Increasing low nitrogen (N) tolerance in maize is an important goal for food security and agricultural sustainability. In order to analyze the population structure of tropical maize lines and identify genomic regions associated with low-N tolerance, a set of 64 inbred lines were evaluated under low-N and optimal-N conditions. The low-N Agronomic Efficiency index (LNAE) of each line was calculated. The maize lines were genotyped using 417,112 SNPs markers. The grouping based on the LNAE values classified the lines into two phenotypic groups, the first comprised by genotypes with high LNAE (named H_LNAE group), while the second one comprised genotypes with low LNAE (named L_LNAE group). The H_LNAE and L_LNAE groups had LNAE mean values of 3,304 and 1,644, respectively. The population structure analysis revealed a weak relationship between genetic and phenotypic diversity. Pairs of lines were identified, having at the same time high LNAE and high genetic distance from each other. A set of 29 SNPs markers exhibited a significant difference in allelic frequencies (Fst > 0.2) between H_LNAE and L_LNAE groups. The Pearson’s correlation between LNAE and the favorable alleles in this set of SNPs was 0.69. These SNPs could be useful for marker-assisted selection for low-N tolerance in maize breeding programs. The results of this study could help maize breeders identify accessions to be used in the development of low-N tolerant cultivars. High levels of nitrogen (N) fertilization is required to achieve high maize (Zea mays L.) grain yield. However, the maize crop utilizes only about 30–40% of the N available in the soil while the remaining is lost through surface runoff, denitrification, volatilization and microbial consumption . N fertiMaize tolerance to low availability of N in the soil is a complex trait influenced by genetic and environmental factors, including soil nitrogen availability, nitrogen uptake, assimilation, transportation, and remobilization . BreedinInformation on the genomic diversity and population structure of tropical maize germplasm can accelerate the genetic gains in maize breeding programs , 10. PreMaize lines from different heterotic groups produced higher-yielding hybrids than those of lines from the same heterotic group. Therefore, the assessment of genetic diversity of germplasm is routinely carried out using different morphological and molecular markers. For this purpose, molecular markers are more beneficial because they are not affected by environmental factors and the plants' developmental stage. Consequently, DNA markers have been an indispensable tool for characterizing genetic resources and providing breeders with more detailed information to assist in selecting diverse parents .With the development of next-generation DNA sequencing methods, the Single Nucleotide Polymorphisms (SNPs) markers have become a useful tool for understanding genetic relationships and population structure of different species , includiThe present study aimed i) at investigating the population structure and genomic diversity of selected tropical maize lines with different levels of tolerance to low-N stress, and ii) at identifying SNP markers under directional selection between groups of lines phenotypically contrasting in low-N tolerance.A diversity panel consisting of 64 Brazilian tropical maize inbred lines with genetic variability for N-stress tolerance was evaluated. Most of these lines originated from the maize breeding program of the Universiade Federal de Viçosa (Brazil) , 18. The−1 of N while the LN field received 60 kg ha−1 of N. In 2015 sites, the doses were 100 kg ha−1 N and 35 kg ha−1 of N, respectively. The top-dressing doses were different from one year to another, but the expected yield ratio between the IN and LN was maintained [The amount of N applied was determined by the expected yield using a given N dosage. Therefore, the IN dose corresponded to 100% expected yield and LN dose to 50% of IN yield . In 2014-1) of each plot and adjusted the yield to 13% grain moisture. The adjusted means were obtained for each inbred line under each nitrogen level and environment, using the following model:y is the grain yield vector, β was the vector of fixed effects of the common constant, the replicate, and the genotype, u was the vector of random effects of blocks with ϵ was the error matrix with ϵ∼, X and Z are the incidence matrix of the fixed and random effects, respectively.We measured the grain yield , for each inbred line in each environment. The LNAE index, introduced by , was caly is the LNAE vector, β is the vector of fixed effects of the common constant, the environment, and the inbred line, ϵ is the error matrix with ϵ∼, X is the incidence matrix of the fixed effects. If there was significant effect of inbred line, we performed the Scott-Knott test using the package ‘Scott-Knott’ [Analysis of variance was performed for LNAE index using the following model:t-Knott’ .Fresh leaves samples were collected from the fourth week old seedlings within each maize line and stored in a deep freezer at -80°C. According to its instruction manual, total genomic DNA extraction was performed using the Qiagen DNeasy Plant Mini Kit (Promega™). The inbred maize lines were genotyped using Affymetrix® Axiom® Maize Genotyping Array, containing 616,201 SNP markers . The qua5 iterations for each K value (ranging from 1 to 10) were performed ten replicate runs of 1 ×105 Markov Chain Monte Carlo iterations. The STRUCTURE 2.3.4 results were summarized using the pophelper R package [Population structure was analyzed using the Bayesian method implemented in STRUCTURE 2.3.4  software package . The num package . Several package  to identvegan [In order to assess and visualize the genetic relationships among the maize lines, we performed principal coordinate analyses (PCoA) via genetic distance matrix with data standardization using the package vegan .The genetic distance among the maize lines was calculated as 1 –IBS (identity by state) using TASSEL 5.0 . IBS is In order to identify SNPs with significant allele frequency differences between contrasting phenotypic groups for LNAE, we calculated the fixation index (Fst) for each SNP  using thBased on the Scott-Knott test, the maize lines were classified into two phenotypic groups, one formed from 29 lines with high LNAE values (named H_LNAE group) and another comprising 35 lines with low LNAE (named L_LNAE group). The distribution of the LNAE values in the H_LNAE and L_LNAE groups is shown in Principal coordinate analysis based on 12,050 SNPs markers  showed nAccording to the Bayesian approach, the population structure was analyzed using STRUCTURE software . Based oThe genetic distances among the 64 maize lines analyzed  ranged fIn order to identify SNPs under directional selection between H_LNAE and L_LNAE groups, we used two approaches, the first one based on Fst  and the We also used the method implemented in the BAYESCAN software to identify SNPs under directional selection between H_LNAE and L_LNAE groups. Through this approach, 4 SNPs were identified under selection between H_LNAE and L_LNAE groups .−10). The R2 coefficient of LNAE linear regression as a function of NFA was 0.46  and the LNAE of the maize lines was 0.68  to measure the tolerance of 64 tropical maize lines to low-N availability in the soil. In the LNAE index calculation, both the absolute GY at low-N availability and the ratio between low-N and optimum-N are considered , 8. TherGenetic variability is essential for success in maize breeding programs. Therefore, genetic variability related to N-utilization has been investigated in maize, wheat, rice, and spring barley , 33–36. A clear understanding of genetic relationships among low-N tolerant inbred lines offers an opportunity to obtain genotypes that could be used in parental combinations to develop simultaneously high-yielding and low-N tolerant maize hybrids. The genetic distance matrix  revealedThat was possible to identify pairwise individuals that possessed high LNAE and were genetically distinct from each other. For example, the genetic distance between the two lines possessing the highest LNAE values (L032 and L014) was relatively high (0.24), indicating that the crossing between them would be promising and could result in transgressive hybrids. In addition, knowledge of genetic distances between pairs of maize lines can be useful for assigning lines to heterotic groups, selecting parental lines, and for estimating of genetic diversity loss during conservation or selection , 38, 39.As a result of the development of next-generation DNA sequencing methods, SNPs molecular markers have become essential for understanding genetic relationships among different species of agronomic interest, including maize . AdditioIn practice, SNPs markers associated with traits can be beneficial when applied to assisted selection in breeding programs. However, from large-scale implementation in large breeding programs, the cost of genotyping large populations with dense SNP panels may still be a limiting factor in this tool –44. ThusIn order to generate an even smaller set of SNPs to use in marker-assisted selection for LNAE, we selected 29 SNPs with Fst values equal to or higher than 0.2 between the two phenotypic groups of maize lines possessing contrasting characteristics (H_LNAE and L_LNAE groups). We observed that four of these SNPs  also showed evidence of having undergone directional selection according to the results of the analysis carried out using the BAYESCAN software  based apConsidering the small set of 29 SNPs under selection between H_LNA and L_LNAE groups, we observed a Pearson’s correlation between the number of favorable alleles for high LNAE and the LNAE index of the maize lines was equal to 0.68. These SNP markers can be useful in marker-assisted selection for low-N tolerance. In addition, the results can help establish breeding programs to improve tolerance of maize to stress due to the low availability of N in the soil.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Pinus quadrifolia has three to four needles per fascicle, suggesting that it is a result of hybridization between the five-needled P. juarezensis and the single-needled P. monophylla. In this study we address the taxonomic validity of P. juarezensis, the hybrid origin of P. quadrifolia and the presence of hybridization and intermediate morphology as a result of interspecific hybridization in this complex.Pinyon pine hybridization is widely acknowledged, but the frequency of and contributors to such interspecific mating remain largely unstudied. We address these questions by combining a genomic and morphological approach. We generated 1868 single nucleotide polymorphisms (SNPs) to detect genetic clusters using principal co-ordinates analyis, discriminant analysis of principal components, fastSTRUCTURE and ADMIXTURE analyses, and performed a morphological analysis of the leaves.P. quadrifolia, reducing the status of P. juarezensis to P. quadrifolia. We also found no evidence that P. quadrifolia is of hybrid origin from P. juarezensis × P. monophylla but is instead a genetically distinct species with natural needle number variation that has yet to be explained. Hybridization does occur in this complex, but mostly between P. quadrifolia and P. californiarum, and less commonly between P. quadrifolia and P. monophylla. Interestingly, some hybrid derivatives were detected between both single-needled taxa, P. monophylla and P. californiarum, a hybrid combination that has not yet been proposed. Hybrids have intermediate morphology when they have similar genetic contributions from both parental species; however, when one parent contributes more, hybrid derivatives resemble the parent with higher genetic contribution, resulting in cryptic introgression.We found that the five-needled pinyons did not differ genetically from the four-needled Our detailed sampling across the distribution of this complex allows us to describe the patterns of hybridization among these taxa, resolves an ancient taxonomic conflict and provides insights into the challenges of exclusively using morphological traits when identifying these taxa with cryptic hybridization and variable morphology. Interspecies hybridization is relatively common across plant taxa and is thought to be the cause of several major speciation events , which nPinus quadrifolia Parl. ex Sudworth, has been proposed to have a hybrid origin based on morphological features and geography , individuals with five needles were another (P. juarezensis) and the three- and four-needled individuals (P. quadrifolia) were intermediate hybrids of the former two.Hybridization in pines has been widely acknowledged . The pineography a. Pinus of P. quadrifolia; however, they have provided inconclusive results. The idea of hybridization was explored using additional morphological features other than needle number, but these studies focused on the potential hybridization between P. monophylla and P. quadrifolia without considering P. juarezensis  was done with internal transcribed spacer (ITS) sequences  to determine the taxonomic validity of P. juarezensis as a species; (2) to assess the hybrid origin of P. quadrifolia; and (3) to test for hybridization and intermediate morphology among species in this complex. We have included samples from the taxa of interest using nuclear genomic data as well as morphological data to assess these questions.With advancements in next-generation sequencing (NGS), the ability to detect reticulated evolution through hybridization has drastically improved  and P. quadrifolia (3–4 needles) occurring in southern California and Baja California. These taxa are characterized by inhabiting small disjunct areas in southern California and Baja California. Ten centimetres of branch tips were cut from each tree, representing 2–3 years of growth and averaging approx. 50 fascicles per tree. Most collections were made from late 2017 to early 2019, with the exception of a few collections made previously (2011–2013). We collected a larger number of individuals in San Jacinto because P. juarezensis and P. monophylla.Samples were taken from 13 locations across the Southwestern USA and Baja California, Mexico ; Fig. 1,P. juarezensis as a species, the hybrid origin of P. quadrifolia and hybridization among species of this complex, we first identified genetic clustering and admixed individuals. Nuclear DNA was extracted using the 2 % cetyltrimethylammonium bromide (CTAB) protocol  to select the most appropriate complexity reduction method, in terms of both the size of the representation and the fraction of a genome selected for assays. DArTseq™ has been successfully applied in genomic studies exploring species boundaries and hybridization in plants and animals (P. lambertiana as the reference genome (GCA_001447015.2) due to its relatively close evolutionary relationship to the pinyon subsection Cembroides. A low-density DArTseq assay resulted in 18 518 SNPs. These SNPs have an index generated by reproducing the data independently, which is ‘the proportion of technical replicate assay pairs for which the marker score is consistent’  forms its own genetic cluster and whether P. quadrifolia  is formed by admixed individuals from the parental species P. monophylla and P. juarezensis or whether there are other pairs of species hybridizing in this complex.The 1868 loci were included in four complementary methods of genetic clustering: principal co-ordinates analysis (PCoA), discriminant analysis of principal components (DAPC), fastSTRUCTURE and ADMIXTURE, which also allowed us to detect admixed individuals between different genetic clusters. We then evaluated whether A PCoA, which considers differences in allele frequencies between individuals, was performed in dartR (gl.pcoa.plot) to examine genetic distance among populations (Population clustering was determined using a DAPC . DAPC usK) was selected using the chooseK command built into fastSTRUCTURE. The resulting Q mean bar plots were visualized using the online application pophelper (K) were reanalysed in fastSTRUCTURE using the logistic prior and five cross-validations to observe lower hierarchical structuring , we used a Bayesian model-based clustering method in the software NewHybrids 1.0 . Runs were initiated at different random starting points using the Jeffrey’s prior for both theta and pi with a burn-in of 10 000 and 100 000 sweeps . All sample populations of P. quadrifolia and purported P. juarezensis clustered together as one group (coloured in purple). All sample populations of P. californiarum (coloured in blue) clustered together as one group.The DAPC analysis  of the SP. monophylla forms one cluster (coloured in red), P. quadrifolia and purported P. juarezensis another cluster (coloured in purple) and P. californiarum the third cluster (coloured in blue). These results show no genetic distinction between individuals with morphological characteristics of P. quadrifolia (three to four needles) and P. juarezensis (five needle); this was further confirmed by the sub-structuring analysis of the cluster containing only the putative P. juarezensis and P. quadrifolia , P. monophylla × P. californiarum (as in Mojave) and P. quadrifolia × P. californiarum  admixture events. These admixture events are evident in the fastSTRUCTURE plot .Interestingly, the NewHybrids analyses  show thasignment . These p1) and hybrid derivatives . All morphological results are listed in P. monophylla and P. californiarum having one needle per fascicle on average but separated by the larger number of resin canals in P. californiarum. In contrast, P. quadrifolia + P. juarezensis formed one group, having the highest number of needles but the lowest number of rows of stomata, and stomata just on the ventral surface. Intermediate forms were detected when hybrids and hybrid derivatives were plotted  present in  unknown .P. quadrifolia, P. californiarum and P. monophylla.P. monophylla. We found that these populations are actually P. californiarum based on the genetic data. Furthermore, the morphology of these populations matches the morphology of other P. californiarum populations, with a high number of resin canals. We found no evidence of P. monophylla in Baja California. While P. quadrifolia is not of P. monophylla × P. juarezensis hybrid origin, it hybridizes with P. californiarum and P. monophylla. Previous studies hypothesized that hybridization occurred between P. quadrifolia and P. monophylla , only one P. californiarum individual was detected, indicating how readily these two species hybridize. This could additionally imply heterosis, with increased fertility of the F1 generation allowing for frequent backcrossing, or selection potentially favouring hybrid phenotypes, promoting the proliferation of individuals with hybrid ancestry. Future studies on the fitness of P. monophylla × P. californiarum crosses must be done to explore these hypotheses.Surprisingly, a majority of the introgression detected in this study was between two species not previously thought to hybridize, a priori bias towards morphological traits. We then generalized the average characteristics of each genetic group to assign morphological qualities to each species. In doing so, our analyses were not influenced by strict morphological cut-offs and we were able to more accurately identify individuals with hybrid ancestry and intraspecific variation compared with other studies in pinyon pines. It is important to note that while some distinguishing morphological patterns were found, these should be used as suggestions for species identification, rather than strict species guidelines/boundaries.Our study delimited species by genetic clusters first, without any P. quadrifolia × P. californiarum hybrid derivatives, individuals with a majority of P. californiarum genotype tend to have one to two needles per fascicle with over ten resin canals; whereas individuals with a majority of P. quadrifolia genotype tend to have two to four needles per fascicle and two to four resin canals. This implies that needle morphology is largely genetically controlled and may be less influenced by environment than previously thought. The only exceptions to this were in recent hybrid derivatives, whose traits were more intermediate between their parents. For example, one P. monophylla × P. quadrifolia hybrid derivative found in San Jacinto had exclusively two needles per fascicle, 12 stomatal rows and two resin canals, resembling P. edulis. It is possible that as these individuals of hybrid ancestry further backcross, the intermediate traits could be recombined or introgressed out to more closely represent one of the parental species. This unfortunately makes the identification of individuals with advanced generation hybrid ancestry quite difficult without using genetic methods because these hybrid derivatives are often indistinguishable from their main genetically contributing parent, concordant with other hybrid systems  Program [grant no. 2018-38422-28614/project accession no. 1016839] from the USDA National Institute of Food and Agriculture, by the National Science Foundation [grant no. DEB-0816675] and by the University Grants Program of San Diego State University.


Cercis canadensis L. (eastern redbud) wild populations on a fine scale within fragmented areas centered around the borders of Georgia–Tennessee, USA. We hypothesized high genetic diversity among the collections of C. canadensis distributed across smaller geographical ranges. Fifteen microsatellite loci were used to genotype 172 individuals from 18 unmanaged and naturally occurring collection sites. Our results indicated presence of population structure, overall high genetic diversity , and moderate genetic differentiation (FST = 0.14) among the collection sites. Two major genetic clusters within the smaller geographical distribution were revealed by STRUCTURE. Our data suggest that native C. canadensis populations in the fragmented area around the Georgia–Tennessee border were able to maintain high levels of genetic diversity, despite the presence of considerable spatial genetic structure. As habitat isolation may negatively affect gene flow of outcrossing species across time, consequences of habitat fragmentation should be regularly monitored for this and other forest species. This study also has important implications for habitat management efforts and future breeding programs.Forest fragmentation may negatively affect plants through reduced genetic diversity and increased population structure due to habitat isolation, decreased population size, and disturbance of pollen‐seed dispersal mechanisms. However, in the case of tree species, effective pollen‐seed dispersal, mating system, and ecological dynamics may help the species overcome the negative effect of forest fragmentation. A fine‐scale population genetics study can shed light on the postfragmentation genetic diversity and structure of a species. Here, we present the genetic diversity and population structure of  Cercis canadensis L. (eastern redbud) wild populations on a fine scale. Our data reveal that current isolated patches and remnant populations are genetically stable and capable of maintaining viable populations at a geographically fine‐scale level. Forest fragmentation may cause a number of negative effects including, but not limited to, reduced genetic diversity and population size, increased population structure due to habitat isolation, and potential disruption of pollen‐seed dispersal.In this study, we used microsatellite loci to estimate genetic diversity of  Our hyp22.1C. canadensis were collected from eastern Tennessee and around the Georgia–Tennessee border, USA, which is located near the center of the current native distribution range of the species . From each tree, five to seven leaves were randomly collected from branches that were at cardinal directions around each tree. Leaves were placed in paper envelopes to dry and geographical coordinates were recorded for each sampled tree. Samples collected from each location were considered a single collection site.Samples of s Figure  and not 2.2S (speed) = 6.00 m/s, T (time) = 30 s. Between each homogenization step, samples were frozen in liquid nitrogen for 5 min to improve the tissue homogenization. Genomic DNA (gDNA) from each sample was isolated using the Qiagen DNeasy Plant Mini Kit (Qiagen) using the manufacturer's protocol with several modifications, which included the following: 2% w/v polyvinylpyrrolidone (PVP) was mixed into lysis buffer (AP1); 8 µl of RNase was added into each sample tube; the time period of incubation at 65°C was increased to 45 min and inverted gently to mix the sample every two minutes; incubation step at −20°C was followed with increased time of incubation to one hour. Additionally, before adding the elution buffer, ethanol was used to wash the spin columns if there was any debris left from the sample tissue. Finally, 50 μl of elution buffer preheated at 65°C was added, and the step was repeated twice, for a total elution of 100 μl. The quality and concentration of the isolated gDNA was assessed using ND1000 Visible (UV–Vis) Spectrophotometer .DNA was isolated from approximately 100 mg of dried leaf tissue. Samples were homogenized four times using a Bead Mill 24 (Fisher Scientific) with the settings of 2.3Cercis and another closely related species Bauhinia faberi Oliver  Gardens to screen all of the primers used in this study. From our initial screening of the 68 microsatellite loci, 15 primer pairs were selected based on polymorphic loci, as well as a successful amplification rate across the five tested samples  amplifications were completed in 10 µl reaction mixture consisting of the following: 1 µl undiluted gDNA, 1 µl of 10 µM of each forward and reverse primer, 0.5 µl of dimethyl sulfide, 4 µl of GoTaq G2 Hot Start Master Mix (Promega Corp), and 2.5 µl water. Both positive control  and negative control (control reaction without any DNA sample) samples were used for every primer tested to ensure validity of the data. Amplification of reactions was completed in 96 well plates using an Eppendorf thermocycler (Eppendorf AG) with the following thermal profile: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s. and an extension at 72°C for 30 s, with a final extension of 72°C for 4 min. Amplified PCR products were visualized using a QIAxcel Capillary Electrophoresis System (Qiagen) and sized with a 15/600 bp internal alignment marker and a 25 to 500 bp DNA size marker. All 180 2.4The FLEXBIN Excel macro version 2 , and expected heterozygosity , genetic differentiation  was estimated which combines both allele richness and evenness of the allele distribution  will provide the number of expected alleles that are evenly distributed in the studied dataset  were escus Nei, . Presenc Wright, ) were alShannon, . H index2.5C. canadensis collection was analyzed with the program STRUCTURE version 2.3.4 . Results were analyzed with STRUCTURE HARVESTER Web version 06.94  and disregards the assumption of Hardy–Weinberg equilibrium within groups of populations. The following parameters were used in this analysis: 20 independent MCMC chains for each K value (1–18) with a burn‐in period of 500,000 and 500,000 repetitions of the thinning interval using the admixture model. An admixture model was implemented to evaluate the proportion of mixed ancestry in an individual and improve the clustering , a model‐free multivariate analysis approach, is a useful tool to investigate and visualize the presence of genetic clusters , a Mantel test , infinite allele model (I.A.M.), and two‐phase mutational model (T.P.M.). Significance of this test under either of these models was evaluated by the Wilcoxon sign‐rank test with 10,000 simulations, as the number of loci for this study was under 20  to 0.98 (127spa). The overall expected heterozygosity (He) across all 15 microsatellite loci was high (He = 0.63), ranging from 0.30 (locus 229a) to 0.84 (locus 1057a). Additionally, the overall Shannon–Wiener diversity index (H) was 1.22 for the 15 loci ranging from 0.58 (locus 229a) to 2.00  considering all the 18 collection sites as one population and (b) dividing the entire dataset into two groups, where the first group (north group) represented collection sites from eastern Tennessee and the second group (south group) represented the collection sites near the Georgia–Tennessee border Figure .n = 15) were detected in 10 out of 18 collection sites (Table He), corrected for the collection site sample size, ranged from 0.33 (Polk Co. 1) to 0.62 (Bradley Co. 2), with an overall value of 0.61 which indicates presence of high genetic diversity in the studied dataset. Comparably, in the two‐group analysis, the north group had lower overall genetic diversity when compared to the south group  to 0.31  of the variation was present between the clusters, whereas 7% (p < .001) of the variation was attributed among collection sites within these two clusters (Table p < .001). When the data were divided into two geographical groups (north and south), 14.88% (p < .001) of observed variation occurred between the groups, whereas only 14.33% (p < .001) of the variation was attributed among collection sites within these two groups  was tested in the following groupings: (a) 18 collection sites into two major clusters on the basis of the STRUCTURE results and (b) 18 collection sites into two geographical groups  with genetic distance of the sampled data .Isolation‐by‐distance analysis suggested that the geographical distance was linearly correlated , Cunninghamia lanceolata (Lamb.) Hook.] (FST = 0.04), and Viburnum rufidulum Raf. (FST = 0.06)   and Cladrastis kentukea (Dum. Cours.) Rudd , showed strong genetic differentiation, which was likely related to their patchy distributions and population structure  harbor seeds with a hard testa that usually fall in close proximity to the parent tree and then germinate within a year or two , white‐tailed deer, quail, pheasants, and several other bird species which are then dispersed in scat  in these fragmented populations can affect the fitness of C. canadensis populations in the future. Therefore, it is important to ensure that healthy and diverse habitats are present for animals responsible for seed and pollen dispersal of C. canadensis. Also, to better understand the effect of habitat fragmentation on C. canadensis, we suggest further studies be conducted on its sexual reproduction and life‐history traits.Our results indicated high genetic diversity and high gene flow, which suggests reproductive isolation of C. canadensis. We suggest that despite the current resilient genetic diversity and high evolutionary potential, C. canadensis populations and other species in the fragmented habitats may suffer severe consequences following further environmental changes and climate threats. Therefore, to better understand the potential consequences of these threats, research on the population genetics of other concurring species is necessary for conservation efforts and habitat management of forest ecosystems. Our results also suggested that wild populations of C. canadensis with high genetic variations can be used as reservoirs of desired genetic variations and can be utilized in breeding programs to improve phenotypic traits in the nursery stocks.Over the past few decades, a number of studies have been conducted to better understand the genetic consequence of habitat fragmentation and anthropogenic disturbances on rare forest species. However, very few studies have been conducted to evaluate the effects of these disturbances on common forest species (Aguilar, Ashworth, Galetto, & Aizen, None declared.DH, WK, and RT conceived and planned the experiments including the main conceptual ideas and proof outline. MO and SB carried out the experiments, MO, MN, SB, and DH troubleshot technical details. All authors contributed to sample collection and preparation. MO, MN, JZ, and DH contributed to data analyses. All authors contributed to the interpretation of the results, manuscript writing, and editing. All authors provided critical feedback and helped shape the research, analysis, and manuscript.SupinfoClick here for additional data file.


Chest blunt trauma (CBT) and the resultant rib fractures often lead to thoracic collapse. The purpose of this study was to explore the effect of displacement of the rib fracture and thoracic collapse on the thoracic volume by using normal chest CT data.n = 630) were reconstructed using 3Dmax 2014. We calculated the thoracic volume and reduction percentage for each thoracic collapse model. Linear regression-based comparisons of thoracic volume reductions were performed.In this retrospective study, seven consecutive normal participants were selected from our hospital between June and July 2018. Normal thoracic models were reconstructed, followed by simulation of lateral fractures through the 4th to 9th ribs under three collapse modes with 1–5 cm of collapse. The thoracic collapse models (P < 0.001). The largest volume reductions in the anterior, posterior, and simultaneous collapse models were in the 6th rib fracture model (P < 0.001), 8th rib fracture model (P < 0.001), and 7th rib fracture model (P < 0.001), respectively.In all three collapse modes, the degree of the collapse was linearly correlated with the mean thoracic volume reduction. The reduction percentage in the posterior collapse mode was higher than that in the anterior collapse mode (The influences of rib fracture displacement and collapse on the thoracic volume in the 6th through 8th ribs are critical in lateral rib fractures. For patients with 6th to 8th rib fractures and posterior rib collapse, surgical intervention to restore thoracic volume may be more essential. Chest blunt trauma (CBT) accounts for more than 15% of emergency trauma cases worldwide, and is second only to head trauma in traffic injury mortality and to head and abdomen injuries in multiple injury mortality . About oChest X-ray (CXR) and chest computed tomography (CT) are conventional imaging methods for blunt trauma evaluation . CXR is Although there are some guidelines for the treatment of rib fractures, most of them are based on expert consensus or clinical experiences , and higThis was a retrospective study conducted using computer simulation and multivariate linear regression analysis to determine the effect of displacement of rib fractures and thoracic collapse on the thoracic volume on the basis of normal chest CT data. This study was approved by the Peking University Research Ethics Committee of our hospital.From June 2018 to July 2018, 7 consecutive participants  were selected from the physical examination center of Peking University People’s Hospital. The selection criteria included 1) normal development without chest deformity; 2) age between 18 and 65 years; 3) no smoking history; and 4) no history of chest trauma or lung or chest disease.All patients underwent chest CT scans with a 64-row Siemens Somatom Definition Flash (128-slice CT). The procedure used for computer simulation and stereo modeling was as follows. First, thin-layer (≤1 mm) data were imported into Materialise Mimics 20.0 software  and a three-dimensional 3D) chest model was built by using the density segmentation tool and hand-drawn selection tool  chest model in Fig. D chest m1 as the number of ribs, the independent variable X2 as the degree of rib collapse (centimeters), and the independent variable X3 as the mode of rib collapse (3 different collapse modes).A total of 630 chest fracture models of rib fractures were simulated from the 7 chest CT datasets, and the thoracic volume in each model was calculated. Multiple linear regression analysis was performed by using R (programming language). We determined the dependent variable Y as the thoracic volume, the independent variable XP < 0.001) and the reduction of thoracic volume in the simultaneous anterior and posterior collapse mode was more significant than the sum of the reductions in the anterior and posterior collapse modes at the same collapse degree (centimeters) (P < 0.001) . The reduction percentage of thoracic volume in the posterior collapse mode was higher than that in the anterior collapse mode (P = 0.69). The largest volume reductions in the anterior, posterior, and simultaneous collapse modes were observed in the 6th rib fracture model (P < 0.001), 8th rib fracture model (P < 0.001), and 7th rib fracture model (P < 0.001), respectively. The collapse of the 4th and 5th ribs caused a relatively small reduction in the thoracic volume, while the anterior collapse of the 9th rib caused almost no change in the thoracic volume. In general, the displacement and collapse of the 6th, 7th, and 8th ribs had a key influence on the thoracic volume in side-rib fractures  . PatientThe anatomical structure of the 24 ribs in the human body is complex because of the irregular shape and different lengths of each rib. As a result, when a rib fracture occurs, the location and type of fracture can be very complex . Clinicap < 0.001). There are two reasons for this. One is that as the length and curvature of the rib on the posterior side are larger than those on the anterior side, when the anterior rib and the posterior rib collapse over the same distance, the posterior lateral collapse of the ribs will reduce thoracic volume to a greater degree. The second reason is that the entire shape of the thorax is narrow in the front and wide at the back, so the posterior part of lateral rib fracture collapse will reduce thoracic volume to a greater extent than the anterior part under the same conditions.The results showed that the reduction in thoracic volume in the posterior collapse mode models was more obvious than that in the anterior collapse mode models . The reason is that when the anterior or posterior part of the rib collapses, the resultant volume reduction decreases from the fracture site to the normal end, but when both anterior and posterior parts of the rib collapse, this situation will not occur.We found that the reduction of thoracic volume in the simultaneous anterior and posterior collapse model was greater than the sum of the reductions in the anterior and posterior collapse models at the same degree of collapse (centimeters)  is quite very novel, which provides a new idea for the similar research in the future. The strengths of this study are as follows. First, simulated rib fractures are free from the influence of pain, pulmonary contusion, and pulmonary infection, which will result in insufficient pulmonary ventilation in actual patients, and the chest volume obtained by CT is less than the actual value , 19. SecThe limitations of this study are as follows. First, the computerized-modelled rib fracture collapse is obtained under ideal conditions, which is different from the actual clinical condition. This study aimed to find out which rib fracture displacement in the lateral rib fracture has the greatest influence on the thoracic volume by computer simulation, not to supply the clinical evidence. The simulated results need to be further verified in clinical practice. Second, we obtained good simulation results for lateral rib fractures in this study. However, we failed to simulate the anterior rib fracture model and posterior rib fractures.In general, the influence of rib fracture displacement and collapse on thoracic volume in fractures of the 6th through 8th ribs are critical in lateral rib fractures. For patients with 6th to 8th rib fractures and posterior rib collapse, operational decisions to perform surgical intervention to restore thoracic volume may be more important. For patients undergoing surgical treatment of flail chest, the collapse of the 6th to 8th ribs and posterior ribs should be first fixed while maintaining the stability of the thoracic cage.Additional file 1. Multiple linear regression results for the three collapse modes.


Oryctolagus cuniculus) is one of the most important breeds of commercial and experimental rabbits in the world, and also one of the most raised rabbit breeds in China. Our goal was to develop a suite of microsatellite markers to aid future conservation genetics research for the Oryctolagus cuniculus breeds. Based on the genetic diversity of 130 New Zealand white rabbits, we obtained a set combination of 22 markers. Then, we performed a genetic analysis of 200 New Zealand white rabbits corresponding to two generations with this combination. It can be used to evaluate the breed conservation of rabbit germplasm resources.The New Zealand white rabbit (p > 0.05), indicating that the characteristics of this excellent rabbit germplasm have been effectively preserved. At the same time, we verified that the established method can be used to evaluate the breed conservation of rabbit germplasm resources.At present, there is an abundance of quality domestic rabbit breeds in China. However, due to the lack of technical standards for the genetic evaluation of rabbit germplasm resources, there have been a number of problems, such as poor breed conservation. By studying the genetic diversity of 130 New Zealand white rabbits (regardless of generation), we obtained the best simple sequence repeat (SSR) marker combination. We found that, when using microsatellite markers for the effective genetic evaluation of domestic rabbits, the number of records should be greater than 60 and the marker number more than 22. Through the comparative analysis of 30 combinations of 22 markers, the optimal combination of 22 markers was determined, and the 22 SSR polymorphic loci were distributed on different chromosomes. We performed a genetic analysis of 200 New Zealand white rabbits corresponding to two generations, using the best SSR polymorphic loci combination. There were no significant differences in the genetic diversity parameters between the two generations of rabbits ( Currently, there is a wealth of domestic rabbit breeds in China, but, due to the lack of standards for the genetic evaluation of rabbit germplasm resources, there have been numerous problems, including the incomplete conservation of breeds. There is insufficient awareness of the necessity to protect the genetic resources of rabbit breeds that are not increasing in numbers, and the current resource protection measures are inadequate. Various factors have led to a sharp decrease in the number of local rabbit breed resource groups in China, and some local rabbit breeds are on the brink of extinction, e.g., Sichuan white rabbits, Himalayan rabbits, and Yunnan flower giant rabbits, and their genetic footprints are now difficult to trace.SOL03, SOL28, SOL08, and SOL30—were found using cloning techniques. The genetic analysis of these four loci in 20 wild rabbits from the East Angola region was used to the advantage of their hybridization. Van et al. [In current molecular genetics research, the main DNA markers used are microsatellites  , restricn et al.  found 18SOL03, SAT2, SOL30, SOL33, SAT5, SAT8, and SAT12) were used to analyze the population structure of European hares from 20 regions [SOL03, SOL08, SOL28, SAT5, SAT7, SOL30, SOL33, SOL44, SAT8, and SAT12) were used to investigate 17 randomly selected hare species from the East Angola region for four consecutive years [Seven SSR polymorphic loci . Operational procedures were stringently conducted in accordance with the Laboratory Animal Requirements of Environment and Housing Facilities (GB14925-2001). All rabbits were from the Pizhou Dongfang Breeding Rabbit Company. We obtained fresh ear tissue from 330 New Zealand white rabbits. DNA was extracted from fresh ear tissue using TIANamp Genomic DNA kits . The DNA was analyzed using agarose gel electrophoresis, and the concentration and purity were checked with a NanoDrop 1000 .2+ free) Buffer, 0.8–1.2 μL of 25 mmol/L MgCl2, l.0 μL of 2.5 mmol/L dNTPs, 1 μL of each 10 μmol/μL unlabeled upstream and downstream primer, 1.0 U of Taq DNA polymerase, and 1.0 μL of 100 ng/μL DNA template, supplemented with 20 μL of ddH2O. The PCR cycles started with 5 min of denaturation at 95 °C, which was followed by 34 cycles of 45 s of denaturation at 94 °C, 40 s of annealing at 50–64 °C (depending on the loci), and 40 s of elongation at 72 °C; the program was ended with a 10 min final elongation at 72 °C.To genotype all the subjects, we used the double PCR approach. By referring to the rabbit gene database and existing references ,11,24,25The PCR products were examined by 1% agarose gel electrophoresis with observation under a UV lamp and photography to record the results. Then, the PCR products were detected using polyacrylamide gel electrophoresis (PAGE), and the ImageQuant300 imaging system was used to photograph and store the most effective images. Using the ONED scan software and referring to the PBR322 DNA/Msp I Marker, the PAGE bands were analyzed to determine the approximate location range of the 43 SSR loci, which laid the foundations for subsequent sequencing analysis.According to the genotypic analysis results from the New Zealand white rabbit PAGE and referring to the sequence sizes of the target fragments of the SSR loci published in GenBank, the SSR loci were used as the parameters, and markers with a difference of more than 20 bp were used. The markers were combined, and each group had three loci. The 5’ ends of each pair of primers were labeled with FAM (blue), HEX (green), or TAMRA (yellow) fluorescent dyes, respectively, resulting in 15 different combinations. Short tandem repeat typing was performed according to the mixed PCR product of each labeled combination. The combination of 43 pairs of fluorescently labeled SSR-labeled primers and PCR reaction conditions are shown in 2+-free) buffer; 0.8–1.2 μL of 25 mmol/L MgCl2; l μL of 2.5 mmol/L dNTPs; 1 μL of each HEX-, FAM-, or TAMRA-labeled forward and unlabeled reverse primer (10 μmol/µL); 1.0 U of Taq DNA polymerase; 1 μL DNA template (100 ng/μL); and 20 μL of ddH2O. The entire loading procedure was carried out on ice, and amplifications were carried out in a 96-well plate. Because the fluorescent SSR primers decompose easily, they were protected from light as much as possible during the sample loading. After the PCR amplifications, each set of amplification products was mixed then covered with tinfoil to prevent exposure to light during storage at −20 °C. The PCR amplification procedure was the same as that for the first PCR.PCRs were performed in 20 μL volumes consisting of 2 μL of l0 × PCR , expected heterozygosity (He), observed heterozygosity (Ho), and polymorphic information content (PIC) were analyzed with GENEPOP 4.2 ,29.SAT2 (HEX), SOL62 (FAM), and INRACCDDV0313 (TRAMER).All the alleles of the 43 SSR loci were detected within 500 bp, with a detection range of between 88 and 456 bp. The agarose electrophoresis results for the PCR amplification products are shown in Using the results of the microsatellite fluorescent labeling capillary electrophoresis, the genetic diversity parameters of New Zealand white rabbit population were analyzed. The results show that a total of 184 alleles were detected in 130 individuals with 43 SSR polymorphic loci . The avep < 0.05) deviated from Hardy–Weinberg equilibrium in 130 individuals. There were 24 SSR loci in the unbalanced state (p < 0.01), and 12 SSR loci (p > 0.05) followed the equilibrium state: SAT8, D7UTR5, SAT2, INRACCDDV0007, INRACCDDV0018, INRACCDDV0313, INRACCDDV0160, D3UTR2, D6UTR4, 12L1C2, SOL03, and 5LIE8 (df = 1). The effective population size of New Zealand white rabbits, which was calculated using the linkage disequilibrium method, was found to be 56.4 (CI95% = 54.0–59.0) in 43 loci of 130 rabbits.The Hardy–Weinberg equilibrium test results for the New Zealand white rabbits are shown in The Ne, He, and average PIC of New Zealand white rabbits was analyzed using different numbers of records A. The reBy analyzing the effect of the loci numbers on the Ne, He, and PIC for each number of records of New Zealand white rabbits, the trend at each locus was revealed B. The reIn order to reduce the cost of the experiment and improve the efficiency of the work, we first numbered the original 43 SSR markers in order from 1 to 43, as shown in L8B5 on Chromosome 7; D7UTR5 and SOL62 on Chromosome 6; SAT2 and INRACCDDV007 on Chromosome 5; INRACCDDV0087 on Chromosome 8; INRACCDDV0192 on Chromosome 2; INRACCDDV0185 on Chromosome 16; INRACCDDV0190 on Chromosome 18; INRACCDDV0346 on Chromosome 13; INRACCDDV0160 on Chromosome 9; INRACCDDV0160 on Chromosome 9; SOL44 on Chromosome 8; 12L4A1 on Chromosome 3; 6L1F10 on Chromosome 4; 6L3F8 on Chromosome 3; D3UTR2, 7L1B10, and 19L1C5 on Chromosome 3; and SOL33, SOL03, and 12LIE11 on Chromosome 9.The positions of the best combination were primarily located on different chromosomes. The positions of the best combination loci were: p > 0.05).Statistical analysis was performed on the genetic diversity parameters of 43 markers and 22 optimal markers for the original generation of New Zealand white rabbits . The resp > 0.05).Statistical analysis was performed on the genetic diversity parameters of the 45 total and 22 optimal SSR polymorphic loci for the first generation of New Zealand white rabbits . The NA,p > 0.05); thus, the germplasm characteristics of the rabbit breeds were effectively preserved.The genetic diversity of the two generations of New Zealand white rabbits was analyzed using the optimal combination of SSR polymorphic loci. p > 0.05). This provides evidence for the feasibility of using the 22 optimal loci combinations for the preliminary genetic evaluation of rabbits.In order to further verify the reliability of the 22 optimal marker loci for evaluating the breeding effect of New Zealand white rabbits, we analyzed the parameters of the genetic diversity of the two generations of New Zealand white rabbits at the original 43 SSR polymorphic loci. As The preservation of the maximum genetic diversity in a population is one of the main objectives within a breed conservation program . When stThis study aimed to establish an effective and fast genetic evaluation system for rabbits using different parameters for the number of records and loci number and to provide a reference for exploring the germplasm resources and genetic structure of rabbit breeds in China. The method used in this study for exploring the effect of different numbers of records and loci numbers on the population genetic structure has been applied to other animal populations. Ceccobelli et al. reduced the loci number from 16 to 12 to distinguish the local goat population, indicating that microsatellite markers are an inexpensive method for discriminating local livestock breeds . A combiThe genetic diversity of a germplasm population can be tested at regular intervals through rapid and efficient detection methods, and, after detection, the population’s germplasm resources can be protected through measures to maintain a rich genetic diversity. The Food and Agriculture Organization of the United Nations (FAO) used SSR polymorphic loci to study the genetic diversity of 14 species of livestock. The research led to the standard conditions for assessing quality germplasm resources: a marker number greater than 30 and a number of records greater than 50. Nei ,36 pointWhen evaluating the genetic diversity of rabbit populations, proper experimental design is especially crucial. The choice of locus type and quantity is equally as important as the number of records. In this study, SSR polymorphic loci were used to study various trends in the genetic diversity parameters of New Zealand white rabbit breeds using different numbers of markers. The investigation showed that the Ne varied with the number of records and loci number, i.e., the Ae tended to be stable at 22–43 loci. Therefore, when the Ae of the New Zealand white rabbits was analyzed, more than 22 loci were selected. The degree of heterozygosity is expected to change with the number of markers for each number of records, i.e., the genetic diversity parameters should stabilize at 22–43 loci. Similarly, the PIC for each number of records showed the same trend associated with loci number. The PIC also tended to begin to stabilize at 22 or more loci, so when New Zealand white rabbit genetics were evaluated for PIC, the loci number selected was 22, but numbers above that are more appropriate. Barker ,38 foundp > 0.05). This shows that the gene bank’s preservation method has effectively preserved the germplasm characteristics of New Zealand white rabbit breeds.In this study, a combination of 22 optimal SSR polymorphic loci were selected and used to detect genetic variation in different generations of New Zealand white rabbit for gene bank conservation. The results provide a theoretical reference for future rabbit conservation. We found that the difference between the genetic diversity parameters of two generations was not significant (For the effective genetic evaluation of rabbits using SSR markers, a record number greater than 60 and loci number of more than 22 are appropriate. The evaluation system based on an optimum combination of 22 loci provides a fast and effective method for the evaluation and establishment of excellent rabbit germplasms. The established method needs to be validated in an outside population of rabbits.


Lamb live weight is one of the key drivers of profitability on sheep farms. Previous studies in Ireland have estimated genetic parameters for live weight and carcass composition traits using a multi-breed population rather than on an individual breed basis. The objective of the present study was to undertake genetic analyses of three lamb live weight and two carcass composition traits pertaining to purebred Texel, Suffolk and Charollais lambs born in the Republic of Ireland between 2010 and 2017, inclusive. Traits (with lamb age range in parenthesis) considered in the analyses were: pre-weaning weight (20 to 65 days), weaning weight (66 to 120 days), post-weaning weight (121 to 180 days), muscle depth (121 to 180 days) and fat depth (121 to 180 days). After data edits, 137 402 records from 50 372 lambs across 416 flocks were analysed. Variance components were derived using animal linear mixed models separately for each breed. Fixed effects included for all traits were contemporary group, age at first lambing of the dam, parity of the dam, a gender by age of the lamb interaction and a birth type by rearing type of the lamb interaction. Random effects investigated in the pre-weaning and weaning weight analyses included animal direct additive genetic, dam maternal genetic, litter common environment, dam permanent environment and residual variances. The model of analysis for post-weaning, muscle and fat depth included an animal direct additive genetic and litter common environment effect only. Significant direct additive genetic variation existed in all cases. Direct heritability for pre-weaning weight ranged from 0.14 to 0.30 across the three breeds. Weaning weight had a direct heritability ranging from 0.17 to 0.27 and post-weaning weight had a direct heritability ranging from 0.15 to 0.27. Muscle and fat depth heritability estimates ranged from 0.21 to 0.31 and 0.15 to 0.20, respectively. Positive direct correlations were evident for all traits. Results revealed ample genetic variation among animals for the studied traits and significant differences between breeds to suggest that genetic evaluations could be conducted on a per-breed basis. This study demonstrated the existence of genetic variation between different breeds of sheep for the three main live weight and two carcass composition traits in the Irish sheep production system suggesting that genetic evaluations should be conducted on a per-breed basis. This would allow for more informed and accurate selection decisions on farm, resulting in superior productivity and profitability within Irish sheep flocks.et al., et al., et al., 2004; Jones et al., et al., et al., et al., et al., et al., et al., et al., Lamb live weight and the rate at which the animal grows have been defined as the key drivers of profitability in Irish . Records pertaining to years 2010 to 2017, inclusive, were retained for analyses. Only purebred lambs (as defined by the data records) of the three aforementioned breeds  were considered in the present study.A full database was extracted across three breeds, namely Texel, Suffolk and Charollais, from Sheep Ireland, the Irish national database . Muscle and fat depth traits were recorded on the same day as post-weaning weight in all lambs. Only muscle depth measurements within the range of 10 to 44 mm and fat depth measurements ranging within 1 to 23 mm were retained.Live weight and carcass composition measurement records were discarded if flock of birth, sire, dam or maternal grandsire were unknown. Dams with no known parity number or a parity number >10 were discarded; parity number was subsequently categorised as 1, 2, 3, 4 or ≥ 5. Age at first lambing was defined based on the age of the ewe at first lambing; ewes were either defined as lambing for the first time as ewe lambs (between 8 and 18 months of age) or those that lambed for the first time as hoggets (between ≥18 and 28 months of age). Birth type was defined as the number of lambs born per lambing event; only birth types between 1 (singles) and 4 (quadruplets) were retained. Rearing type was defined as the number of lambs reared per litter; only rearing type between 1 and 3 were retained for analysis. Lambs that were recorded as artificially reared or reared by a non-genetic dam were not included for further analysis.For all traits, each lamb was allocated to a contemporary group of breed-by-flock-by-week of weighing. Only contemporary groups containing at least five records were retained for analysis. Following all edits described above, 33 721 pre-weaning weight records, 32 623 weaning weight records, 28 140 post-weaning weight records, 21 468 muscle depth records and 21 442 fat depth records were retained for genetic analysis; the breakdown of records per breed is shown in Table et al., Y = lamb live weight or carcass composition record, CG = contemporary group, AFL = age at first lambing of the dam, Parity = parity of the dam, Gender*Age = the interaction between the gender and age of the lamb, Birth type*Rearing type = the interaction between the birth type and rearing type of the lamb, Animal = random animal direct additive genetic effect, Dam = random maternal genetic effect, DamPE = random dam permanent environmental effect associated with multiple lambing records of the same dam, Litter = common environmental effect reflecting the non-genetic covariance among members of the same litter and e = random residual effect.Variance components were estimated for each lamb live weight trait  and each carcass composition trait  using linear mixed animal models in ASReml  were calculated for each trait and genetic trends were produced from these results by estimating the slope of the average ram EBV per year of birth. Genetic trends were only produced for sires with at least 10 progeny and ranged from 3 to 61 sires per year across all traits and breeds.Direct heritability was calculated as the ratio of the direct additive genetic variance to the observed total phenotypic variance. Maternal heritability was estimated as the ratio of the maternal genetic variance to the total phenotypic variance. Common environmental effect was calculated as the ratio of the litter variance to the total phenotypic variance. Dam repeatability was calculated as the ratio of maternal genetic variance plus permanent environment to the total phenotypic variance. The correlation between the direct additive and maternal genetic effects was also estimated where applicable. Genetic correlations between the studied traits were estimated pairwise using the model previously described in a series of bivariate analyses. Estimated breeding values  as shown in Table Variance components were estimated (Table P < 0.05) positive genetic correlations between the direct additive genetic effects on pre-weaning and subsequent weights for each of the three breeds were calculated  trends were observed for all live weight traits in the Texel breed, pre-weaning weight in the Suffolk breed and weaning weight in the Charollais breed. Muscle depth had a strong positive significant trend for all breeds, while fat depth had weakly positive significant trends for both the Suffolk and Charollais breeds. There was considerable variation in genetic trends estimated for the same trait among the three studied breeds with higher rates of genetic gain being achieved in the Texel breed for live weight traits and muscle depth in comparison to the other two breeds.Genetic trends based on EBVs of rams with ≥10 progeny  to 0.32 (Texel). Genetic parameter estimates have not previously been reported in Ireland on a per-breed basis. One previous study reported genetic parameter estimates within a multi-breed analysis (McHugh , et al.  where a  (et al. . In the  et al.,  and resu, et al.  suggesteet al. (et al., The strong positive direct genetic correlations among the three live weight traits were as expected, indicating that lambs that are genetically heavier early in life are also more likely to be genetically heavier later on. While these figures corresponded well with the literature, some of the estimates in the present study were outside the ranges previously reported with weaker correlations observed in the present study compared to those previously reported (Safari and Fogarty, et al.  that sho et al.,  was baseet al., et al., et al., As with the live weight traits, strong positive correlations were also seen among the two carcass composition traits and post-weaning weight. Very few previous studies have estimated correlations among these traits at the similar time points to the present study; however, the direct correlations estimated here are broadly within the range previously reported (Atkins et al., et al., et al., For pre-weaning and weaning weight, a negative correlation was observed between the direct additive and maternal genetic effects. Although this corresponded with the majority of the literature for growth and live weight traits (Notter, To our knowledge, this is the first time genetic trends on Irish sheep are reported for the studied traits. Genetic trends varied between the three breeds for all traits in the present study. From the genetic trends, the Texel breed appears to be achieving the most genetic gain as significantly positive trends were recorded for all live weight traits as well as the muscle depth trait. No significant trend was found for fat depth in the Texels, indicating that this trait is remaining relatively static which is more desirable than the increasing trend observed for the Suffolk and Charollais breeds. The muscle depth trait showed a positive trend for all three breeds. These results are indicative of the ongoing genetic selection programme in Ireland based on the emphasis that is being placed on muscle depth for all breeds as well as the increase in genetic gain in live weight that has been seen in all three breeds.Variance components and genetic parameters derived in the present study for five live weight and carcass traits may be used to support the breeding programme of sheep in Ireland. Considerable differences in genetic analysis results were found between the Texel, Suffolk and Charollais breeds for each of the five traits examined in the present study. Differences were observed in both heritability and genetic correlation estimates suggesting that current genetic improvement systems may benefit by considering these breeds separately in future genetic evaluations.


The indigenous cattle populations from Greece and Cyprus have decreased to small numbers and are currently at risk of extinction due to socio-economic reasons, geographic isolation and crossbreeding with commercial breeds. This study represents the first comprehensive genome-wide analysis of 10 indigenous cattle populations from continental Greece and the Greek islands, and one from Cyprus, and compares them with 104 international breeds using more than 46,000 single nucleotide polymorphisms (SNPs).Bos indicus introgression in almost all the investigated local cattle breeds along the route from Anatolia up to the southern foothills of the Alps, as well as in most cattle breeds along the Apennine peninsula to the southern foothills of the Alps.We estimated several parameters of genetic diversity  that indicated a severe loss of genetic diversity for the island populations compared to the mainland populations, which is mainly due to the declining size of their population in recent years and subsequent inbreeding. This high inbreeding status also resulted in higher genetic differentiation within the Greek and Cyprus cattle group compared to the remaining geographical breed groups. Supervised and unsupervised cluster analyses revealed that the phylogenetic patterns in the indigenous Greek breeds were consistent with their geographical origin and historical information regarding crosses with breeds of Anatolian or Balkan origin. Cyprus cattle showed a relatively high indicine ancestry. Greek island populations are placed close to the root of the tree as defined by Gir and the outgroup Yak, whereas the mainland breeds share a common historical origin with Buša. Unsupervised clustering and D-statistics analyses provided strong support for All investigated Cyprus and Greek breeds present complex mosaic genomes as a result of historical and recent admixture events between neighbor and well-separated breeds. While the contribution of some mainland breeds to the genetic diversity pool seems important, some island and fragmented mainland breeds suffer from a severe decline of population size and loss of alleles due to genetic drift. Conservation programs that are a compromise between what is feasible and what is desirable should focus not only on the still highly diverse mainland breeds but also promote and explore the conservation possibilities for island breeds. Bos taurus) and indicine (Bos indicus) species arose, sharing the aurochs (Bos primigenius) as common ancestor ~ 250,000 years ago ) (Table fpA = 0.376) as well as the highest average inbreeding coefficient (F = 0.457). For GRB, most of the diversity estimates had the highest values but the frequency of private alleles (fpA = 0.021) and the inbreeding coefficient (F = 0.108) were low. Generally, all the analyzed island populations except the KEA breed, which was sampled on the island of Kea and on mainland, had very high levels of inbreeding and very low diversity parameters (Table HO) and bi-allelic SNPs (HO[SNP]) side-by-side in Fig. HO[SNP] suggests a high level of genetic diversity in some Alpine and North West European breeds, whereas HO highlights breeds from South East Europe and Anatolia as having the highest level of diversity. The ascertainment bias of SNP chip data was further highlighted by the scatterplot of HO[SNP] versus HO in Fig. HO[SNP] and HO are estimators of the true diversity. Therefore, the diversity of the breeds placed above the overall trend line (e.g. North West Europe) is overestimated by HO[SNP] and the diversity of the breeds placed below this line (e.g. Minor Asia) is underestimated by HO[SNP].In total, 590 common alleles were detected among the 115 breeds studied, which represents only 0.7% of the total number 80,720) of alleles. All estimators of genetic diversity for the breeds studied here were highly differentiated among the predefined geographical groups  is relatively small and ranges from 28 in the Iberian to 67 in the French geographic group. The Greek and Cyprus group showed a small average value (Ne5 = 40). Only GRB showed a larger Ne5(Ne5 = 85) than the weighted average of the group (Table Ne50 250 years ago), the effective population size was clearly larger for the South East Europe Buša (Ne50 = 399) group than for other European breed groups. Only the Minor Asian group showed a larger value (Ne50 = 549) during pre-industrial times. A comparable trend was also observed for effective population size 10,000 years ago (Ne2000). The values for the GRB and Buša groups were comparable and differed from those for the other Greek cattle breeds. The 11 indigenous CRT animals sampled on the island of Crete showed the highest inbreeding level and the smallest effective population size  in the entire dataset  ranged from 0.001 to 0.462. The breeds and breed groups with high allelic diversity and high heterozygosity showed a very low level of differentiation. For example, five breeds of the Minor Asia group showed an average differentiation to each other of only 0.006 and the Buša breeds a value of 0.050. On the other hand, the Greek and Cyprus breeds were highly differentiated  and high values were obtained between two island breeds i.e. CRT-AGT (DEST = 0.413). It is remarkable that the CRT breed showed the highest differentiation level among all the investigated breeds  between animals within a breed as a measure of the breed-level differentiation. As shown in Additional file PS values were observed for most of the Buša and Anatolian breeds together with the Greek GRB and the Italian PODO breeds, whereas the lowest values were observed for the Greek island breeds.In addition, we used average allele sharing distance . In addition, we used the allele counts of bi-allelic SNPs and reconstructed the phylogeny with the TreeMix program  among 2858 animals and projected these by multidimensional scaling (MDS) on a two-dimensional (2D) plane. The MDS projection of 115 breeds is shown in Fig. Bos indicus and the remaining European cattle breeds. CYP, AGT and KAS cluster together with the Anatolian breeds, except TRG. Subsequently, the mainland Greek breeds  and the Nisyros island breed (NSY) cluster in the geographic region corresponding to some of the Buša breeds , some of the breeds from the East Podolian geographic group  and some Italian breeds (Tyrrhenian group) of South and Central Italy  including all Italian podolic breeds . CRT is isolated from the other two main Greek breed clusters. KEA is positioned in the geographic region of some Alpine and Italian breeds, showing a closer relationship to these breeds than to its own (Greek) geographic cluster. The second dimension of MDS (MDS2) separates the breeds of North Europe and Alpine geographic area from the breeds of central, west and southern Europe.We used multi-allelic SNP blocks to estimate the allele sharing distance matrix (Dcv error = 0.5358) was determined at K = 78. Figure K = 4, 10, 24 and 78. The reasons for choosing K = 78 are that: (i) with a K larger than 4, animals from highly selected breeds start to separate into distinct clusters; (ii) the clusters at K = 10 represent the number of pre-defined geographical breed groups plus 1, i.e. nine groups of European Bos taurus plus NDA and GIR; (iii) the cross-validation error decreases almost linearly from K = 1 to K = 24; and (iv) the clustering with the lowest cross-validation error is at K = 78. Additional file K = 2 till 26. All breeds from Minor Asia and most of the South East geographic groups, as well as GRB, showed a high level of complex admixture even at K = 78. All highly selected breeds and isolated breeds formed their own cluster. GRB and KTR, and SYK to a lesser degree, shared the same pattern with most of the breeds belonging to the South East group with evidence of shared ancestry between these and the Anatolian breeds. The highly inbred and differentiated island breeds assigned to separate clusters. The same was also observed for small-sized mainland populations, such as PRG and ROG. At K = 24, a significant proportion of the Alpine ancestry  was estimated for KEA, which is also retained at the most probable true K = 78.The Admixture analysis presents the second unsupervised clustering method applied to 2832 animals (26 Mongolian yak excluded) using the genotypes of bi-allelic SNPs. The lowest cross-validation error  = 0.72) and low negative correlation between F and npA  = − 0.21).In spite of the ascertainment bias of the BovineSNP50 chip data , 43, it LL Table . We obseDEST levels . For the whole territory of the Dodecanese islands, on which the AGT, NSY, and KAS breeds are raised, the same source identifed 2434 of the 6210 recorded animals (39%) as local. Today, the population size of all the above-mentioned cattle breeds is very small  were observed for the breed groups from Minor Asia and South East Europe and for some mainland Greek breeds [e.g. GRB, PRG, SYK, and KTR  suggests that compared to western European breeds, modern South East European cattle breeds had larger founder population sizes as a result of their proximity to the center of domestication. In fact, up to ~ 50 generations ago, cattle breeds from southeastern Europe had larger Ne than those from northwestern Europe. However, the current Ne indicates that the population size of the South East European cattle breeds such as Buša and GRB, has decreased substantially in the last 50 generations or so. Furthermore, this observation implies that such populations have accumulated a small number of common but long haplotypes, which contribute to the high LD. The industrial revolution, modern breeding practices and changes in customers’ preference have led to the replacement of local cattle breeds with commercial cattle breeds, which mostly originate from northwestern Europe. This factor, coupled with uncontrolled breeding in the remaining fragmented populations due to the absence of modern reproduction and breeding management practices contributed to population shrinkage of most of the previously highly diverse cattle breeds from southeastern Europe.Estimating the Bos indicus influence was found is SIC (Z < |3|), with a D-value close to zero. The D-values clearly decrease as the spatial distance to the origin of Bos indicus increases. Interestingly, three breeds from the East Podolian roup also show a significant indicine introgression, among which UKP that was sampled east of the Carpate Mountains. As discussed recently by Verdugo et al. [Bos indicus alleles. It should be noted that we cannot distinguish the proportion of diversity caused by introgression from that caused by other evolutionary forces, e.g. low pressure of artificial selection. We also note that some estimators of allelic diversity, such as the number of private and semi-private alleles measures the proportion of unique alleles not present even in GIR or other neighbor breeds. The fact that we sampled and genotyped some of the close neighboring breeds intrinsically reduced the number of private and semi-private alleles. However, although GIR and many neighbor breeds from South East Europe and Greece were sampled  code assigned to the pre-defined groups within this paper, breed names, breed code, number of sampled and genotyped individuals (N), number of genotyped and unrelated individuals used to estimate the diversity parameters (Nd), current breeding purposes as well as sporadic or recent past breeding purposes in parenthesis, breed origin, and source of the samples or genotypes used in this study or from previous studies [Table S2. Phenotypic, productive and reproductive traits. Description of phenotype, productive and reproductive traits of the 11 Greek and Cypriot analyzed breeds. Table S3. Evolution of bovine population sizes on the islands during the last 60 years. Statistical information regarding the evolution of cattle population on the Greek island based on [ studies , 67–70. based on .Additional file 2. Detailed description of the indigenous Greek and Cypriot cattle. Compilation of information on Greek indigenous cattle breeds from diverse sources [ sources , 71–87. Additional file 3: Figure S1. Tessellated projection. Spatial geographic presentation of the herein estimated diversity parameters , npA, nspA).Additional file 4: Figure S2. Tessellated projection. Spatial geographic presentation of the estimated effective population number .Additional file 5: Table S4. Genetic differentiation index. Pairwise values for DEST, per analyzed breed. Maximum and minimum values within each of the predefined geographical groups are depicted in bold letters in gray rectangle. Maximum and minimum values considering all 115 breeds are in bold letters in green and yellow, respectively.Additional file 6: Figure S3. Tessellated projection. Spatial geographic presentation of the estimated allele sharing distance matrix (DPS) among breeds using multi-allelic SNP-blocks.Additional file 7: Figure S4. Phylogenetic tree. Neighbor-joining tree based on Nei’s genetic distance DA using multi-allelic SNP blocks. Mongolian yak (YAK) was used as a root. Dotted lines indicate the reduced length of YAK and GIR to improve visibility. Special square marks represent the influence of East-Podolian (grey), Alpine (green) and North-West (olive green) group.Additional file 8: Figure S5. Admixture analysis. Population structure of the 114 studied breeds/populations presented for each of K inferred ancestral population value, using a genome‐wide set of bi-allelic SNPs. All the analyzed individuals are presented as colored vertical bars, divided at most K segments and with proportional heights, according to their genotype membership. In the depicted plots (K = 2 to K = 26), a different color is presented for each cluster. From left to right within each group the presented breeds are as follows: Minor Asia: ATER, ATBC, ATSR, ATSY, TRG; Greece and Cyprus: CYP, AGT, CRT, NSY, GRB, KAS, KEA, PRG, ROG, SYK, KTR; South East Europe: RHS, MKB, SRB, PRE, RMB, SHB, DGB, DBB, MAB, LKB, SKB, MNB, BHB, HRB; East Podolian: HRI, HRP, UKP; Tyrrhenian: PODO, CINI, MOSI, RSIC, MOSA, SARD, SBRU, CORS, AGER, MARE, CHI, MPIS, CALV, MCH, RMG, GARF, PONT, MODE, CABA, REGG, PMT, BURL; Alpine: PRDO, OVAR, REND, BPUS, PUST, SIC, PIN, TGV, MWF, OBV, BBV, DFV, FGV, VOG, ABO, MON, TAR; France: RDBI, SAL, AUB, LIM, CHR, PAR, BAQ, GAS; Iberian: MNRQ, MALL, NGAN, CANA, MARI, ALEN, BAR, MARO, SYG; North West Europe: BPN, NOR, MAN, BBB, LKF, HF, GNS, JSY, HER, SHR, KRY, DXT, GLW, AAN, HGL, NRC, SERC, FJL, FIAY, FINE, FINW, FINN, YARO.


Coregonus artedi complex in the Apostle Islands of Lake Superior, the largest lake in the Laurentian Great Lakes. Using 29,068 SNPs, we were able to clearly distinguish among the three most common forms for the first time, as well as identify putative hybrids and potentially misidentified specimens. Population assignment rates for these forms using our RAD data were 93%–100% with the only mis‐assignments arising from putative hybrids, an improvement from 62% to 77% using microsatellites. Estimates of pairwise differentiation (FST: 0.045–0.056) were large given the detection of hybrids, suggesting that reduced fitness of hybrid individuals may be a potential mechanism for the maintenance of differentiation. We also used a newly built C. artedi linkage map to look for islands of genetic divergence among forms and found widespread differentiation across the genome, a pattern indicative of long‐term drift, suggesting that these forms have been reproductively isolated for a substantial amount of time. The results of this study provide valuable information that can be applied to develop well‐informed management strategies and stress the importance of re‐evaluating conservation units with genomic tools to ensure they accurately reflect species diversity.Effective resource management depends on our ability to partition diversity into biologically meaningful units. Recent evolutionary divergence, however, can often lead to ambiguity in morphological and genetic differentiation, complicating the delineation of valid conservation units. Such is the case with the “coregonine problem,” where recent postglacial radiations of coregonines into lacustrine habitats resulted in the evolution of numerous species flocks, often with ambiguous taxonomy. The application of genomics methods is beginning to shed light on this problem and the evolutionary mechanisms underlying divergence in these ecologically and economically important fishes. Here, we used restriction site‐associated DNA (RAD) sequencing to examine genetic diversity and differentiation among sympatric forms in the  In addition, we included a small number of available samples from two rare forms of cisco—zenithicus and the nigripinnis‐like—from Minnesota, Michigan, and Ontario waters in Lake Superior collected by the GLSC in summers of 2007, 2012–2015. All samples were identified to putative form using a suite of standardized morphological characteristics  and normalized to a concentration of approximately 50 ng/µl for a 2‐µl digestion reaction with the restriction enzyme SbfI followed by ligation with barcoded adaptors. Individually barcoded libraries were pooled into master libraries of 96 and fragmented to ~300‐500bp with 12–14 30‐s cycles in a Q500 sonicator . Fragmented DNA containing library adapters was bound to Dynabeads™ M‐280 Streptavidin magnetic beads (Invitrogen) and washed with buffer to remove nontarget fragments before an incubation step to release DNA from the beads. Following purification with AMPure XP beads , master libraries were input into the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® at the End Prep step for ligation of master library barcodes, a 250‐bp insert size‐selection, and a 12‐cycle PCR enrichment. Successful size selection and enrichment were confirmed with visualization of products on a 2% agarose E‐Gel (Invitrogen). Products underwent a final AMPure XP purification cleanup followed by quantification with a Qubit® 2.0 Fluorometer. All prepared libraries were sent to Novogene  for sequencing on the Illumina NovaseqS4 platform.Restriction site‐associated DNA (RAD) libraries were prepared following the BestRAD protocol . Filtered reads for each individual were aligned to create matching stacks with ustacks following guidelines suggested from empirical testing to avoid under‐ or over‐merging loci from RAD datasets . Locus stacks for each individual were matched to the catalog using sstacks (parameter flag: ‐‐disable_gapped), data were oriented by locus in tsv2bam, and reads were aligned to loci and SNPs were called with gstacks. SNPs genotyped in greater than 30% of individuals (parameter flag: ‐r 0.3) were exported with the subprogram populations in genepop and variant call format (vcf) files. We isolated SNP calling to single‐end reads to ensure equal coverage across sequences, but paired‐end assemblies for each locus are available from Blumstein et al.  and lower (L: 163‐193bp) size ranges; therefore, Sfo8 is considered two loci. Fragment analysis was performed using a Genetic Analyzer 3.0 (Life Technologies), and genotypes were assigned at each locus using GeneMapper 3.7 (Life Technologies). We used Genepop v4 . Three loci were removed for being out of Hardy–Weinberg Equilibrium in all three forms , and no loci showed significant linkage disequilibrium.We used the methods described in Stott et al.  to genotRousset,  to condu2.4FST‐related measures of population‐level differences in allele frequencies and was run on our data with default parameters. Pcadapt takes an individual‐level approach and uses principal component analysis (PCA) for outlier detection. A scree plot of the first 20 principal components (termed K in pcadapt) indicated that the optimal K was 2 for computing correlations between our RAD loci and K principal components. We used Benjamini and Hochberg's , so we proceeded using the full dataset of RAD loci.To examine the influence of selection on estimates of differentiation and diversity with our RAD dataset, we employed two statistical tools for the identification of putative outlier loci—BayeScan v2.1  to examine support for each K. ADMIXTURE analysis was only conducted on the RAD dataset because this program is not compatible with microsatellite data.To ensure that our putative form designations were appropriate, we used two approaches to assess genetic similarity among individuals. First, we conducted a PCA in the R package “adegenet” Jombart,  for bothhoyi fell within the cluster of kiyi samples. Given the discrete clustering of the remaining of hoyi and kiyi, there is strong possibility that these three hoyi samples were misidentified in the field, so we removed these individuals from both RAD and microsatellite datasets for all further analyses to prevent bias in estimates of diversity, differentiation, and Ne.PCA and ADMIXTURE analysis with RAD genotypes revealed that three individuals originally identified as artedi—ART, hoyi—HOY, kiyi—KIY, nigripinnis—NIG, and zenithicus—ZEN) including percentage of total observed alleles, observed and expected heterozygosity, and an inbreeding coefficient (GIS). Summary statistics for each locus and populations were calculated using both microsatellite and RAD datasets in GenoDive v2.0b23 . To compare genetic differentiation between RAD and microsatellite datasets, we used GenoDive to estimate standardized pairwise genetic differentiation, G’ST  that an individual genotype is from a specific group given the allele frequencies  was estimated for all forms with n > 10 using RAD and microsatellite datasets with the bias‐corrected linkage disequilibrium method , equal proportions of female and male migration, biallelic loci with free recombination, a mutation rate equivalent to that measured in human and fish nuclear genomes , (b) approximately twice the amount observed (FST ≈0.10), and (c) approximately four times the amount observed (FST ≈0.20). Preliminary levels of differentiation were achieved with low migration rates (m) set over 1,000 generations  and followed by 5, 10, or 15 generations of one of three higher migration  rates  for every initial level of differentiation. Each unique parameter combination was run using a dataset of 1,000 loci and replicated 50 times. Estimates of FST for each replicate were generated in the R package “diveRsity”  over an extended period of time , and we allowed populations to hybridize for 2, 5, or 10 generations with m = 0.05, which was equal to roughly the proportion of hybrids observed in our wild populations between ART‐HOY and HOY‐KIY. Since no putative ART‐KIY hybrids were observed in our RAD dataset, we chose to implement a one‐dimensional stepping‐stone model of migration . We then plotted overall FST across the genome and constructed a bubble plot to visualize the number of significant windows on each LG for each comparison. We found over 50 significant windows for each comparison (see results). Conducting an in‐depth investigation of all significant windows was not feasible; therefore, we isolated in‐depth analysis to one LG (Cart21) that contained the most significant windows and outlier loci in the dataset.We examined genetic differentiation across the genome by pairing our data with the n et al. . Catalogn et al.  that incn et al. . Highly Salmo salar), which is syntenic to Cart21 in artedi  were also placed on the linkage map. Since RAD sequencing and microsatellite amplification were performed on the same hoyi and kiyi samples, microsatellite genotypes used in our analyses were restricted to the same individuals that successfully genotyped with our RAD loci. An additional 30 artedi from Stockton Island were genotyped at the 9 microsatellite loci.A total of 137 individuals were RAD sequenced producing more than 455 million reads and an average of 3,346,457 reads per sample. After filtering, 119 individuals with representatives from all five putative cisco forms in Lake Superior were genotyped at 29,068 loci (Table 3.2FST) decreased by an average of only 0.0039 with the neutral dataset. Results from exploratory analyses with the neutral dataset are included . Of the rare forms, nigripinnis (NIG) loosely grouped in the center of the PCA, with five of the six samples falling out between the ART, HOY, and KIY clusters  or the KIY cluster (n = 3). Low representation of both NIG and ZEN in our RAD dataset reduces our ability to draw strong conclusions based on these PCA results, so both groups were unaltered for estimates of diversity, inbreeding, and differentiation and dropped for the remaining analyses.PCA showed a sharp contrast in resolution between marker sets with microsatellites producing one large cluster of overlapped forms across the first two principal components and the RAD dataset producing three major clusters primarily composed of ART, HOY, and KIY Figure . The ARTs Figure . The sixK) estimated using the cross‐validation procedure in ADMIXTURE was two , the major genetic ancestries differentiate the ART, HOY, and KIY clusters as seen on PC2. The three putatively misidentified hoyi first noted in the PCA all had Q estimates of 100% for the KIY cluster and were removed from further analyses. Additional Ks did not differentiate either the NIG or ZEN cluster but begin to differentiate small subsets of individuals within groups, which was likely statistical noise , and the rest were between −0.028 and 0.196. All estimates of pairwise genetic differentiation among forms with n > 10  with the RAD dataset were significant (Table FST = 0.049–0.056). This pattern remained the same with a standardized measure of genetic differentiation in the RAD and microsatellite datasets (Table G’ST generated using microsatellites (0.110–0.122) than SNPs (0.060–0.75). See Tables Observed and expected heterozygosity ranged from 0.186–0.246 and 0.242–0.273, respectively, in the RAD dataset and 0.534–0.629 and 0.607–0.659, respectively, in the microsatellite dataset . In the PCA generated from the same data, these individuals were oriented between the main clusters. In the microsatellite dataset with the same hoyi and kiyi samples, neither of the two potentially misclassified hoyi assigned to the HOY group, with one being assigned to ART and one to KIY. Assignment scores are reported in Tables Population self‐assignment rate using the microsatellite dataset ranged from 61.9% to 76.7% with Ne with the microsatellite dataset ranged from 73 in HOY to 659 in KIY , whereas the estimates for HOY and KIY produced confidence intervals with an “infinite” upper bound. An “infinite” upper bound is typically an indication that the data are not powerful enough to produce an accurate estimate of Ne given the sample size, population size, and/or marker resolution . Simulated hybridization over a 15‐generation period resulted in declines in genetic differentiation among populations for all tested levels of migration Figure . When thQ‐scores between 0.1 and 0.2 for alternative forms observed in our empirical dataset could be the result of statistical noise. Results from the simulations with 2 generations of migration (G2), where only F1 migrants are possible, demonstrated that these Q‐scores in the range of 0.1–0.2 can be generated by statistical noise as they were present in this simulation , followed by the ART‐HOY comparison (101), ART‐KIY comparison (98), and HOY‐KIY comparison (75). Significantly differentiated windows were found on all but five LGs . LG Cart21 displayed the highest number of differentiated windows, prompting us to conduct an in‐depth investigation of this LG to investigate patterns and potential drivers of divergence , ART‐HOY comparison (4), and HOY‐KIY comparison (1). We were able to place 351 loci on Cart21, and 12 of these loci displayed overall FST values > 0.3 , followed by the overall 4C. artedi species complex, estimates of Ne and diversity in Apostle Islands populations do not suggest the three major extant forms are experiencing bottleneck effects. Genetic differentiation among forms was notably high despite the presence of hybrids, and simulations suggest either that hybridization at the rates we observed between forms is a relatively recent phenomenon or that the fitness of hybrids is reduced. This second scenario is further supported by the discovery of widespread differentiation between forms across the genome, indicating that much of the divergence observed may be driven by long‐term reproductive isolation and drift.The detection of genetic structure in recently diverged species complexes has proved challenging with traditional genetic methods, prompting the reevaluation with genomics of a taxonomically uncertain species complex in the Laurentian Great Lakes. Using a genome‐wide panel of SNPs to examine differentiation and diversity of sympatric coregonines in the Apostle Islands of Lake Superior, we were able to unambiguously assign individuals to the three major forms as well as identify putative hybrids and misidentified individuals. Despite a century of anthropogenic impacts in the Great Lakes that has seen the extirpation and extinction of historically documented forms in the 4.1artedi, hoyi, kiyi) was relatively high compared to previous research in cisco using allozymes, mtDNA, microsatellites, AFLPs, and RAD data, which has largely suggested that forms are not diverged within lakes  using RAD data, which were much lower than the FST values of ~ 0.05 observed in our study. Unfortunately, genetic data for cisco in the Great Lakes are relatively sparse; however, a previous study using mtDNA and microsatellites did not find strong evidence of differentiation among forms  and brook trout  display significant life history polymorphisms in the Great Lakes, with lake trout exhibiting morphologically distinct ecotypes related to depth and brook trout exhibiting both fluvial and adfluvial life histories. Two recent studies used RAD sequencing to investigate life history polymorphism in these species, and neither was able to document strong signals of divergence among forms  from two lakes in Switzerland and found high divergence on all chromosomes, and Gagnaire, Pavey, et al.  and that units of conservation should be designed to preserve environments that have facilitated the evolution of different forms  rather than on forms at a larger scale (Turgeon & Bernatchez, We documented high genetic differentiation among the three major cisco forms in Lake Superior and, based on this information, we suggest that separate conservation units could be constructed for each form. This strategy differs from the current conservation recommendation that the entire nigripinnis and zenithicus to the three major forms was limited by low sample size; therefore, genotyping additional contemporary and/or historic specimens may help resolve the placement these forms.Our results suggest that “last generation” markers may be insufficient for capturing differentiation in evolutionarily young species, such as cisco, and highlight the utility of genomic data for designating conservation units in these species. However, it is important to note that we only surveyed animals from one lake, and a larger genotyping effort across the Great Lakes is necessary to accurately inform conservation units for Great Lakes ciscoes. Additionally, our ability to draw conclusions regarding the relationships of the rare forms The potential of genomic data to revolutionize construction of conservation units has been frequently discussed (reviewed in Allendorf et al., 4.4C. artedi species complex within the Great Lakes. The ability to differentiate forms with genomics provides researchers with a powerful tool for ground truthing morphological phenotypes and identifying cisco species at any life stage. In particular, the ability to identify larval ciscoes will allow researchers to estimate recruitment, which is vital for management and conservation, and will also significantly improve our understanding of early life history characteristics and reproductive dynamics in this species. Finally, our results suggest that some management units created using last generation markers may not adequately reflect species diversity and could be re‐evaluated with genomic data.Our study provides some of the first evidence that cisco forms within the Great Lakes are genetically differentiated. We documented high genetic differentiation among the three major forms in Lake Superior, and highly differentiated markers were distributed across the genome, with islands of divergence found on nearly every linkage group. Additionally, we identified putative hybrids but hypothesize that fitness breakdown of backcrosses may aid in maintaining differentiation among these forms. The results of this study provide the foundation for a new understanding of the ecology and evolution of the None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.