Restriction Site-associated DNA Research Articles

Identification of Insertion and Deletion (InDel) Markers for Chickpea (Cicer arietinum L.) Based on Double-Digest Restriction Site-Associated DNA Sequencing.

Enhancing the marker repository and the development of breeder-friendly markers in chickpeas is important in relation to chickpea genomics-assisted breeding applications. Insertion-deletion (InDel) markers are widely distributed across genomes and easily observed with specifically designed primers, leading to less time, cost, and labor requirements. In light of this, the present study focused on the identification and development of InDel markers through the use of double-digest restriction site-associated DNA sequencing (ddRADSeq) data from 20 chickpea accessions. Bioinformatic analysis identified 20,700 InDel sites, including 15,031 (72.61%) deletions and 5669 (27.39%) insertions, among the chickpea accessions. The InDel markers ranged from 1 to 25 bp in length, while single-nucleotide-length InDel markers were found to represent the majority of the InDel sites and account for 79% of the total InDel markers. However, we focused on InDel markers wherein the length was greater than a single nucleotide to avoid any read or alignment errors. Among all of the InDel markers, 96.1% were less than 10 bp, 3.6% were between 10 and 20 bp, and 0.3% were more than 20 bp in length. We examined the InDel markers that were 10 bp and longer for the development of InDel markers based on a consideration of the genomic distribution and low-cost genotyping with agarose gels. A total of 29 InDel regions were selected, and primers were successfully designed to evaluate their efficiency. Annotation analysis of the InDel markers revealed them to be found with the highest frequency in the intergenic regions (82.76%), followed by the introns (6.90%), coding sequences (6.90%), and exons (3.45%). Genetic diversity analysis demonstrated that the polymorphic information content of the markers varied from 0.09 to 0.37, with an average of 0.20. Taken together, these results showed the efficiency of InDel marker development for chickpea genetic and genomic studies using the ddRADSeq method. The identified markers might prove valuable for chickpea breeders.

Conservation genomic study of Hopea hainanensis (Dipterocarpaceae), an endangered tree with extremely small populations on Hainan Island, China.

Hopea hainanensis Merrill & Chun is considered a keystone and indicator species in the tropical lowland rainforests of Hainan Island. Owing to its high-quality timber, H. hainanensis has been heavily exploited, leading to its classification as a first-class national protected plant in China and a plant species with extremely small populations (PSESPs). This study analyzed genome-wide single nucleotide polymorphisms obtained through restriction site-associated DNA sequencing from 78 adult trees across 10 H. hainanensis populations on Hainan Island. The nucleotide diversity of the sampled populations ranged from 0.00096 to 0.00138, which is lower than that observed in several other PSESPs and endangered tree species. Bayesian unsupervised clustering, principal component analysis, and neighbor-joining tree reconstruction identified three to five genetic clusters in H. hainanensis, most of which were geographically widespread and shared by multiple populations. Demographic history analysis based on pooled samples indicated that the decline in the H. hainanensis population began approximately 20,000 years ago, starting from an ancestral population size of approximately 10,000 individuals. The reduction in population size accelerated approximately 4,000 years ago and has continued to the present, resulting in a severely reduced population on Hainan Island. Intensified genetic drift in small and isolated H. hainanensis populations may contribute to moderate differentiation between some of them, as revealed by pairwise F st. In conclusion, our conservation genomic study confirms a severe population decline and an extremely low level of nucleotide variation in H. hainanensis on Hainan Island. These findings provide critical insights for the sustainable management and genetic restoration of H. hainanensis on Hainan Island.

Genome-wide discovery of selection signatures in four Anatolian sheep breeds revealed by ddRADseq

High-density genomic data analyzed by accurate statistical methods are of potential to enlighten past breeding practices such as selection by unraveling fixed regions. In this study, four native Turkish sheep breeds (80 samples) were genotyped via 296.097 single nucleotide polymorphisms (SNPs) detected by double-digest restriction site-associated DNA (ddRADseq) library preparation combined with the Illumina HiSeq X Ten instrument in order to identify genes under selection pressure. A total of 32, 136, 133, and 119 protein-coding genes were detected under selection pressure by runs of homozygosity (ROH), integrated haplotype score (iHS), the ratio of extended haplotype homozygosity (Rsb), and fixation index (FST) approaches, respectively. Of these, a total of 129 genes were identified by at least two statistical models which overlapped with a total of 52 quantitative trait loci (QTL)-associated SNPs, known to be related to fiber diameter, milk content, body weight, carcass traits, some blood parameters, and entropion. A total of six genes under selection pressure were validated by three statistical approaches five of which are of potential to be integrated into animal breeding since they were associated with wool fiber diameter (ZNF208B), behaviors related to neurocognitive development (CBX1 and NFE2L1), adaptation to high-altitude (SDK1), and anxiety causing internal stress (GSG1L). The sixth gene (COPZ1) turned out to play an important role in coping with different types of cancer in mammals. In particular, ROH analysis uncovered significant findings that the Güney Karaman (GKR) had experienced different selection practices than the Akkaraman (AKR) breed. Moreover, some genes specifically under selection in the GKR breed turned out to be associated with olfaction (OR6K6, OR6N1, OR6N2, and OR4C16), survival during the gestation period (PRR15L), and heat stress (CDK5RAP9). The results of this study imply that GKR may become genetically different from the AKR breed at the genome level due to most probably experiencing different adaptation processes occurring in raised climatic conditions. These differences should be conserved to face future challenges, while other native Turkish sheep breeds could be monitored via genome-wide high-density SNP data to obtain deeper knowledge about the effects of natural selection.

Adaptive radiation of the Callicarpa genus in the Bonin Islands revealed through double-digest restriction site-associated DNA sequencing analysis.

The Bonin Islands, comprised of the Mukojima, Chichijima, and Hahajima Islands, are known for their isolated and distinctive habitats, hosting a diverse array of endemic flora and fauna. In these islands, adaptive radiation has played a remarkable role in speciation, particularly evident in the Callicarpa genus that is represented by three species: Callicarpa parvifolia and C. glabra exclusive to the Chichijima Islands, and Callicarpa subpubescens, distributed across the entire Bonin Islands. Notably, C. subpubescens exhibits multiple ecotypes, differing in leaf hair density, flowering time, and tree size. In this study, we aimed to investigate species and ecotype diversification patterns, estimate divergence times, and explore cryptic species within Callicarpa in the Bonin Islands using phenotypic and genetic data (double-digest restriction site-associated DNA sequencing). Genetic analysis revealed that C. parvifolia and C. glabra both formed single, distinct genetic groups. Conversely, C. subpubescens consisted of six genetic groups corresponding to different ecotypes and regions, and a hybrid group resulting from the hybridization between two of these genetic groups. Population demography analysis focusing on six Chichijima and Hahajima Islands-based species/ecotypes indicated that all species and ecotypes except one ecotype diverged simultaneously around 73-77 kya. The star-shaped neighbor-net tree also suggests the simultaneous divergence of species and ecotypes. The species and ecotypes that simultaneously diverged adapted to dry environments and understory forests, suggesting that aridification may have contributed to this process of adaptive radiation. Moreover, leaf morphology, flowering time, and genetic analyses suggested the presence of two cryptic species and one hybrid species within C. subpubescens.

Climate change vulnerability and conservation strategies for tertiary relict tree species: Insights from landscape genomics ofTaxus cuspidata.

The unprecedented habitat fragmentation or loss has threatened the existence of many species. Therefore, it is essential to understand whether and how these species can pace with the environmental changes. Recent advantages in landscape genomics enabled us to identify molecular signatures of adaptation and predict how populations will respond to changing environments, providing new insights into the conservation of species. Here, we investigated the pattern of neutral and putative adaptive genetic variation and its response to changing environments in a tertiary relict tree species, Taxus cuspidata Sieb. et Zucc, which is distributed in northeast China and adjacent regions. We investigated the pattern of genetic diversity and differentiation using restriction site-associated DNA sequencing (RAD-seq) and seven nuclear microsatellites (nSSRs) datasets. We further explored the endangered mechanism, predicted its vulnerability in the future, and provided guidelines for the conservation and management of this species. RAD-seq identified 16,087 single nucleotide polymorphisms (SNPs) in natural populations. Both the SNPs and nSSRs datasets showed high levels of genetic diversity and low genetic differentiation in T. cuspidata. Outlier detection by F ST outlier analysis and genotype-environment associations (GEAs) revealed 598 outlier SNPs as putative adaptive SNPs. Linear redundancy analysis (RDA) and nonlinear gradient forest (GF) showed that the contribution of climate to genetic variation was greater than that of geography, and precipitation played an important role in putative adaptive genetic variation. Furthermore, the genetic offset and risk of non-adaptedness (RONA) suggested that the species at the northeast edge may be more vulnerable in the future. These results suggest that although the species has maintained high current genetic diversity in the face of recent habitat loss and fragmentation, future climate change is likely to threaten the survival of the species. Temperature (Bio03) and precipitation (Prec05) variables can be potentially used as predictors of response of T. cuspidata under future climate. Together, this study provides a theoretical framework for conservation and management strategies for wildlife species in the context of future climate change.

High-Density Genetic Map Construction and Quantitative Trait Locus Analysis of Fruit- and Oil-Related Traits in Camellia oleifera Based on Double Digest Restriction Site-Associated DNA Sequencing.

Camellia oleifera, an important tree species and source of edible oil in China, has received significant attention owing to the oil's high unsaturated fatty acid content, which has benefits for human health. However, the mechanisms underlying C. oleifera yield and oil quality are largely unknown. In this study, 180 F1 progenies were obtained from two parents with obvious differences in fruit- and oil-related traits. We constructed a high-density genetic map using a double digest restriction site-associated DNA sequencing (ddRAD-Seq) strategy in C. oleifera. This map spanned 3327 cM and anchored 2780 markers in 15 linkage groups (LGs), with an average marker interval of 1.20 cM. A total of 221 quantitative trait loci (QTLs) associated with fruit- and oil-related traits were identified across three years' worth of phenotypic data. Nine QTLs were detected simultaneously in at least two different years, located on LG02, LG04, LG05, LG06, and LG11, and explained 8.5-16.6% of the phenotypic variation in the corresponding traits, respectively. Seventeen major QTLs were obtained that explained 13.0-16.6% of the phenotypic variance. Eleven and five flanking SNPs of major QTLs for fruit- and oil-related traits were detected which could be used for marker-assisted selection in C. oleifera breeding programs. Furthermore, 202 potential candidate genes in QTL regions were identified based on the collinearity of the genetic map and the C. oleifera "CON" genome. A potential regulatory network controlling fruit development and oil biosynthesis was constructed to dissect the complex mechanism of oil accumulation. The dissection of these QTLs will facilitate the gene cloning underlying lipid synthesis and increase our understanding in order to enhance C. oleifera oil yield and quality.

Neutral markers reveal complex population structure across the range of a widespread songbird.

Understanding how both contemporary and historical physical barriers influence gene flow is key to reconstructing evolutionary histories and can allow us to predict species' resilience to changing environmental conditions. During the last glacial maximum (LGM), many high latitude North American bird species were forced into glacial refugia, including mountain bluebirds (Silia currucoides). Within their current breeding range, mountain bluebirds still experience a wide variety of environmental conditions and barriers that may disrupt gene flow and isolate populations. Using single nucleotide polymorphisms (SNPs) obtained through restriction site-associated DNA sequencing, we detected at least four genetically distinct mountain bluebird populations. Based on this structure, we determined that isolation-by-distance, the northern Rocky Mountains, and discontinuous habitat are responsible for the low connectivity and the overall history of each population going back to the last glacial maximum. Finally, we identified five candidate genes under balancing selection and three loci under diversifying selection. This study provides the first look at connectivity and gene flow across the range of these high-altitude and high latitude songbirds.

Striking genetic homogeneity in the widespread South American bracken.

Bracken (Pteridium, Dennstaedtiaceae) is a cosmopolitan genus of aggressive disturbance colonizers that are toxic to agricultural livestock. The taxonomy of Pteridium has been treated in multiple schemes, ranging from one to six species worldwide, with numerous subspecies and varieties. Recent work has focused on the worldwide distribution and systematics of the bracken fern, but South America has been poorly represented. We present the first continent-wide sampling and analysis of Pteridium esculentum, a Southern Hemisphere diploid species. Within South America, P. esculentum has several morphotypes, distinguished into subspecies by variation in indument and lamina architecture. We used double digest restriction site-associated DNA sequencing (ddRADSeq) to assess the phylogenetic relationships of P. esculentum subspecies. We found a striking genetic homogeneity in the species, being able to support only two morphotypes from molecular data: P. e. arachnoideum and P. e. campestre. We had high confidence for shallow and deep phylogenetic relationships, but less support for relationships among crown groups. We describe an east-west geographic pattern that would explain the relationships between populations; and, in contrast to previous studies, we detected differences with P. esculentum from Australia. These results will lay the foundations for studying variations in this species' behavior as a weed, as well as its impact on the production of agricultural livestock in South America.

Genetic diversity and population structure of Piper nigrum (black pepper) accessions based on next-generation SNP markers.

Despite the economic importance of Piper nigrum (black pepper), a highly valued crop worldwide, development and utilization of genomic resources have remained limited, with diversity assessments often relying on only a few samples or DNA markers. Here we employed restriction-site associated DNA sequencing to analyze 175 P. nigrum accessions from eight main black pepper growing regions in Sri Lanka. The sequencing effort resulted in 1,976 million raw reads, averaging 11.3 million reads per accession, revealing 150,356 high-quality single nucleotide polymorphisms (SNPs) distributed across 26 chromosomes. Population structure analysis revealed two subpopulations (K = 2): a dominant group consisting of 152 accessions sourced from both home gardens and large-scale cultivations, and a smaller group comprising 23 accessions exclusively from native collections in home gardens. This clustering was further supported by principal component analysis, with the first two principal components explaining 35.2 and 12.1% of the total variation. Genetic diversity analysis indicated substantial gene flow (Nm = 342.21) and a low fixation index (FST = 0.00073) between the two subpopulations, with no clear genetic differentiation among accessions from different agro-climatic regions. These findings demonstrate that most current black pepper genotypes grown in Sri Lanka share a common genetic background, emphasizing the necessity to broaden the genetic base to enhance resilience to biotic and abiotic stresses. This study represents the first attempt at analyzing black pepper genetic diversity using high-resolution SNP markers, laying the foundation for future genome-wide association studies for SNP-based gene discovery and breeding.

Analysis of genetic diversity by the SLAF-seq among the farmed Onychostoma macrolepis populations

ObjectiveThe objective of this study was to examine the genetic diversity within and between farmed populations of Onychostoma macrolepis, and to establish a foundation for enhancing the genetic resources of breeding groups through the introduction of new individuals and crossbreeding. A total of 49 individuals were subjected to sequencing using Specific-Locus Amplified Fragment Sequencing (SLAF-seq), one of the restriction site-associated DNA sequencing technologies. The single nucleotide polymorphisms(SNPs)were identified to conduct the analyzation of phylogeny population structure, principal component and genetic diversity.ResultsA total of 853,067 SNPs were identified. The results of the phylogenetic analysis revealed that each sample was genetically clustered into three distinct groups: ZhenPing (ZP), LanGao parents (LG), and their progeny population (LG-F1). Each population was observed to be clustered together. Analysis of population genetic diversity revealed that the observed heterozygosity (Ho) ranged from 0.200 to 0.230, the expected heterozygosity (He) ranged from 0.280 to 0.282, and the polymorphic information content (PIC) ranged from 0.228 to 0.230. These results indicate that the genetic diversity of the population is low and the signs of long-term interbreeding are obvious, but there are differences between the populations, and the genetic diversity of the population can be improved by hybridization in different regions.

Genetic structure of a narrowly distributed species Chimonobambusa tumidissinoda in the Yunnan-Guizhou Plateau, and its implications for conservation

Chimonobambusa tumidissinoda is a narrowly distributed and endangered bamboo species, which is endemic to the Yunnan-Guizhou Plateau and possesses significant ecological and economic value, particularly due to its delectable shoots. In this study, we employed the restriction site-associated DNA sequencing (RAD-seq) method to elucidate the population structure, genetic diversity, and demographic history of C. tumidissinoda across its entire distribution range. Unlike the genetic pattern revealed previously, three distinct genetic groups within the C. tumidissinoda samples were consistently discriminated based on multiple analyses, and low levels of genetic diversity (HO = 0.085, HE = 0.115, π = 0.116) were found. Furthermore, moderate genetic differentiation (FST = 0.070 - 0.095; PhiPT = 0.129 - 0.156) was detected among these groups with Mantel tests suggesting that isolation by distance (IBD) could potentially drive population genetic differentiation in this species. Moreover, demographic analyses incicated that C. tumidissinoda likely experienced a bottleneck event since the Pliocene, which may be attributed to drier and colder winters in Yunnan resulting from intensified Asian monsoon activity. Based on these findings, we suggest that the endangered status of C. tumidissinoda has been underestimated. Conservation measures should be implemented both in situ and ex situ to enhance its adaptability towards future climate fluctuations. Additionally, seed collection for germplasm storage is recommended.

Genomic insights into the critically endangered King Island scrubtit.

Small, fragmented or isolated populations are at risk of population decline due to fitness costs associated with inbreeding and genetic drift. The King Island scrubtit Acanthornis magna greeniana is a critically endangered subspecies of the nominate Tasmanian scrubtit A. m. magna, with an estimated population of < 100 individuals persisting in three patches of swamp forest. The Tasmanian scrubtit is widespread in wet forests on mainland Tasmania. We sequenced the scrubtit genome using PacBio HiFi and undertook a population genomic study of the King Island and Tasmanian scrubtits using a double-digest restriction site-associated DNA (ddRAD) dataset of 5,239 SNP loci. The genome was 1.48 Gb long, comprising 1,518 contigs with an N50 of 7.715Mb. King Island scrubtits formed one of four overall genetic clusters, but separated into three distinct subpopulations when analysed independently of the Tasmanian scrubtit. Pairwise FST values were greater among the King Island scrubtit subpopulations than among most Tasmanian scrubtit subpopulations. Genetic diversity was lower and inbreeding coefficients were higher in the King Island scrubtit than all except one of the Tasmanian scrubtit subpopulations. We observed crown baldness in 8/15 King Island scrubtits, but 0/55 Tasmanian scrubtits. Six loci were significantly associated with baldness, including one within the DOCK11 gene which is linked to early feather development. Contemporary gene flow between King Island scrubtit subpopulations is unlikely, with further field monitoring required to quantify the fitness consequences of its small population size, low genetic diversity and high inbreeding. Evidence-based conservation actions can then be implemented before the taxon goes extinct.

Whole-genome selection signature differences between Chaohu and Ji'an red ducks.

Assessing the genetic structure of local varieties and understanding their genetic data are crucial for effective management and preservation. However, the genetic differences among local breeds require further explanation. To enhance our understanding of their population structure and genetic diversity, we conducted a genome-wide comparative study of Chaohu and Ji'an Red ducks using genome sequence and restriction site-associated DNA sequencing technology. Our analysis revealed a distinct genetic distinction between the two breeds, leading to divided groups. The phylogenetic tree for Chaohu duck displayed two branches, potentially indicating minimal impact from artificial selection. Additionally, our ROH (runs of homozygosity) analysis revealed that Chaohu ducks had a lower average inbreeding coefficient than Ji'an Red ducks. We identified several genomic regions with high genetic similarity in these indigenous duck breeds. By conducting a selective sweep analysis, we identified 574 candidate genes associated with muscle growth (BMP2, ITGA8, MYLK, and PTCH1), fat deposits (ELOVL1 and HACD2), and pigmentation (ASIP and LOC101797494). These results offer valuable insights for the further enhancement and conservation of Chinese indigenous duck breeds.

Which contributes more to the relict flora distribution pattern in East Asia, geographical processes or climate change? New evidence from the phylogeography of Rehderodendron kwangtungense

BackgroundRelict species are important for enhancing the understanding of modern biogeographic distribution patterns. Although both geological and climatic changes since the Cenozoic have affected the relict flora in East Asia, the contributions of geographical processes remain unclear. In this study, we employed restriction-site associated DNA sequencing (RAD-seq) and shallow genome sequencing data, in conjunction with ecological niche modeling (ENM), to investigate the spatial genetic patterns and population differentiation history of the relict species Rehderodendron kwangtungense Chun.ResultsA total of 138 individuals from 16 populations were collected, largely covering the natural distribution of R. kwangtungense. The genetic diversity within the R. kwangtungense populations was extremely low (HO = 0.048 ± 0.019; HE = 0.033 ± 0.011). Mantel tests revealed isolation-by-distance pattern (R2 = 0.38, P < 0.001), and AMOVA analysis showed that the genetic variation of R. kwangtungense occurs mainly between populations (86.88%, K = 7). Between 23 and 21 Ma, R. kwangtungense underwent a period of rapid differentiation that coincided with the rise of the Himalayas and the establishment of the East Asian monsoon. According to ENM and population demographic history, the suitable area and effective population size of R. kwangtungense decreased sharply during the glacial period and expanded after the last glacial maximum (LGM).ConclusionOur study shows that the distribution pattern of southern China mountain relict flora may have developed during the panplain stage between the middle Oligocene and the early Miocene. Then, the flora later fragmented under the force of orogenesis, including intermittent uplift during the Cenozoic Himalayan orogeny and the formation of abundant rainfall associated with the East Asian monsoon. The findings emphasized the predominant role of geographical processes in shaping relict plant distribution patterns.

Low-coverage sequencing and Wahlund effect severely bias estimates of inbreeding, heterozygosity and effective population size in North American wolves.

vonHoldt etal. ((2024), Molecular Ecology, 33, e17231) (vH24) used low-coverage (average ~ 7X read depth) restriction site-associated DNA sequence data to estimate individual inbreeding and heterozygosity, and recent effective population size (Ne), in Great Lakes (GL) and Northern Rocky Mountain (RM) wolves. They concluded that RM heterozygosity rapidly declined between 1991 and 2020, and that Ne declined substantially in GL and RM over the last 50 generations. Here, we evaluate the effects of low sequence coverage and sampling strategy on vH24's findings and provide general recommendations for using sequence data to evaluate inbreeding, heterozygosity and Ne. Low-coverage sequencing resulted in downwardly biased estimates of individual inbreeding and heterozygosity, and an erroneous large temporal decline in RM heterozygosity due to declining read depth through time. Additionally, vH24's sampling strategy-which combined individuals from several genetically differentiated populations and across at least eight wolf generations-is expected to have resulted in severe downward bias in estimates of recent Ne for RM. We recommend using high-coverage sequence data ( 15-20X) to estimate inbreeding and heterozygosity. Carefully filtering individuals, loci and genotypes, and using genotype imputation or likelihoods can help to minimise bias when low-coverage sequence data must be used. For estimation of contemporary Ne, the marginal benefits of using more than 103-104 loci are small, so aggressive filtering of loci with low average read depth potentially can retain most individuals without sacrificing much precision. Individuals are relatively more valuable than loci because analyses of contemporary Ne should focus on roughly single-generation samples from local breeding populations.

Development of 51 K liquid-phased probe array for Loblolly and Slash pines and its application to GWAS of Slash pine breeding population

Loblolly pine (Pinus taeda) and Slash pine (Pinus elliottii) are important economically and ecologically tree species worldwide. To implement genomic based breeding strategy, genotyping large number of samples is needed. However, huge and complex pines genome make it difficult to obtain abundant and cost-effectively markers. In this study, we designed a SNP array containing 51 K liquid-phased probes based on the published SNPs and probes of the two species, novel Slash pine-specific long-read transcript, and SNPs obtained from a double digest Restriction-Site Associated DNA sequencing (ddRADseq). We performed targeted capture sequencing using the developed SNP array on three pine species of Loblolly, Slash, and Caribbean pine (Pinus caribaea). The probe capture had an efficiency of 68.26% on average. Principal Component Analysis (PCA), phylogenetic analysis, and genetic structure analysis of 28,670 SNPs genotyped for the three pine species demonstrated the transferability of the SNP array for the three pines. Furthermore, we genotyped 221 range-wide Slash pine genotypes, analyzed their genetic diversity and performed a genome-wide association study (GWAS). We identified 95 SNPs significantly associated with growth and wood quality traits of Slash pine. We have identified potential roles of the MYB2 and MYB46 transcription factors in regulating cell cycle and differentiation in Slash pine, while also suggesting that AP2/ERF and Glycosyl hydrolase may play significant roles in wood formation processes. This new SNP array can be widely applied in future molecular breeding research of conifers.

Genomic evaluation of mud sunfish in the atlantic slope drainage

Mud Sunfish (Acantharchus pomotis, Baird), a cryptic species native to the Atlantic and Gulf slope drainages of North America, have experienced a documented reduction in their geographic range. Atlantic Slope populations of Mud Sunfish were evaluated to determine the population structure, assess genetic diversity, and to identify presence of evolutionary significant units (ESU) in order to develop fundamental understandings of its occurrence. Tissue samples were collected from across the Atlantic Slope for genomic comparison. Restriction site Associated DNA Sequencing (RAD-Seq) was performed on 95 individuals from 14 populations. Mantel test provided evidence of a correlation between genetic and geographical distances, suggesting a pattern of isolation-by-distance. Differences among populations of Mud Sunfish largely occur along a north-south latitudinal gradient. Bayesian clustering of putatively outlier SNP loci, suggests that regional adaptive pressure is occurring. Despite a large geographic distance and significant differences between the majority of the populations of Mud Sunfish, Bayesian clustering analysis suggests that two unresolved clades are persisting within the Atlantic Slope Drainage: a northern clade of Mud Sunfish ranging from New Jersey to North Carolina and a southern clade ranging from South Carolina to Florida. A population of Mud Sunfish in Florida appears distinct from all others in the Atlantic Coastal plain and likely represents a distinct management unit.

Genome-wide association study reveals candidate genes critical for skin pigmentation in common carp (Cyprinus carpio) strains including koi

The differentiation and variation of body color in fish is an important component of biological evolution and phylogenetic research, and is influenced by several factors such as heredity, nutrition, physiological hormones and rearing conditions, with heredity being the most critical factor. An improved comprehension of the genetic regulation in different carp populations will facilitate future applications of precision breeding and pigment cell development-related genes editing. In this study, we used restriction site-associated DNA sequencing (2d-RAD) to determine the distribution characteristics of single nucleotide polymorphisms (SNPs) in eight common carp strains including six koi strains, i.e., Kohaku (Hb), Taisho (Dz), Showa (Zh), Golden (Hj), Matsukawabake (Sch) and Five-color (Ws), and two genetically improved breeding strains, i.e., FFRC No. 2 strain (Fr) and red FFRC strain (Hfr), and then SNP markers to resolve genetic information on the population structure and skin color formation of these strains. We found that most individuals of eight strains were clearly divided into eight clades. Genetic diversity within Fr, Sch and Ws is relatively high, while it is low within Dz, Hb and Hfr. A comparison between the populations revealed that Hfr and Ws have the highest degree of genetic differences, while Hb and Dz have the lowest. The GWAS analysis for two strategies identified several SNPs that are significantly related to skin color variation and affect melanin synthesis (ephrinb2, slc22a23, herc2, ephb2), carotenoid metabolism (oca2, bcmo1, bco2) and circadian rhythm(cipc). To summarize, our study provides some information on the genetic structure and diversity of Japanese koi and Chinese FFRC carp. Furthermore, based on the data obtained on relevant genes related to skin color differentiation and pigmentation, it can provide a theoretical basis for the subsequent integration of dominant traits and precision breeding in carps.

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.

A Diverging Species within the Stewartia gemmata (Theaceae) Complex Revealed by RAD-Seq Data.

Informed species delimitation is crucial in diverse biological fields; however, it can be problematic for species complexes. Showing a peripatric distribution pattern, Stewartia gemmata and S. acutisepala (the S. gemmata complex) provide us with an opportunity to study species boundaries among taxa undergoing nascent speciation. Here, we generated genomic data from representative individuals across the natural distribution ranges of the S. gemmata complex using restriction site-associated DNA sequencing (RAD-seq). Based on the DNA sequence of assembled loci containing 41,436 single-nucleotide polymorphisms (SNPs) and invariant sites, the phylogenetic analysis suggested strong monophyly of both the S. gemmata complex and S. acutisepala, and the latter was nested within the former. Among S. gemmata individuals, the one sampled from Mt. Tianmu (Zhejiang) showed the closest evolutionary affinity with S. acutisepala (which is endemic to southern Zhejiang). Estimated from 2996 high-quality SNPs, the genetic divergence between S. gemmata and S. acutisepala was relatively low (an Fst of 0.073 on a per-site basis). Nevertheless, we observed a proportion of genomic regions showing relatively high genetic differentiation on a windowed basis. Up to 1037 genomic bins showed an Fst value greater than 0.25, accounting for 8.31% of the total. After SNPs subject to linkage disequilibrium were pruned, the principal component analysis (PCA) showed that S. acutisepala diverged from S. gemmata along the first and the second PCs to some extent. By applying phylogenomic analysis, the present study determines that S. acutisepala is a variety of S. gemmata and is diverging from S. gemmata, providing empirical insights into the nascent speciation within a species complex.