Double-digest Restriction Site-associated DNA Research Articles

Genetic diversity and population structure of four Anatolian sheep revealed by genome-wide ddRADseq data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between −0.034 (AKR) to −0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK and MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

Genomic signatures of hybridization between Ixodes ricinus and Ixodes persulcatus in natural populations.

Identifying hybridization between common pathogen vectors is essential due to the major public health implications through risks associated with hybrid's enhanced pathogen transmission potential. The hard-ticks Ixodes ricinus and Ixodes persulcatus are the two most common vectors of tick-borne pathogens that affect human and animal health in Europe. Ixodes ricinus is a known native species in Finland with a well-known distribution, whereas I. persulcatus has expanded in range and abundance over the past 60 years, and currently it appears the most common tick species in certain areas in Finland. Here we used double-digest restriction site-associated DNA (ddRAD) sequencing on 186 ticks (morphologically identified as 92 I. ricinus, and 94 I. persulcatus) collected across Finland to investigate whether RAD generated single nucleotide polymorphisms (SNPs) can discriminate tick species and identify potential hybridization events. Two different clustering methods were used to assign specific species based on how they clustered and identified hybrids among them. We were able to discriminate between the two tick species and identified 11 putative hybrids with admixed genomic proportions ranging from approximately 24 to 76 percent. Four of these hybrids were morphologically identified as I. ricinus while the remaining seven were identified as I. persulcatus. Our results thus indicate that RAD SNPs are robust in identifying both species of the ticks as well as putative hybrids. These results further suggest ongoing hybridization between I. ricinus and I. persulcatus in their natural populations in Finland. The unique ability of RAD markers to discriminate between tick species and hybrids adds a useful aspect to tick evolutionary studies. Our findings align with previous studies and suggest a shared evolutionary history between the species, with instances of individuals possessing a considerable proportion of the other species' genome. This study is a significant step in understanding the formation of hybridization zones due to range expansion potentially associated with climate change.

Development and utilization of genome-wide InDel markers in Sorghum [Sorghum bicolor (L.) Moench

Sorghum (Sorghum bicolor L. Moench) has become an increasingly valuable crop for food, feed, and especially bioenergy feedstock production, which makes the crop extremely attractive for studying genomics and genetic diversity. Molecular markers and genomics play essential roles in sorghum breeding. The rapid development of next-generation sequencing technology has facilitated the identification of genome-wide insertion-deletion (InDel) polymorphisms, enabling the efficient construction of InDel markers that are suitable for user-friendly PCR. This study was conducted with the objective of discovering and developing InDel markers using double digest restriction site-associated DNA sequencing (ddRAD-Seq) data. A total of 19,226 InDels distributed across 10 chromosomes in the sorghum genome was identified. Of those, deletions constituted 65.7% while the remain was insertions. A comprehensive analysis of all the chromosomes revealed a total of 80 InDel sites with a minimum length of 10 bp. For a good conversion of the InDel regions to beneficial molecular markers, specific primers were designed for the amplification of 47 InDel regions that were selected for further investigation. A diverse panel of sorghum consisting of 16 accessions served a source for the developed InDel markers validation. Of the 47 InDel markers, 14 were tested across 16 sorghum accessions and were demonstrated their helpfulness for marker-assisted selection in sorghum. The polymorphic information content (PIC) values of the 16 markers varied between 0.11 and 0.38, with an average of 0.28. The findings of this study indicated that the identification of InDels and the development of molecular markers for sorghum were accomplished using the ddRAD-Seq data.

Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Phylogenetic insights into Central European Chorthippus and Pseudochorthippus (Orthoptera: Acrididae) species using ddRADseq data

The evolution of several orthopteran groups, especially within the grasshopper family Acrididae, remains poorly understood. This is particularly true for the subfamily Gomphocerinae, which comprises cryptic sympatric and syntopic species. Previous mitochondrial studies have highlighted major discrepancies between taxonomic and phylogenetic hypotheses, thereby emphasizing the necessity of genome-wide approaches.In this study, we employ double-digest restriction site-associated DNA sequencing (ddRADseq) to reconstruct the evolution of Central European Chorthippus and Pseudochorthippus species, especially C.smardai, P.tatrae and the C.biguttulus group.Our phylogenomic analyses recovered deep discordance with mitochondrial DNA barcoding, emphasizing its unreliability in Gomphocerinae grasshoppers. Specifically, our data robustly distinguished the C.biguttulus group and confirmed the distinctiveness of C.eisentrauti, also shedding light on its presence in the Berchtesgaden Alps. Moreover, our results support the reclassification of C.smardai to the genus Pseudochorthippus and of P.tatrae to the genus Chorthippus.Our study demonstrates the efficiency of high-throughput genomic methods such as RADseq without prior optimization to elucidate the complex evolution of grasshopper radiations with direct taxonomic implications. While RADseq has predominantly been utilized for population genomics and within-genus phylogenomics, its application extends to resolve relationships between deeply-diverged clades representative of distinct genera.

Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch (Sitta nagaensis) based on ddRAD-seq data

Extra-pair paternity (EPP) is commonly found in socially monogamous birds, especially in small passerine birds, and there are interspecific and intraspecific variations in the extent of EPP. The Chestnut-vented Nuthatch (Sitta nagaensis) is a socially monogamous passerine bird, and verifying whether this species has EPP relies on parentage testing—S. nagaensis is not known to have EPP. In this study, we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing (ddRAD-seq) data. A panel consisting of 50 SNP markers, with a mean heterozygosity of 0.343, was used to resolve 95% of nestlings to fathers. The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999, respectively. This panel of SNP markers is a powerful tool for parentage assignments in S. nagaensis. In addition, we found that three offspring (7.9%) from three nests (23.1%) were the result of extra-pair fertilization out of 38 offspring in 13 nests. Our study provided information on parentage analysis that has not been reported before in S. nagaensis. It also supplemented the understudied EPP behavior of birds in Asia, contributing to a general understanding of the EPP behaviors of birds.

DdRAD Sequencing and DNA Barcoding.

Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including single-nucleotide polymorphisms (SNPs). These SNPs can be utilized to gain evolutionary insights into patterns observed in DNA barcodes, to infer population structure and phylogenies, to detect gene flow and introgression, and to perform species delimitation analyses. The protocol includes chemically shearing genomic DNA with restriction enzymes, unique tagging, size selection, and amplification of the resulting DNA fragments. Here we provide a detailed description of each step of the protocol, as well as information on essential equipment and common issues encountered during laboratory work.

Rapid seasonal changes in phenotypes in a wild Drosophila population

Seasonal environmental change is one of the most rapid and striking environmental variables. Although relatively rapid adaptation to environmental changes over several years or several decades has been described in many taxa, rapid responses to seasonal environments are delicate, and therefore, the detection of the evolutionary responses requires sensitive methods. In this study, we examined seasonal changes in phenotypes related to thermal tolerance and morphological traits of Drosophila lutescens collected at the spring and autumn periods from a single location. We first demonstrated that flies in the two seasonal periods were almost genetically identical using double-digest restriction site-associated DNA sequencing and analysis. Using an experimental design to eliminate the effect of possible confounding factors that influence phenotypes (i.e., maternal effects and the environmental conditions in which each phenotype was analyzed), we showed that the heat tolerance of D. lutescens was significantly higher in the autumn population than in the spring population. Furthermore, cold tolerance was slightly higher in the spring population than in the autumn one. Although wing length and thorax length did not change significantly between seasons, the ratio of wing length to thorax length changed significantly between them. These results suggest that seasonal environmental heterogeneity induces rapid phenotypic changes within a year. Finally, we discuss the possibility of rapid evolutionary responses to seasonal changes.

Implications of methodologies for integrating empirical kinships into ex situ population management using PMx: A case study of Baer's Pochard (Aythya baeri) in North America.

The application of molecular tools to population management can improve the long-term genetic viability of ex situ populations. In this study, we aimed to understand the implications of integrating empirical kinships into the genetic management of an ex situ population of the endangered waterfowl, Baer's pochard (Aythya baeri), in North America. Single nucleotide polymorphism data were generated for 141 Baer's pochard using double digest restriction site-associated DNA sequencing and empirical kinships were derived and integrated into the population management software PMx. Analyses suggested 37.7% of pairwise relationships previously assumed to be unrelated were first, second, or third-order relatives. We determined that most genetic summary statistics were impacted through the calculation of the population's mean kinship, which increased from MK¯=0.0772 to MK¯=0.2074 after empirical kinships were integrated into our analyses. Our results also revealed the importance of understanding how molecular kinships derived from a particular estimator are scaled, if the scale differs significantly from pedigree-based kinships. We describe the theory behind the genetic metrics impacted and provide general guidance on incorporating empirical kinships into ex situ population management as well as provide suggestions for sampling strategies to minimize the biases inherent in merging two types of kinship estimators.

Origins of cryophilous lineages and the role of Beringia: evidence from the cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae)

Abstract The cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae) occurs across the Arctic and is most diverse in the high southern Asian mountains, including presence at the most extreme latitudinal and elevational limits of vascular plants. Using the primary fossil record and double digest restriction site-associated DNA sequencing data, we estimated divergence times of Stellaria and relatives across the Caryophyllaceae. We then used ancestral area and ancestral state reconstructions to interpret the biogeographical history of the group. We found that Stellaria originated in the Miocene and gave rise to repeated New World lineages, mostly from temperate Old World regions and probably via Bering Land Bridges. Circumboreal lineages were recovered of recent, Pleistocene origin and several might have originated in southerly mountains of both the Old World and the New World before subsequently colonizing the Arctic. Ancestral state reconstruction of inhabitance of cold vs. temperate climates and of wet vs. dry habitats revealed repeated evolutionary transitions across these extremes by members of the genus worldwide. Our study, which samples nearly all species within a diverse and cosmopolitan lineage of flowering plants, recovers a group characterized by niche lability and helps to support prior findings of temperate origins of many cryophilous plant lineages. Pre-adaptation to cold might have been a prerequisite for colonization of the Arctic.

Revelation of genetic diversity and genomic footprints of adaptation in Indian pig breeds

In the present study, the genetic diversity measures among four Indian domestic breeds of pig namely Agonda Goan, Ghurrah, Ghungroo, and Nicobari, of different agro-climatic regions of country were explored and compared with European commercial breeds, European wild boar and Chinese domestic breeds. The double digest restriction site-associated DNA sequencing (ddRADseq) data of Indian pigs (102) and Landrace (10 animals) were generated and whole genome sequencing data of exotic pigs (60 animals) from public data repository were used in the study. The principal component analysis (PCA), admixture analysis and phylogenetic analysis revealed that Indian breeds were closer in ancestry to Chinese breeds than European breeds. European breeds exhibited highest genetic diversity measures among all the considered breeds. Among Indian breeds, Agonda Goan and Ghurrah were found to be more genetically diverse than Nicobari and Ghungroo. The selection signature regions in Indian pigs were explored using iHS and XP-EHH, and during iHS analysis, it was observed that genes related to growth, reproduction, health, meat quality, sensory perception and behavior were found to be under selection pressure in Indian pig breeds. Strong selection signatures were recorded in 24.25–25.25 Mb region of SSC18, 123.25–124 Mb region of SSC15 and 118.75–119.5 Mb region of SSC2 in most of the Indian breeds upon pairwise comparison with European commercial breeds using XP-EHH. These regions were harboring some important genes such as EPHA4 for thermotolerance, TAS2R16, FEZF1, CADPS2 and PTPRZ1 for adaptability to scavenging system of rearing, TRIM36 and PGGT1B for disease resistance and CCDC112, PIAS1, FEM1B and ITGA11 for reproduction.

Genomic diversity of wild and cultured Yesso scallop Mizuhopecten yessoensis from Japan and Canada.

The Yesso scallop Mizuhopecten yessoensis is an important aquaculture species that was introduced to Western Canada from Japan to establish an economically viable scallop farming industry. This highly fecund species has been propagated in Canadian aquaculture hatcheries for the past 40 years, raising questions about genetic diversity and genetic differences among hatchery stocks. In this study, we compare cultured Canadian and wild Japanese populations of Yesso scallop using double-digest restriction site-associated DNA (ddRAD) sequencing to genotype 21,048 variants in 71 wild-caught scallops from Japan, 65 scallops from the Vancouver Island University breeding population, and 37 scallops obtained from a commercial farm off Vancouver Island, British Columbia. The wild scallops are largely comprised of equally unrelated individuals, whereas cultured scallops are comprised of multiple families of related individuals. The polymorphism rate estimated in wild scallops was 1.7%, whereas in the cultured strains, it ranged between 1.35 and 1.07%. Interestingly, heterozygosity rates were highest in the cultured populations, which is likely due to shellfish hatchery practices of crossing divergent strains to gain benefits of heterosis and to avoid inbreeding. Evidence of founder effects and drift was observed in the cultured strains, including high genetic differentiation between cultured populations and between cultured populations and the wild population. Cultured populations had effective population sizes ranging from 9 to 26 individuals whereas the wild population was estimated at 25,048-56,291 individuals. Further, a depletion of low-frequency variants was observed in the cultured populations. These results indicate significant genetic diversity losses in cultured scallops in Canadian breeding programs.

Phylogenomic data reveal cryptic diversity and deep phylogeographical structure within the common chuckwalla, Sauromalus ater (Squamata: Iguanidae)

Abstract Understanding how historical geological processes drive diversification and shape the contemporary distribution of species is fundamental to phylogeography. We take a genomic approach in order to elucidate the deep phylogeographical history and species limits of chuckwallas (Sauromalus), a conspicuous group of lizards of the arid lands of south-western North America. Phylogenetic and population genomic analyses of double digest restriction site-associated DNA sequencing data confirm the presence of at least two major lineages, peninsular and continental groups, within the widespread and morphologically variable common chuckwalla (Sauromalus ater). These lineages diversified in the vicinity of the head of the Gulf of California in north-eastern Baja California in the early Pliocene to late Miocene, during the formation of the northern gulf. The peninsular lineage of S. ater subsequently gave rise to the four insular endemic species of Sauromalus associated with the Baja California peninsula. Genomic analyses strongly support the continued recognition of the insular gigantics Sauromalus varius and Sauromalus hispidus as distinct species, although their relationship as sister species remains unresolved. Weaker phylogenetic signal for the insular species Sauromalus slevini and Sauromalus klauberi is provided by the genomic data; thus, it is advocated to continue recognizing these species until additional data can be analysed to evaluate their distinctiveness.

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.

Genomic signatures of ecological divergence between savanna and forest populations of a Neotropical tree.

In eastern Neotropical South America, the Cerrado, a large savanna vegetation, and the Atlantic Forest harbour high biodiversity levels, and their habitats are rather different from each other. The biomes have intrinsic evolutionary relationships, with high lineage exchange that can be attributed, in part, to a large contact zone between them. The genomic study of ecotypes, i.e. populations adapted to divergent habitats, can be a model to study the genomic signatures of ecological divergence. Here, we investigated two ecotypes of the tree Plathymenia reticulata, one from the Cerrado and the other from the Atlantic Forest, which have a hybrid zone in the ecotonal zone of Atlantic Forest-Cerrado. The ecotypes were sampled in the two biomes and their ecotone. The evolutionary history of the divergence of the species was analysed with double-digest restriction site-associated DNA sequencing. The genetic structure and the genotypic composition of the hybrid zone were determined. Genotype-association analyses were performed, and the loci under putative selection and their functions were investigated. High divergence between the two ecotypes was found, and only early-generation hybrids were found in the hybrid zone, suggesting a partial reproductive barrier. Ancient introgression between the Cerrado and Atlantic Forest was not detected. The soil and climate were associated with genetic divergence in Plathymenia ecotypes and outlier loci were found to be associated with the stress response, with stomatal and root development and with reproduction. The high genomic, ecological and morphophysiological divergence between ecotypes, coupled with partial reproductive isolation, indicate that the ecotypes represent two species and should be managed as different evolutionary lineages. We advise that the forest species should be re-evaluated and restated as vulnerable. Our results provide insights into the genomic mechanisms underlying the diversification of species across savanna and forest habitats and the evolutionary forces acting in the species diversification in the Neotropics.

Using genomics and morphometrics to monitor data-poor and commercially exploited octopod populations

Over 150 species of benthic octopods have been described within the ‘catch-all’ Octopus genus (Family: Octopodidae) and yet, many Octopus species harvested by fisheries remain unidentified to species-level due to a lack of distinguishing traits. Within species, there is also limited information on how populations differ genetically and the level of connectivity between populations. Therefore, we sampled octopods from commercial fisheries in southeast Australia, in order to identify the species, examine the phylogeographic relationships among species and the level of population genetic structuring within species, as well as to look for any adaptive genetic variation. The mitochondrial gene, cytochrome oxidase subunit III (COIII), was sequenced in 346 octopods along with single nucleotide polymorphisms using double digest restriction site-associated DNA sequencing (ddRADseq). Morphometric traits were also measured in mature specimens. The southern keeled octopus (‘Octopus’berrima) and pale octopus (‘Octopus’pallidus) were identified using COIII data. For ‘Octopus’berrima, we found that some populations whilst being morphologically similar were genetically distinct. In contrast, ‘Octopus’pallidus populations were both morphologically and genetically distinct across the studied regions. Our results provide key information to better inform conservation and management decisions for developing octopod fisheries in southeast Australia and highlight the importance of genomics tools in the conservation management of commercially and recreationally important species.

Extensive gene flow suggests lack of reproductive barriers between the two subspecies of the green whip snake, Hierophis viridiflavus (Squamata: Colubridae)

Abstract Speciation is the fundamental process that underlies the diversification of species. Different steps of speciation, such as reproductive isolation or ecological niche separation, can evolve in different orders and over different time scales. The two subspecies of the green whip snake, Hierophis viridiflavus viridiflavus and Hierophis viridiflavus carbonarius, sometimes considered as separate species, are ideal to study speciation because they diverged in isolated glacial refugia. So far, little is known about whether these two subspecies are reproductively isolated from each other. Furthermore, it is unknown whether the common melanism of the subspecies H. v. carbonarius might be genetically encoded. We quantified gene flow along two transects across the contact zone of the two subspecies using double-digest restriction site-associated DNA. Our analyses implicated the existence of two well-defined clades that experience substantial gene flow along a contact zone that is ~300 km wide. We detected a clear association between the genetic assignment and the quantitative extent of melanism within the contact zone, which is likely to be genetically polygenic. Given the level of gene flow, it seems that strong postzygotic reproductive barriers have not evolved. We conclude that the two subspecies represent evolutionarily significant units for conservation and suggest treating them taxonomically as subspecies.

Mapping of a major QTL for increased robustness and detection of genome assembly errors in Asian seabass (Lates calcarifer)

BackgroundFor Asian seabass (Lates calcarifer, Bloch 1790) cultured at sea cages various aquatic pathogens, complex environmental and stress factors are considered as leading causes of disease, causing tens of millions of dollars of annual economic losses. Over the years, we conducted farm-based challenges by exposing Asian seabass juveniles to complex natural environmental conditions. In one of these challenges, we collected a total of 1,250 fish classified as either ‘sensitive’ or ‘robust’ individuals during the 28-day observation period.ResultsWe constructed a high-resolution linkage map with 3,089 SNPs for Asian seabass using the double digest Restriction-site Associated DNA (ddRAD) technology and a performed a search for Quantitative Trait Loci (QTL) associated with robustness. The search detected a major genome-wide significant QTL for increased robustness in pathogen-infected marine environment on linkage group 11 (ASB_LG11; 88.9 cM to 93.6 cM) with phenotypic variation explained of 81.0%. The QTL was positioned within a > 800 kb genomic region located at the tip of chromosome ASB_LG11 with two Single Nucleotide Polymorphism markers, R1-38468 and R1-61252, located near to the two ends of the QTL. When the R1-61252 marker was validated experimentally in a different mass cross population, it showed a statistically significant association with increased robustness. The majority of thirty-six potential candidate genes located within the QTL have known functions related to innate immunity, stress response or disease. By utilizing this ddRAD-based map, we detected five mis-assemblies corresponding to four chromosomes, namely ASB_LG8, ASB_LG9, ASB_LG15 and ASB_LG20, in the current Asian seabass reference genome assembly.ConclusionAccording to our knowledge, the QTL associated with increased robustness is the first such finding from a tropical fish species. Depending on further validation in other stocks and populations, it might be potentially useful for selecting robust Asian seabass lines in selection programs.

Source tracing of Anthonomus grandis captured in areas of the USA where the species had previously been eradicated

Anthonomus grandis Boheman (Coleoptera: Curculionidae) is comprised of two subspecies: the boll weevil (A. g. grandis) and the thurberia weevil (A. g. thurberiae). The thurberia weevil is generally regarded as a non-pest variant of the species, whereas the boll weevil is an important economic pest of commercial cotton. In the USA, a nationwide eradication program has been largely successful against the boll weevil, but reintroductions of eradicated zones remain a threat. In 2015, A. grandis was trapped in the functionally eradicated Winter Garden growing region in Texas, where it had not been found since 2012. Weevils established in the area and triggered a multi-year, multi-million-dollar mitigation effort. In 2017, the species was trapped in Hidalgo County, New Mexico, another area where it had been previously eradicated. In this study, we obtained specimens from both trapping events and used double digest restriction site-associated DNA sequencing to obtain genetic data in homology with previously published A. grandis population genetic data. We conducted source tracing to match the captured weevils to previously described populations to determine a likely source population. We also compared the accuracy of the approach to other diagnostic methods currently used by the United States Department of Agriculture. The results suggested that a small founder population of weevils dispersed from the Lower Rio Grande Valley population in southern Texas and northern Tamaulipas and established in the Winter Garden. In Hidalgo Co., NM, the weevils were found to be genetically indistinct from non-pest populations of thurberia weevil associated with Arizona wild cotton (Gossypium thurberi). Thurberia weevils in Arizona remain unlikely to threaten commercial growers. Improved rapid diagnostic tools may be desirable to enable better assessment of trap captures and allow for a reduction in the application of mitigation efforts when the non-pest variant is identified.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targetedGENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.