-species amplification for all 33 loci in four additional breams: M. amblycephala, M. skolkovii, M. terminalis, and Sinibrama wui. The cross-species amplification showed a significant high level of transferability (79%-97%), which might be due to their dramatically close genetic relationships. The polymorphic microsatellites developed in the current study will not only contribute to further conservation genetic studies and parentage analyses of this endangered species, but also facilitate future work on the other closely related species.

Development of new microsatellite markers for Salvia officinalis L. and its potential use in conservation-genetic studies of narrow endemic Salvia brachyodon Vandas.

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (H(E)) was compared to the expected genetic diversity at mutation-drift equilibrium (H(EQ)) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Individual and collective protective responses during the early phase of the COVID-19 pandemic in 10 different countries: Results from the EUCLID online survey.

BACKGROUND: In times of unprecedented infectious disease threats, it is essential to understand how to increase individual protective behaviors and support for collective measures. The present study therefore examines factors associated with individual and collective pathways. METHODS: Data was collected through an online survey from 4483 participants (70.8% female, M = 41.2 years) across 10 countries from April 15, 2020 to June 2, 2020 as part of the "EUCLID" project (https://euclid.dbvis.de). Structural equation modeling was used to examine individual and collective pathways across and within countries. RESULTS: Overall, the adoption of individual protective behaviors and support for collective measures were high. Risk perception on the individual level and perceived effectiveness at the collective level were positively associated with both individual protective behaviors and support for collective measures. Furthermore, the model explained considerable variance in individual (40.7%) and collective protective behaviors (40.8%) and was largely replicated across countries. CONCLUSIONS: The study extends previous research by demonstrating that individual risk perception and perceived effectiveness of collective measures jointly affect individual protective health behaviors and support for collective measures. These findings highlight the need to jointly consider a variety of behavioral actions against infectious disease threats, acknowledging interactions between individual and collective pathways.

Cross-Amplification in Strigiformes: A New STR Panel for Forensic Purposes.

Strigiformes are affected by a substantial decline mainly caused by habitat loss and destruction, poaching, and trapping. Moreover, the increasing trend in bird trade and the growing interest in wild-caught rather than captive-bred birds are expected to encourage illegal trade. The biomolecular investigation represents a valuable tool to track illegal trade and to explore the genetic variability to preserving biodiversity. Microsatellite loci (STRs) are the most used markers to study genetic variability. Despite the availability of species-specific microsatellite loci in Strigiformes, a unique panel permitting the description of the genetic variability across species has not been identified yet. We tested 32 highly polymorphic microsatellite markers to evaluate the reliability of a unique microsatellite panel in different species of Strigiformes and its use for conservation and forensic purposes. We included in the study 84 individuals belonging to 28 parental groups and 11 species of Strigiformes. After screening polymorphic microsatellite loci, the description of genetic variability, and the kinship assessment, we characterized a final panel of 12 microsatellite loci able to identify individuals in 9 Strigiformes species. This STR panel might support the authorities in the forensic investigation for suspected smugglers and false parental claims; moreover, it can be useful to evaluate relatedness among individuals in captive-bred populations and to implement research projects finalized to the description of the genetic variability in wild populations.

Competitive activation cross amplification combined with smartphone-based quantification for point-of-care detection of single nucleotide polymorphism.

In this study, we firstly propose a novel smartphone-assisted visualization SNP genotyping method termed competitive activation cross amplification (CACA). The mutation detection strategy depends on the ingenious design of both a start primer and a verification probe with ribonucleotide insertion through competitive combination and perfect matching with the target DNA, Meanwhile, the RNase H2 enzyme was utilized to specifically cleave ribonucleotide insertion and achieve extremely specific dual verification. Simultaneously, the results allow both colorimetric and fluorescence product dual-mode visualization by using self-designed 3D-printed dual function cassette. We validated this novel CACA by analyzing the Salmonella Pullorum rfbS gene at the 237th site, successfully solve the current bottleneck of specific identification and visual detection of this pathogen. The concentration detection limits of the plasmid and genomic DNA were 1500 copies/µL and 3.98 pg/µL, respectively, and as low as the presence of 0.1% mutant-type can be distinguished from 99.9% wild-type. Combined with a powerful hand-warmer, which can provide heating more than 60 °C for 20 h to realize power-free, dual function cassette and smartphone quantitation, our novel CACA platform firstly realizes user-friendly, cost-effective, portable, rapid, and accurate POC detection of SNP.

Development of EST-based new SSR markers in seabuckthorn.

EST-based SSR markers were developed by screening a collection of 1584 clustered ESTs of seabuckthorn (Hippophae rhamnoides). PCR primers were designed for the amplification of 30 microsatellite loci. Two to five allelic bands were displayed by nine primer pairs in H. rhamnoides genotypes and by eleven primer pairs in H. salicifolia genotypes. None of the thirty primer pairs detected polymorphism in H. tibetana genotypes. Considering the high polymorphism detected in the tested genotypes and their direct origin from the genic regions, these EST-SSR markers hold immense promise in seabuckthorn genome analysis, molecular breeding and population genetics.

Development of genome-wide polymorphic microsatellite markers for Trichinella spiralis.

BACKGROUND: Trichinella nematodes are globally distributed food-borne pathogens, in which Trichinella spiralis is the most common species in China. Microsatellites are a powerful tool in population genetics and phylogeographic analysis. However, only a few microsatellite markers were reported in T. spiralis. Thus, there is a need to develop and validate genome-wide microsatellite markers for T. spiralis. METHODS: Microsatellites were selected from shotgun genomic sequences using MIcroSAtellite identification tool (MISA). The identified markers were validated in 12 isolates of T. spiralis in China. RESULTS: A total of 93,140 microsatellites were identified by MISA from 9267 contigs in T. spiralis genome sequences, in which 16 polymorphic loci were selected for validation by PCR with single larvae from 12 isolates of T. spiralis in China. There were 7-19 alleles per locus (average 11.25 alleles per locus). The observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.325 to 0.750 and 0.737 to 0.918, respectively. The polymorphism information content (PIC) ranged from 0.719 to 0.978 (average 0.826). Among the 16 loci, markers for 10 loci could be amplified from all 12 international standard strains of Trichinella spp. CONCLUSIONS: Sixteen highly polymorphic markers were selected and validated for T. spiralis. Primary phylogenetic analysis showed that these markers might serve as a useful tool for genetic studies of Trichinella parasites.

Cross-amplification and characterization of microsatellite loci for the Neotropical orchid genus Epidendrum.

In this study we tested the cross-amplification of 33 microsatellite loci previously developed for two closely related Neotropical orchid genera (Epidendrum and Laelia). A set of ten loci were polymorphic across five examined species (20 individuals each) with 2 to 15 alleles per locus. The mean expected and observed heterozygosity (average across species) ranged from 0.34 to 0.82 and from 0.27 to 0.85, respectively. In addition we tested all loci in 35 species representative of the genus Epidendrum. Of these, 26 loci showed successful amplification. Cross-application of these loci represent a potential source of co-dominant markers for evolutionary, ecological and conservation studies in this important orchid genus.