Restriction Site-associated DNA Sequencing Research Articles

Spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), exhibits large-scale spatial genetic structure across Canada but not fruit host–associated genetic structure

Abstract Spotted-wing drosophila, Drosophila suzukii, is a global pest of soft fruits that is capable of reproducing on a wide range of cultivated and wild plant species. In Canada, D. suzukii was first reported in British Columbia in 2009 and is now widespread across the country. Understanding the genetic structure of D. suzukii populations could be important for pest management if there are phenotypic differences between genetically distinct populations. For example, insect pest populations could respond differently to directional selection imposed by insecticides, differ in their host plant preferences, and vary in their susceptibility to biological control agents. Here, we used double-digest restriction site–associated DNA sequencing to examine large- and fine-scale patterns of the genetic structure of D. suzukii reared from fruit hosts in Canada. We found that this species has a large-scale spatial genetic structure; the flies collected formed two distinct genetic clusters, one of which was distinct to western Canada and the other to eastern Canada. At the local scale, D. suzukii populations showed no evidence of host-associated structuring in British Columbia, suggesting that pest management tactics may be best applied at the landscape level. Our results highlight the need to investigate phenotypic differences between western and eastern D. suzukii populations in Canada.

Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq) Technology.

Double digest restriction-site associated DNA sequencing (ddRADseq) technology combines genome reduced representation by digestion with two restriction enzymes and next generation sequencing (NGS) to obtain thousands of markers (SNP, SSR, and InDels) and genotype tens to hundreds of samples simultaneously. In this chapter, we describe a 96-plex derived ddRADseq protocol that can be set up to obtain different depth of coverage per locus and can be exploited to model and non-model plant species.

Genetic Assessment of Fertile F1 Hybrids between Two Hercules Beetles, Dynastes maya Hardy and D. grantii Horn (Scarabaeidae).

Although hybridization may complicate taxonomic practices, it can be common between animal species. Animal hybridization not only can help with generating phenotypic and species diversity in nature, but also with understanding the genetic and genomic basis of phenotypic evolution in the laboratory. We assessed the genetic composition of captive bred F1 hybrids between two Hercules beetle species using mitochondrial CO1 and nuclear loci from a double-digest restriction-site associated DNA sequencing (ddRADseq) library. We showed that the F1 hybrids were genetically clustered with samples from the maternal species, D. grantii, based on CO1 data. Nuclear genome data, on the other hand, clearly showed that the F1 individuals were genetically intermediate between D. maya, the paternal species, and D. grantii, based on a principal component analysis. Our results also revealed that sampling design may have a major impact on the inferred genetic structure and hybrid individuals using ddRADseq data sets. We discuss the importance and potential from studying the genomics of this hybrid progeny in terms of understanding the origin and maintenance of both intraspecific and interspecific phenotypic divergence and convergence.

Multispectral Phenotyping and Genetic Analyses of Spring Appearance in Greening Plant, Phedimus spp.

The change in appearance during the seasonal transitions in ornamental greening plants is an important characteristic. In particular, the early onset of green leaf color is a desirable trait for a cultivar. In this study, we established a method for phenotyping leaf color change by multispectral imaging and performed genetic analysis based on the phenotypes to clarify the potential of the approach in breeding greening plants. We performed multispectral phenotyping and quantitative trait locus (QTL) analysis of an F1 population derived from 2 parental lines of Phedimus takesimensis, known to be a drought and heat-tolerant rooftop plant species. The imaging was conducted in April of 2019 and 2020 when dormancy breakage occurs and growth extension begins. Principal component analysis of 9 different wavelength values showed a high contribution from the first principal component (PC1), which captured variation in the visible light range. The high interannual correlation in PC1 and in the intensity of visible light indicated that the multispectral phenotyping captured genetic variation in the color of leaves. We also performed restriction site-associated DNA sequencing and obtained the first genetic linkage map of Phedimus spp. QTL analysis revealed 2 QTLs related to early dormancy breakage. Based on the genotypes of the markers underlying these 2 QTLs, the F1 phenotypes with early (late) dormancy break, green (red or brown) leaves, and a high (low) degree of vegetative growth were classified. The results suggest the potential of multispectral phenotyping in the genetic dissection of seasonal leaf color changes in greening plants.

Genomic differentiation with isolation by distance along a latitudinal gradient in the spotted-leg treefrog Polypedates megacephalus.

The patterns of isolation by distance (IBD) entailing increased genetic differentiation among populations have aroused extensive concerns for evolutionary biologists. Although the IBD may act on spatial processes contributing to the genetic differentiation among populations in anuran species, the factors shaping the IBD of frogs among populations in natural systems are largely unknown. Here, we studied the genetic differentiation among six populations with 24 individuals of the spotted-leg treefrog along a latitudinal gradient (1860.31 km) based on 1020 single nucleotide polymorphisms from restriction site-associated DNA sequencing. The results showed that the genetic diversity differed significantly among populations and that the insular populations had higher genetic diversity than the mainland populations. Furthermore, we also found a significant genetic differentiation among populations (FST = 0.277) and no sign of inbreeding (FUNI = -0.145). The IBD was detected for all populations, and a higher degree of the IBD was indicated when controlling for the effects of the isolation between Hainan and mainland populations caused by the Qiongzhou Strait. Our findings suggest that the form of the Qiongzhou Strait plays a key role in shaping the genetic diversity and population differentiation in treefrogs.

Morphology, nuclear SNPs and mate selection reveal that COI barcoding overestimates species diversity in a Mediterranean freshwater amphipod by an order of magnitude.

DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito-nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence=1.8-20.3%), co-occurring in syntopy at two-thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co-occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI-based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs.

Multiple genetic sources facilitate the northward range expansion of an intertidal oyster along China's coast.

Coastal artificial structures on the former mudflats provide available habitats for the rocky intertidal species which can establish new populations in these emerging habitats over their former distribution range limits. As a former southern species, the oyster Crassostrea sikamea has become a pioneer and rapidly invaded the artificial shorelines in northern China. We used a seascape genomics approach to investigate the population structure and genetic sources of C. sikamea on the coastal artificial structures, which is crucial for understanding the genetic mechanisms driving species distribution range expansion and invasion pathway of intertidal species. Five C. sikamea populations, including two artificial substrate populations (WGZ and ZAP), one oyster reef population (LS), and two natural rocky shore populations (ZS and XM), were measured using single nucleotide polymorphism (SNPs) obtained from double digest restriction-site associated DNA sequencing (ddRAD-Seq). Redundancy analyses (RDA) were implemented for investigating the relationship between local temperature variables and the temperature adaptability of C. sikamea. Genetic diversity, direction and strength of gene flow, and population structure all revealed that the LS and ZS populations were the genetic sources for the oyster populations on the emerging northern coastal artificial structures. Results of RDA showed that there were different adaptive potentials for northern and southern populations to local temperature variables and the oyster reef population which frequently suffers from heat stress owned high heat adaptability. The ZS population as a genetic source nearby the Yangtze River estuary provided mass larvae for the northern populations, and the other genetic source, the heat-tolerant LS population, in the oyster reef played an important role in the post-settlement success by providing preadapted genotypes. These results highlight the importance of multiple sources with divergent adaptative capabilities for biological invasion, and also emphasize the importance of the oyster reef in coastal biodiversity and conservation.

Haploid gynogens facilitate disomic marker development in paleotetraploid sturgeons.

Acipenseriformes (sturgeons and paddlefishes) are of substantial conservation concern, and development of genomic resources for these species is difficult due to past whole genome duplication. Development of disomic markers for polyploid organisms can be challenging due to difficulty in resolving alleles at a single locus from those among duplicated loci. In this study, we detail the development of disomic markers for the endangered pallid sturgeon (Scaphirhynchus albus) found in North America. One of the strategies for pallid sturgeon conservation is to stock U.S. rivers with offspring of pure pallid sturgeon, but introgression with the sympatric shovelnose sturgeon (S.platorynchus) threatens pallid sturgeon genetic integrity. Currently, 19 microsatellite loci are used to differentiate between both species and their hybrids, but the markers are insufficient to robustly identify backcrosses. We performed double digest restriction site-associated DNA sequencing (ddRADseq) on shovelnose sturgeon haploid gynogens to produce a reduced-representation genomic reference. Contiguous sequences that were heterozygous within a haploid individual were flagged as potentially encompassing multiple loci. Approximately 60 individuals of each species from two management units were sequenced, and reads were mapped to the haploid reference to identify single nucleotide polymorphisms (SNPs) at individual loci. The final data set contained 11,082 microhaplotyped loci which offer at least an order of magnitude greater resolution for species discrimination than the current panel of 19 microsatellites. These markers will be used to examine a larger sample of Scaphirhynchus individuals throughout their ranges to determine the extent and trajectory of hybridization.

Unequal Reproduction Early in a Social Transition: Insights from Invasive Wasps.

AbstractIn eusocial insects, nestmate queens can differ in their reproductive output, causing asymmetries in the distribution of mutual benefits. However, little is known about how reproductive success is partitioned in incipiently polygynous species, which would provide clues about the evolutionary forces shaping the emergence of polygyny. Here, we leverage a recent transition from predominantly single-queen (monogyne) to multiple-queen (polygyne) colonies in an invasive yellowjacket species to investigate whether queens in incipiently polygyne colonies invest equally in reproductive effort or vary in their relative investment in each caste. We excavated nine polygyne Vespula pensylvanica colonies in Hawaii and used restriction site-associated DNA sequencing to infer the parentage of worker, male, and gyne (daughter queen) pupae from each nest comb. In four colonies with at least eight gyne pupae, a single queen produced most or all gynes. These queens had no male offspring and few worker offspring, suggesting that a subset of nestmate queens might exploit the collective benefits of newly polygyne societies. In contrast to most queens, gyne producers had offspring distributed nonrandomly across nest combs. Nestmate queens generally exhibited low relatedness levels. Our results suggest that rapid, ecologically driven transitions to polygyny among unrelated queens may, at their onset, be vulnerable to reproductive asymmetries that are likely evolutionarily unstable. More broadly, this study contributes to the understanding of social evolution by uncovering asymmetric partitioning of reproduction in a population with newly evolved polygyny and raises questions about the future trajectories of introduced populations.

Molecular ecology of the sleeper shark subgenus Somniosus (Somniosus) reveals genetic homogeneity within species and lack of support for S. antarcticus.

Inferences made from molecular data support regional stock assessment goals by providing insights into the genetic population dynamics of enigmatic species. Population genomics metrics, such as genetic diversity and population connectivity, serve as useful proxies for species health and stability. Sleeper sharks (genus Somniosus) are ecologically important deep-sea predators, estimated to reach ages of 250 to 300 yr and taking decades to reach sexual maturity. The subgenus Somniosus (Somniosus) is comprised of 3 species: S. pacificus, S. microcephalus, and S. antarcticus. Given the life history strategy of somniosids, they are vulnerable to overfishing and population declines. Further, data to assess the stocks of these species are limited. To address this deficiency, we used the reduced representation library method Restriction-site Associated DNA sequencing (RADseq) to conduct phylogenomic and population genomics analyses, providing novel information for use in stock assessments. Our results strongly support the species status of S. microcephalus (N = 79), but recover S. antarcticus (N = 2) intermixed within the S. pacificus (N = 170) clade. Population genomics analyses reveal genetic homogeneity within S. pacificus and S. microcephalus, and estimates of effective population size were in the hundreds for both species. Kinship analysis identified 2 first-degree relative pairs within our dataset (1 within each species). Our results contribute new information for stock assessments of these uniquely long-lived species by providing the strongest molecular evidence to date for the synonymization of S. antarcticus and S. pacificus, as well as estimating population genomic metrics for each supported species within the Somniosus (Somniosus) subgenus.

Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification

We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

Detecting a novel locus associated with pellicle peelability in Japanese chestnut populations preselected for a major peelability gene

• The distribution of phenotypic values of peelability in Japanese chestnut populations preselected for a major peelability gene was biased toward maximum values. • Use of rank-based inverse normal transformation and categorization of peelability phenotypes was applied for GWAS to further identify QTLs associated with peelability. • A single QTL for peelability was identified at CCR1.0F_56177061 on chromosome F. Peelability is one of the most important traits in Japanese chestnut ( Castanea crenata Sieb. et Zucc.) breeding. Previous studies revealed that the peelability of Japanese chestnut is controlled by a major recessive peeling gene (MRPG). This locus has been used effectively for marker-assisted selection in chestnut breeding programs, but there are also likely to be minor loci that would further improve the efficiency of breeding for peelability. We conducted a genome-wide association study (GWAS) based on a variational approximation version of BayesB using 27 cultivars and MRPG-preselected populations consisting of 520 F 1 seedlings from 22 families. Using double-digest restriction site-associated DNA sequencing (ddRAD-Seq), we obtained 9164 tag single-nucleotide polymorphisms. Because the phenotypic values of peelability were distributed very asymmetrically, with a peak at the high end due to the preselection for MRPG, we converted the original data set to both inverse normal transformed values and categorical scores, and we analyzed both sets of converted data by GWAS. In both analyses, a quantitative trait locus (QTL) for peelability at CCR1.0F_56177061 was identified with posterior probability values of 0.68 and 1.00, respectively. The average values of genotypes at CCR1.0F_56177061 for cultivars and seedlings in this study were 60.4% (peelability rate) for CC, 63.7% for CT, and 86.5% for TT. This analysis reveals a significant association between CCR1.0F_56177061 genotype and peelability, indicating that this newly identified QTL will be useful for improving peelability in chestnut breeding programs.

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales.

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Evidence of backcross inviability and mitochondrial DNA paternal leakage in sea turtle hybrids.

Hybridization is known to be part of many species' evolutionary history. Sea turtles have a fascinating hybridization system in which species separated by as much as 43million years are still capable of hybridizing. Indeed, the largest nesting populations in Brazil of loggerheads (Caretta caretta) and hawksbills (Eretmochelys imbricata) have a high incidence of hybrids between these two species. A third species, olive ridleys (Lepidochelys olivacea), is also known to hybridize although at a smaller scale. Here, we used restriction site-associated DNA sequencing (RAD-Seq) markers, mitogenomes, and satellite-telemetry to investigate the patterns of hybridization and introgression in the Brazilian sea turtle population and their relationship with the migratory behaviours between feeding and nesting aggregations. We also explicitly test if the mixing of two divergent genomes in sea turtle hybrids causes mitochondrial paternal leakage. We developed a new species-specific PCR-assay capable of detecting mitochondrial DNA (mtDNA) inheritance from both parental species and performed ultra-deep sequencing to estimate the abundance of each mtDNA type. Our results show that all adult hybrids are first generation (F1) and most display a loggerhead migratory behaviour. We detected paternal leakage in F1 hybrids and different proportions of mitochondria from maternal and paternal species. Although previous studies showed no significant fitness decrease in hatchlings, our results support genetically-related hybrid breakdown possibly caused by cytonuclear incompatibility. Further research on hybrids from other populations in addition to Brazil and between different species will show if backcross inviability and mitochondrial paternal leakage is observed across sea turtle species.

Conservation genomics of Agave tequilana Weber var. azul: low genetic differentiation and heterozygote excess in the tequila agave from Jalisco, Mexico.

Genetic diversity is fundamental for the survival of species. In particular, in a climate change scenario, it is crucial that populations maintain genetic diversity so they can adapt to novel environmental conditions. Genetic diversity in wild agaves is usually high, with low genetic differentiation among populations, in part maintained by the agave pollinators such as the nectarivorous bats. In cultivated agaves, patterns of genetic diversity vary according to the intensity of use, management, and domestication stage. In Agave tequilana Weber var. azul (A. tequilana thereafter), the plant used for tequila production, clonal propagation has been strongly encouraged. These practices may lead to a reduction in genetic diversity. We studied the diversity patterns with genome-wide SNPs, using restriction site associated DNA sequencing in cultivated samples of A. tequilana from three sites of Jalisco, Mexico. For one locality, seeds were collected and germinated in a greenhouse. We compared the genomic diversity, levels of inbreeding, genetic differentiation, and connectivity among studied sites and between adults and juvenile plants. Agave tequilana presented a genomic diversity of HT = 0.12. The observed heterozygosity was higher than the expected heterozygosity. Adults were more heterozygous than juveniles. This could be a consequence of heterosis or hybrid vigor. We found a shallow genetic structure (average paired FST = 0.0044). In the analysis of recent gene flow, we estimated an average migration rate among the different populations of m = 0.25. In particular, we found a population that was the primary source of gene flow and had greater genomic diversity (HE and HO ), so we propose that this population should continue to be monitored as a potential genetic reservoir. Our results may be the consequence of more traditional management in the studied specific region of Jalisco. Also, the exchange of seeds or propagules by producers and the existence of gene flow due to occasional sexual reproduction may play an important role in maintaining diversity in A. tequilana. For populations to resist pests, to continue evolving and reduce their risk of extinction under a climate change scenario, it is necessary to maintain genetic diversity. Under this premise we encourage to continue acting in conservation programs for this species and its pollinators.

Development and identification of 131 SNP markers in Sthenoteuthis pteropus (Steenstrup)

The orangeback flying squid, Sthenoteuthis pteropus, is a species of significant potential value that is widely distributed in the tropical and temperate waters of the Atlantic Ocean. There have been no reports of the population genetics and effective molecular markers for this species due to a lack of reliable information regarding its genetic structure and its many individual differences, as well as its complex and changeable life history. Therefore, the development of auxiliary molecular markers would contribute to the development, sustainable utilization, and protection of the species. In this study, 131 novel single nucleotide polymorphism (SNP) markers were developed by double digest restriction-site associated DNA sequencing (ddRAD-Seq). The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.00 to 0.80 and 0.18 to 0.50, respectively. The polymorphism information content (PIC) value ranged from 0.18 to 0.50. None of the marker locations significantly deviated from the Hardy–Weinberg equilibrium (p > 0.05) after a Bonferroni correction. These polymorphic SNPs will be important in the further analysis of the population heredity of S. pteropus and its scientific management.

Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

DdRADseq-mediated detection of genetic variants in sugarcane.

The article presents an optimization of the key parameters for the identification of SNPs in sugarcane using a GBS protocol based on two Illumina NextSeq and NovaSeq platforms. Sugarcane (Saccharum sp.), a world-wide known feedstock for sugar production, bioethanol, and energy, has an extremely complex genome, being highly polyploid and aneuploid. A double-digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-fibre biotype for the detection of single nucleotide polymorphisms (SNPs). In this work we tested two Illumina sequencing platforms, read size (70 vs. 150bp), different sequencing coverage per individual (medium and high coverage), and single-reads versus paired-end reads. We also explored different variant calling strategies (with and without reference genome) and filtering schemes [combining two minor allele frequencies (MAFs) with three depth of coverage thresholds]. For the discovery of a large number of novel SNPs in sugarcane, we recommend longer size and paired-end reads, medium sequencing coverage per individual and Illumina platform NovaSeq6000 for a cost-effective approach, and filter parameters of lower MAF and higher depth coverages thresholds. Although the de novo analysis retrieved more SNPs, the reference-based method allows downstream characterization of variants. For the two best performing matrices, the number of SNPs per chromosome correlated positively with chromosome length, demonstrating the presence of variants throughout the genome. Multivariate comparisons, with both matrices, showed closer relationships among commercial hybrids than with the high-fibre biotype. Functional analysis of the SNPs demonstrated that more than half of them landed within regulatory regions, whereas the other half affected coding, intergenic and intronic regions. Allelic distances values were lower than 0.07 when analysing two replicated genotypes, confirming the protocol robustness.

Genome-wide SNPs reveal novel patterns of spatial genetic structure in Aedes albopictus (Diptera Culicidae) population in China.

Since the second half of the 20th century, Aedes albopictus, a vector for more than 20 arboviruses, has spread worldwide. Aedes albopictus is the main vector of infectious diseases transmitted by Aedes mosquitoes in China, and it has caused concerns regarding public health. A comprehensive understanding of the spatial genetic structure of this vector species at a genomic level is essential for effective vector control and the prevention of vector-borne diseases. During 2016-2018, adult female Ae. albopictus mosquitoes were collected from eight different geographical locations across China. Restriction site-associated DNA sequencing (RAD-seq) was used for high-throughput identification of single nucleotide polymorphisms (SNPs) and genotyping of the Ae. albopictus population. The spatial genetic structure was analyzed and compared to those exhibited by mitochondrial cytochrome c oxidase subunit 1 (cox1) and microsatellites in the Ae. albopictus population. A total of 9,103 genome-wide SNP loci in 101 specimens and 32 haplotypes of cox1 in 231 specimens were identified in the samples from eight locations in China. Principal component analysis revealed that samples from Lingshui and Zhanjiang were more genetically different than those from the other locations. The SNPs provided a better resolution and stronger signals for novel spatial population genetic structures than those from the cox1 data and a set of previously genotyped microsatellites. The fixation indexes from the SNP dataset showed shallow but significant genetic differentiation in the population. The Mantel test indicated a positive correlation between genetic distance and geographical distance. However, the asymmetric gene flow was detected among the populations, and it was higher from south to north and west to east than in the opposite directions. The genome-wide SNPs revealed seven gene pools and fine spatial genetic structure of the Ae. albopictus population in China. The RAD-seq approach has great potential to increase our understanding of the spatial dynamics of population spread and establishment, which will help us to design new strategies for controlling vectors and mosquito-borne diseases.