Single-molecule microscopy allows for the investigation of the dynamics of individual molecules and the visualization of subcellular structures at high spatial resolution. For single-molecule imaging experiments, and particularly those that entail the acquisition of multicolor data, calibration of the microscope and its optical components therefore needs to be carried out at a high level of accuracy. We propose here a method for calibrating a microscope at the nanometer scale, in the sense of determining optical aberrations as revealed by point source localization errors on the order of nanometers. The method is based on the imaging of a standard sample to detect and evaluate the amount of geometric aberration introduced in the optical light path. To provide support for multicolor imaging, it also includes procedures for evaluating the geometric aberration caused by a dichroic filter and the axial chromatic aberration introduced by an objective lens. Two of Localization of single molecules with statistical uncertainties of tens of nanometers is routinely achievable in single-molecule microscopy ,12, and The complexity of advanced microscopy setups, optical imperfections, and possible misalignments of mirrors or lenses can all contribute to geometric aberrations in the image produced by a microscope. Errors due to such non-ideal conditions can lead to erroneous answers to biological questions, even when they are on the nanometer scale. For example, when imaging the dynamics of a single molecule that interacts with an endosome, the position of a single molecule may be determined to be inside rather than outside of the endosome, or vice versa, when the localization error is on the order of only nanometers. Errors of this nature can obviously lead to vastly different interpretations of the biological phenomenon. Therefore, to ensure that data of reliably good quality is produced, the performance of the imaging system must be routinely assessed prior to performing single-molecule imaging experiments. This assessment requires defined samples that can be used as standards and calibration methods that are suited for single-molecule localization microscopy. The calibration methods should be system-independent and yield reproducible results, such that the same standards can be used to calibrate different microscopes.Several calibration standards have been introduced over the years to characterize the performance of an imaging system, including ones that utilize fluorescent beads ,16 and DTo overcome the various limitations of approaches that use beads or DNA origami, in recent years techniques have been developed to create slides imprinted with defined patterns for system calibration. For example, Argolight uses a laser to induce into glass substrates fluorescent materials that are stable and have a broadband emission spectrum. An example of an Argolight slide contains different fluorescent patterns in two and three dimensions, the elementary structure of which is an empty cylinder with a diameter of about 0.7 µm. A second technique is the lithography system used by Miraloma Tech to produce calibration slides containing defined patterns. A particularly useful pattern consists of an array of regularly spaced sub-wavelength-sized apertures, allowing for uniform sampling of the imaging field. These apertures are empty and imaged using trans-illumination. Hence the imaging of these slides is not affected by fluorophore photobleaching and can produce extremely high signal-to-noise ratio images even with short exposure times.Recent studies have used arrays of regularly spaced points  or other patterns or features to evaluate the performance of fluorescence microscopes in terms of lateral resolution, field distortion, chromatic aberration, etc. In , arrays We will similarly make use of an array of sub-wavelength-sized apertures in this study. The basis of our microscope calibration method is the evaluation of geometric aberrations by way of determining the difference between the true and the measured positions of the apertures. An important issue we take into account is that there can be manufacturing errors associated with the positions and sizes of the apertures. Our method determines the true aperture positions as a set of reference positions, as relying on the nominal aperture positions would make it difficult to determine whether the geometric aberrations observed in the acquired images are due to defects in the optical system or manufacturing errors in the calibration sample.Recently, simultaneous multicolor single-molecule tracking has been widely used for the observation of the fast dynamics of individual molecules in living cells ,22,23. FTherefore, the calibration method that we present here is optimized for multicolor single-molecule microscopy. This method couples the use a lithographically fabricated aperture array with novel analysis algorithms to evaluate the performance of the microscope and its optical components with high accuracy.The proposed method comprises two major components. The first component concerns the assessment of the performance of a microscope through determining the level of geometric aberration in its optical path. As the extent of the aberration is reflected in the deviation of the imaged positions of microscopic apertures in the calibration sample from their true positions, the key here, as mentioned above, is to establish a set of accurately determined reference aperture positions that serve as a benchmark for comparison in subsequent calibrations of a microscope using the same sample. The approach therefore entails procedures for the accurate estimation of imaged aperture positions and the determination of the similarity between a given set of aperture position estimates and the reference aperture positions.The second component of the proposed calibration method comprises approaches for evaluating two critical microscope components, namely the objective lens and the dichroic filter. The quality of the objective lens is evaluated based on how well it corrects for chromatic aberration along the optical axis. This assessment is important because there is no guarantee that even apochromat objectives will be able to fully correct for the aberration over a wide spectral range. The performance of the dichroic filter is evaluated by investigating the deviation between corresponding aperture positions estimated from images simultaneously acquired of the filter’s transmitted and reflected light paths. This type of evaluation is of particular importance because it is often the case that suboptimal properties of a dichroic filter lead to distortions in the light that it reflects.The proposed calibration method is supported by a core set of analysis tools for carrying out a number of important tasks. These tasks comprise the determination of the in-focus position of a 3D data set, the registration of images in two dimensions, the estimation of the position of an aperture, the assessment of the horizontality of the microscope stage and calibration sample, and the quantitative comparison of two data sets.To demonstrate the evaluation of the performance of a microscope, we apply our calibration method to both a microscope of poor quality (one with a damaged objective thread adapter) and a microscope of good quality. Our results show that our method is capable of distinguishing between the two microscopes in terms of the level of geometric aberration.In addition, we apply our method to an apochromat objective lens to evaluate its axial chromatic aberration over a wide spectral range. We also demonstrate the ability of our method to distinguish between dichroic filters that differ in terms of their flatness.The proposed method enables meaningful and repeatable calibration data acquisitions that are reproducible across different microscopes. Also, our approach provides useful insights in the selection of an appropriate microscope and suitable optical elements for a given application. It allows researchers, ranging from novice microscope users to expert microscopists, to evaluate their microscopes and optical elements with high accuracy.2.We describe in this section the type of sample, instrumentation, and data that are required by our calibration method, as well as the specific implementations that are used in Section 2.1A NanoGrid slide  is used as a calibration standard for the microscope performance evaluation. The slide consists of a 20 by 20 array of sub-wavelength-sized apertures with a regular spacing of 4 µm between adjacent apertures. The apertures, which we will also refer to as holes, are approximately 200 nm in diameter. The numbers for the spacing and diameter are taken from specifications provided by the manufacturer. Of the 20 by 20 array of holes, only the center 10 by 10 holes are used for the proposed analyses  consists of a standard inverted microscope  and an EMCCD camera  operated in CCD readout mode. The camera exposure time is set to 0.1 s. The plan-apochromatic objective lens  has a 63× magnification and a numerical aperture of 1.4 and is used with an immersion medium with an index of refraction of 1.51. A piezo objective positioner  is used to translate the objective lens in the z direction. This device has a positioning repeatability  of ±5 nm. A microscope objective thread adapter  is installed between the objective lens and the piezo objective positioner. This adapter allows microscope objectives with RMS  objective mounts to be used on piezo objective positioners with M27 (metric 27-millimeter) threads. To provide an example of a microscope with geometric aberrations, the adapter that is installed is replaced with one whose thread is damaged. The damaged thread causes the objective lens to deviate from its alignment with the optical axis, resulting in aberrations in the image formed.The second microscope configuration (“microscope 2”) consists of a standard inverted microscope  and a CCD camera . The camera exposure time is set to 0.2 s. The plan-apochromatic objective lens  has the same magnification and numerical aperture as the objective lens for the first setup and is used with the same immersion medium. A motorized focus drive included in the microscope stand is used to translate the objective lens along the optical axis. For chromatic aberration analysis, microscope 2 is equipped with filter sets for FITC , Cy3 , and Cy5 .The third microscope configuration (“microscope 3”) is assembled by adding an emission image splitter  to microscope 1. Two identical EMCCD cameras , both operated in CCD readout mode, are connected to the output ports of the image splitter unit. The exposure time is set to 0.1 s for each camera. For the evaluation of dichroic filters, either a standard flatness dichroic filter  or an improved flatness dichroic filter  is mounted in a filter cube and placed in the image splitter unit.Briefly, after adding the image splitter to the microscope and attaching the two cameras, the following procedure is performed to align the in-focus positions of the two cameras. A filter cube with a 50:50 beam splitter is inserted into the image splitter, and a NanoGrid slide is positioned so that its image is placed in the center of the field of view of the camera in the transmitted light path. If necessary, the xy position of the camera in the reflected light path is then adjusted (using position adjustment knobs that are part of the image splitter) so that the image of the slide is also centered in its field of view. To check whether the two cameras share a focal plane, z-stacks of the NanoGrid sample are simultaneously acquired using the cameras, and the procedure of Section The image splitter used in microscope 3 is a discontinued product. A similar product that could be used is the Double Adapter Duolink  .In all three microscope setups, the NanoGrid calibration slide is trans-illuminated with a light-emitting diode (LED) . Also, the mechanical stage  is equipped with a mounting frame  that can hold a petri dish (max. diameter 68 mm) or a sample slide (max. length 120 mm). The mounting frame can be detached from the stage and rotated 180 degrees.2.3In this paper, three types of calibration data sets are utilized. The first type  consists of a z-stack of the NanoGrid slide, the second type  comprises multicolor z-stacks of the slide, and the third type  consists of 200 in-focus images of the slide.Calibration data set 1 is acquired as follows. The aperture array on the calibration slide is placed at the center of the imaging field. A z-stack is then acquired by moving the objective lens along the optical axis to obtain a series of images centered about the visually determined in-focus position. More specifically, the z-stack is obtained by moving the objective lens with a piezo nanopositioner or a motorized focus drive in 50-nm steps and acquiring an image at each position of the objective lens. Calibration data set 2 is acquired using the same procedure, except an image of each of the color channels is taken per z-step using a motorized filter cube turret.Calibration data set 3 is acquired as follows. A z-stack is first acquired using the procedure for the acquisition of calibration data set 1. The in-focus frame number of the z-stack is determined as described in Section 2.4N are the height and width of the image in pixels, and The Brenner gradient method ,26 is us2.5A can be written as R is rigid  as kth pair-matched coordinates from the second data set (target data set) as kth pair of hole positions is then defined as The following analysis is carried out to measure the difference, in terms of the estimates of hole positions, between two calibration data sets acquired of the same NanoGrid sample under different conditions, including the use of different microscopes, different sample orientations, different illumination angles, and different detectors. In this analysis, calibration data set 3 is acquired using each setup and analyzed for comparison. The lateral position of each hole is estimated from each data set as described in Section K pairs of hole positions are represented with arrows as illustrated in the top panel of For visualization of the difference pattern, the differences between all Q. The quality score represents the overall difference between the two data sets in terms of the deviations between the positional estimates obtained for the NanoGrid holes. If there are significant deviations between corresponding hole positions from the two data sets, the quality score will be high. Conversely, if there are only small deviations between the corresponding hole positions, the quality score will be low.Finally, the root mean squared difference, which we call the “quality score” in this paper, is defined as 2.9kth grid point. The randomly distorted true position kth grid point is then modeled as The following simulation is carried out to generate simulated calibration data sets. We first generate 100 points that form a 10 by 10 grid with a uniform spacing of 4 µm between adjacent points . Let sk∈jth repeat measurement Subsequently, 200 repeat measurements of each distorted grid point position are generated from a normal distribution. Specifically, for 2.10Q is then determined for the two data sets as described in Section Q determined for data from such repeat experiments is considered the lowest score Experimental errors intrinsic to the performance of repeat data acquisitions, such as errors associated with the focusing of a sample before each acquisition, places a practical limit on the best  quality score that is attainable for a given microscopy setup. To determine this best quality score for a microscope, the following procedure is carried out. The horizontality of the microscope stage and the calibration sample is first determined and adjusted as described in Section  Section .Note that in the calculation of 2.11Q is low enough, the estimated hole positions are considered independent from the illumination angle.Two sets of 200 in-focus images of the NanoGrid slide  are acquired with two different angles of the illuminating light source, namely 0 and 45 degrees. The independence of the estimated hole positions from the illumination angle is then assessed by comparing the two data sets according to the procedure of Section 2.12Q is then determined for these two data sets using the procedure of Section Q is low enough  to verify the absence of any significant field-dependent aberrations. Specifically, the slide should be positioned at locations that well sample the field of view. In each case, the quality score Reference hole positions could alternatively be determined using higher-resolution imaging techniques such as atomic force microscopy and electron microscopy. The approach presented here, however, relies solely on an optical microscope and therefore has the advantage that no additional instrumentation is required.2.132.13.1To analyze axial chromatic aberration for an objective lens, calibration data set 2 is acquired with three different color channels whose wavelengths approximately cover the range 400 nm to 700 nm. For example, the three channels commonly employed in fluorescence imaging experiments, namely the FITC, Cy3, and Cy5 channels used in microscope 2 Section , are sui2.13.2.Q is then computed for the two data sets using the procedure of Section Q indicates little difference between the images produced by the transmitted and reflected light paths, and the dichroic filter/dichroic filter set or beam splitter is considered to be of high quality. Note that it is important for this analysis that in-focus images are simultaneously acquired by the two cameras. Even though the cameras have been aligned so that they share a focal plane  as well as the image analysis software Lumio (Astero Technologies LLC).3.In this section, we demonstrate our calibration method by applying its constituent tools and protocols to specific microscope setups and optical components. Details pertaining to the calibration sample, the instrumentation, the data sets, and the tools and protocols are all as provided in Section 3.1The process of acquiring a z-stack of a NanoGrid sample Section  is essen3.2Determining an in-focus position is important for precisely estimating the lateral positions of the NanoGrid holes. This is because the closer the image is to focus, the higher the localization precision . The det3.3Our calibration method entails analyses that rely on the precise estimation of the positions of NanoGrid holes. In order to estimate the lateral position of the holes with high precision, we need to acquire a significant number of photons. Therefore, using microscope 1, 200 images of the NanoGrid slide  were acquired at the in-focus position determined in Section 3.4As described in Section The 3D position of each hole was then given by its estimated z position and its lateral position. As specified in Section 3.5Wide-field microscopy typically requires the microscope stage to be perpendicular to the optical axis. Therefore, it is necessary to determine the horizontality of the microscope stage and make any needed adjustments. The horizontality of the stage on microscope 1 was measured as described in Section The above results show that while the sign of the tilt angle was reversed after the 180-degree rotation of the mounting frame, the magnitude of the angle had decreased. This indicated that the stage or the sample or both were not horizontal. Therefore, we adjusted the horizontality of the stage and sample as described in Section 3.6Q , two calibration data sets were generated as described in Section Q for all scenarios considered. The x-axis of the plot represents the overall geometric aberration level Q. Different colors are used to denote different values of the localization uncertainty Q increases with the overall geometric aberration level Q increases with the localization uncertainty Q significantly. Also shown in the plot is the line Q is effectively an estimate of Q is an estimate of the quantity X and Y are Gaussian random variables with variance Q is relatively close to Q is very close to the The second simulation study (simulation study 2) was carried out the same way as the first study, but with a different range of localization uncertainties. For the second study, the localization uncertainty Q also increases. However, Q.Similar to The results of our simulation studies indicate that in order to obtain quality scores that accurately reflect the difference in the level of geometric aberration between two data sets, it is important that the NanoGrid hole positions are estimated with high precision  in both data sets.3.7To demonstrate the calculation of the baseline quality score 3.8As the NanoGrid slide was illuminated with a LED light source, we wanted to make sure that specifics of the illumination did not impact the results. We therefore imaged the same slide using two different illumination angles of the light source and compared the hole positions estimated from the two data sets.The procedure followed is as described in Section Q for the two data sets is 0.96 nm, which is lower than The quality score 3.9We propose the use of a NanoGrid slide as a calibration standard to evaluate the performance of a microscope system or optical components such as the objective lens and the dichroic filter. The approach is to determine whether the microscope or optical component in question causes geometric aberrations in the light path that are reflected in deviations of hole position estimates from their true positions. As we can expect manufacturing errors in the positions of the NanoGrid holes, we cannot take the holes’ nominal positions to be their true positions. Instead, it is necessary for us to determine accurate locations of the holes ourselves, and use them as a reference for comparison. Specifically, we need to be able to very precisely estimate the positions of the holes using a “high-quality” microscope. A high-quality microscope is one that does not introduce significant distortions in the measured hole locations. Therefore, the hole position estimates obtained using such a microscope can be used as reference hole positions for subsequent calibration analyses.The approach used to establish whether a microscope is suitable for the determination of reference hole positions is as follows. Suppose the calibration sample is imaged before and after a rotation or translation. Any specific pattern associated with the sample should move with the sample, but a geometric aberration pattern should not change with the position or orientation of the sample. This is because geometric aberrations are caused by defects or misalignments in the optics, and not due to variations in the sample. Provided that we use a high-quality microscope, the imaged pattern after the rotation or translation should coincide with the pattern before the rotation or translation. On the other hand, when we use a microscope that distorts the measured locations of the holes, the imaged patterns before and after the rotation or translation will not match well.Using this concept, the reference positions of the NanoGrid holes were determined as described in Section Q for the two data sets is 1.42 nm, which is very close to the baseline score The left panel of 3.10Many microscope imaging systems make use of an objective thread adapter that allows an objective lens to be used on a microscope with a mounting thread that is not of the same pitch or diameter as the objective lens. Such adapters are also often used to increase the axial position of the objective lens. If an inappropriate adapter, such as one that is damaged or one whose thread is incompatible with the thread of the objective lens, is used in a setup, geometric aberrations could be introduced.To illustrate the analysis of a microscope configuration that exhibits geometric aberration , we installed a damaged thread adapter for use with the objective lens on microscope 1. The evaluation of the microscope system was then carried out as follows, using the reference NanoGrid hole positions determined in Section Q for the two sets of lateral positions is a large 8.67 nm, indicating that the microscope configuration indeed produces significant geometric aberrations.An arrow plot showing the differences between the estimated hole positions and their corresponding reference positions is provided in the left panel of Q for the two sets of lateral positions is a significantly smaller 1.76 nm. The small quality score confirms that this microscope configuration introduces only minor geometric aberrations.We carried out the same analysis to evaluate a different microscope configuration  The results are shown in 4.The advent of single-molecule microscopy has enabled researchers to estimate the position of single molecules at the nanoscale, allowing the tracking of the dynamics of single molecules and the visualization of fine details of subcellular structures. At such high resolutions, calibration of the microscope system plays an important role in guaranteeing the appropriate level of accuracy for the results obtained. Calibration should be one of the essential steps in the design and implementation of single-molecule imaging experiments, and it needs to be carried out with great care. We therefore developed a calibration method that is optimized for single-molecule microscopy, and in particular for multicolor applications.Our calibration method is based on detecting geometric distortions in a microscope’s light path by imaging a NanoGrid slide, which comprises an array of regularly spaced sub-wavelength-sized holes. The quality of a microscope is assessed by estimating the hole locations and evaluating the deviation of the locations from reference hole positions that have been obtained using a high-quality microscope. We showed here that when enough light is collected, hole positions can be determined with a sub-nanometer standard deviation, thereby allowing calibration at the nanometer scale. By applying the method to microscopes of good and poor quality, we demonstrated that our approach can indeed be used to evaluate microscope performance.The proposed calibration method also includes approaches for evaluating the quality of optical components. The dichroic filter is a critical component in multicolor single-molecule microscopy, and any distortions it introduces can be highly problematic for multicolor single-molecule experiments. The use of a dichroic filter of insufficient quality can lead to significant distortions in the reflected light path. In our approach, the quality of a dichroic filter is evaluated by comparing the positions of NanoGrid holes estimated from data acquired of the filter’s transmitted and reflected light paths. We applied our method to dichroic filters of differing flatness, and showed that it can in fact discern their performance.In addition, we presented an approach for assessing the axial chromatic aberration introduced by an objective lens. Axial chromatic aberration refers to focus shift as a function of the wavelength of the collected light. In multicolor imaging experiments, even slight focus shifts between colors can result in misinterpretation of the acquired data. This is especially the case in the single-molecule context. Since most objective lenses are designed to correct for chromatic aberration , the importance of this evaluation is often overlooked. Even apochromatic objective lenses do not necessarily provide the level of correction needed over a wide spectral range, and our demonstration shows that such lenses can in fact introduce significant chromatic aberration.The proposed calibration method is relatively easy to implement, and therefore suitable for routine assessments of single-molecule imaging setups in a laboratory. By imaging and localizing the sub-wavelength-sized holes of a NanoGrid slide or similar calibration standard, the user can easily follow the protocol to obtain a quality score that quantifies the level of geometric aberration that is produced by the given imaging configuration. At the core of the method is a general procedure that can be applied not only to the microscope as a whole, but also to a specific optical element as we have demonstrated with dichroic beam splitters.


Genetic diversity and population structure assessment in crops is essential for marker trait association, marker assisted breeding and crop germplasm conservation. We analyzed a set of 285 durum wheat accessions comprising 215 Ethiopian durum wheat landraces, 10 released durum wheat varieties, 10 advanced durum wheat lines from Ethiopia, and 50 durum wheat lines from CIMMYT. We investigated the genetic diversity and population structure for the complete panel as well as for the 215 landraces, separately based on 11,919 SNP markers with known physical positions. The whole panel was clustered into two populations representing on the one hand mainly the landraces, and on the other hand mainly released, advanced and CIMMYT lines. Further population structure analysis of the landraces uncovered 4 subgroups emphasizing the high degree of genetic diversity within Ethiopian durum landraces. Population structure based AMOVA for both sets unveiled significant (P < 0.001) variation between populations and within populations. Total variation within population accessions  was higher than total variation between populations  for both sets. Population structure analysis based genetic differentiation (FST) and gene flow (Nm) for the whole set and the Ethiopian landraces were 0.19 and 0.24, 1.04, and 0.81, respectively indicating high genetic differentiation and limited gene flow. Diversity indices verify that the landrace panel was more diverse with  than the advanced lines . Similarly, differences within the landrace clusters were observed. In summary a high genetic diversity within Ethiopian durum wheat landraces was detected, which may be a target for national and international wheat improvement programs to exploit valuable traits for biotic and abiotic stresses. Triticum turgidum ssp. durum (Desf.) Husn.] was domesticated from wild emmer (Triticum turgidum ssp. dicoccoides) to emmer (Triticum turgidum ssp. dicoccum) followed by a secondary domestication, i.e. from emmer to naked forms and durum wheat (Triticum turgidum ssp. durum) [T. urartu (genome AA) [Aegilops speltoides (genome BB) [n = 4x = 28, seven homoeologous groups—13,000 M bp) [Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor . Recently, the reference sequence of the genome of cv. Svevo led to the identification of 66,559 high confidence (HC) genes enabling genome-wide genetic diversity analyses in tetraploid durum wheat [Durum wheat [. durum) . The allnome AA) , 3 and anome BB) , 5. Thus00 M bp) . A high-00 M bp)  and comp00 M bp)  developeum wheat .Durum wheat is one of the ten most important crops worldwide with an annual production of 37 million tons  and EthiThe Ethiopian Biodivesity Institute (EBI) hosts more than 7000 landraces collected from durum wheat growers for genetic conservation and for the exploitation of genetic diversity , 21. BasAnalysis of genetic diversity in populations is an important topic in breeding as well as conservation and evolutionary genetics studies , 33. ExpUp to now only a small part of the huge collection of durum wheat landraces hosted at EBI was characterized using SSR –48 and Shttp://www.ebi.gov.et/). Landraces were selected based on the acreage in each seed source region (origin). Thus, more samples were taken from major growing regions (Oromia and Amhara) and some samples from minor growing regions. 105 ETDWL were sampled from Oromia, 88 from Amhara, 1 from Benishangul Gumuz, 16 from Tigray and 5 from South Nation Nationalities and Peoples (SNNP), representing different seed sources (origin), seed collection zones and geographic regions  was used for the analysis of genetic diversity. The SP included, 215 Ethiopian durum wheat landraces assigned as ETDWL, 10 released durum wheat varieties, 10 advanced durum wheat lines from Ethiopia and 50 durum wheat lines from CIMMYT . The ETD regions . Online uly 2020 . For the® ND-1000 Spectrophotometer . 50 ng of DNA per sample was used for SNP analysis using the 90K iSelect chip . Genotyping was conducted by Trait Genetics, Gatersleben (Germany). SNPs with a low minor allele frequency (MAF) are generally considered as rare with less power in detecting marker trait associations (MTAs) and are prone to genotyping error [The durum wheat SP was grown in the greenhouse at Quedlinburg, Germany for 15 days under standard growing conditions, i.e. 20 to 22°C during day time and 17 to 19°C at night  with autng error . Thus, Sng error . Physicang error  to constGenotypic data were used to describe the genetic diversity within the durum wheat study panel. We analyzed the population structure and genetic diversity of the ETDWL separately, and compared this with the population structure and genetic diversity of the SP. The underlying genetic population structure was estimated with STRUCTURE 2.3.4 software . SNP marGenetic distance between populations was determined using Nei’s Genetic Distance  based onPi^2 is the sum of squared population allele frequencies and, PPL = ∑Pi/N, where Pi is the proportion of loci polymorphic in a population and N the number of populations.In addition, Shannon’s Information Index (I) , expecteAfter filtering, 11,919 SNPs were used for genetic analysis. These were continuously distributed across the A and B genome of durum wheat for the SP . In all Population structure analysis for the durum wheat SP revealed ΔK at K = 2 i.e two populations hereafter considered as Pop1 and Pop2 . Pop1 coEthiopian durum wheat landraces (ETDWL) comprise accessions collected from major wheat producing regions of the country  . PopulatAnalysis of molecular variance (AMOVA) for the durum wheat SP and ETDWL was conducted taking the respective population structure clusters (ΔK) into account . In bothWe investigated the genetic diversity of the SP and ETDWL based on population structure analysis results of ΔK at k = 2 and k = 4, respectively . The genHybridization arrays are believed to represent a significant fraction of SNPs distributed across genomes. In wheat they represent SNPs between populations of diverse geographical origin , 44, 45.In this study, population structure and Neighbor-Joining (NJ) analysis showed two populations (Pop1 and Pop2) for the study panel (SP). Concerning Pop1, 206 (99.5%) accessions were from ETDWL and only 1, DZ005 (0.5%) from the advance durum wheat lines . This duT. durum subs. abyssinicum or T. aethiopicum [It has been reported that, Ethiopian durum wheat landraces are distinct and have no kinship with the Middle-East, which is the primary region of origin of durum wheat . Therefohiopicum , 23. Addhiopicum .Population structure analysis of ETDWL alone uncovers four populations , which is in agreement with and NJ analysis . MengistDurum wheat is one of the important cereal crops grown in Ethiopia and the country is endowed with a wealth of genetic diversity for tetraploid wheat. Phenotypic and morphological analysis , 67–69 aFixation index (differentiation = FST) measures population differentiation due to genetic structure  and FST We employed high quality SNP markers to analyze the population structure and genetic diversity of a durum wheat study panel comprising 285 accessions of which 215 accessions were ETDWL. AMOVA (P < 0.001) unveiled that genetic variation within population accessions was higher than genetic variation between populations for the SP and ETDWL. Structure analysis of SP revealed two distinct populations (Pop1 and Pop2). Genetic diversity indices for the SP illustrated higher genetic diversity in Pop1, which constituted 99.5% of the ETDWL in comparison to Pop2, which comprised only 11.5% ETDWL. Further population structure analysis of the ETDWL alone uncovered four populations emphasizing the high degree of genetic diversity that exists in ETDWL. Genetic diversity indices for the ETDWL indicated Pop1-3 was the most diverse followed by Pop1-2. Therefore, the high genetic diversity detected in ETDWL showed the existence of plentiful variability that could be utilized for future wheat breeding programs.S1 Tablen = 285 with Delta K at k = 2 and ETDWL, n = 215 with Delta K at k = 4.(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.


Piaractus mesopotamicus from the breeding program located in Brazil by analyses of the mitochondrial DNA control region (mtDNA). Moreover, broodstocks from eight commercial fish farms were used for comparative evaluation, four from Brazil (Br1-Br4) and four from Argentina (Ar1-Ar4). The descriptive results revealed 47 polymorphic sites and 51 mutations, which evidenced 34 haplotypes. Ten haplotypes were shared among fish farms and 24 were exclusive. The nucleotide diversity (π) ranged from 0.00031 to 0.01462 and haplotype diversity (Hd) from 0.125 to 0.868. The analysis of molecular variance (AMOVA) indicated high structure present in the analyzed stocks (FST = 0.13356 and ФST = 0.52707). The genetic diversity was high in most of the commercial broodstocks, especially those from Brazil. We observed seven haplotypes in the genetic breeding population, of which four were exclusive and three shared among the commercial fish farms. The genetic diversity was moderate (π = 0.00265 and Hd = 0.424) and considered appropriated for this breeding population of pacu. Our results provide support for the genetic diversity maintenance and mtDNA traceability of pacu commercial broodstocks.The main objective of this study was to estimate the genetic diversity levels and haplotype traceability in pacu  Piaractus mesopotamicus, popularly known as pacu, is a Serrasalmidae fish of commercial importance distributed in the Paraná, Paraguay, and Uruguay River basins, mainly in the Pantanal plains (P. mesopotamicus and Piaractus brachypomus), with an estimated annual production of 13,276 tons , the different degrees of inbreeding depression significantly affected the percentage of hatching, fecundity and larval survival, as well as generating individuals with morphological deformities  and microsatellites in different fish farms from Brazil for a pre-breeding program in The main objectives of this study were: 1) to assess the genetic diversity by mtDNA in the breeding population of pacu from Caunesp ; 2) to identify unique haplotypes in this breeding population for traceability of products resultant from genetic improvement by mtDNA; 3) to compare farmed stocks from different Brazilian and Argentinian commercial fish farms by mtDNA in order to detect non-contemporary genetic differences of founder stocks and to understand the haplotype diversity and distribution between these countries.Piaractus mesopotamicus from the breeding nucleus belonging to the Caunesp  were used for the genetic analysis. These breeders are resulting from the base population established with purposes of genetic selection for growth performance and disease resistance in pacu   anesthesia and all efforts were made to minimize suffering. Fin samples were stored in 95% ethanol at −20 °C.Fin samples were collected from each fish under benzocaine solution (200 mgDNA extraction was carried out using the Wizard Genomic DNA Purification Kit (Promega). DNA integrity was evaluated on 1% agarose gel and its purity was assessed using a NanoDrop One spectrophotometer . The DNA concentration was quantified using the Qubit dsDNA BR Assay kit  and measured in a Qubit 3.0 Fluorometer .P. mesopotamicus (NC_024940) as reference. PCR assays were performed at a final volume of 12 μl: 1X Taq polymerase buffer; 1.5 mM MgCl 2; 100 μM of each dNTP; 0.1 μM of each primer; 10-50 ng of genomic DNA and 0.5 U of Taq polymerase (Invitrogen). The following amplification cycle was used: denaturation 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 1 min; annealing at 62 °C for 45, extension to 72 °C for 1 min, and a final extension at 72 °C for 10 min. PCR assays were performed on the ProFlex ™ PCR System (Life technologies) and the final product checked in 1.5% agarose gel.Genetic diversity was assessed through the partial sequencing of the mitochondrial DNA control region (D-loop). The primers PM01 (5’GATCCCAGTACATTATATGTAT3’) and PM02 (5’CCTTGTTAATCATTACRCTGA3’) were designed using the software Geneious V.7.1.3, using the mitochondrial genome of PCR products were cleaned with EXO-SAP Kit (USB® ExoSAP-IT® PCR Product Cleanup) and then underwent PCR sequencing, using BigDYE, Terminator Cycle Sequencing Kit version 3, 1 . The sequencing was performed in the Center of Biological Resources and Genomic Biology (CREBIO), in UNESP - Jaboticabal, Brazil, using the ABI 3730 XL DNA Analyzer (Applied Biosystems).The assembled sequences were analyzed manually using the program CLUSTALW . The nucThe results revealed 47 polymorphic sites from approximately 400 sequenced base pairs (bp), characterizing 34 haplotypes (GenBank accession numbers MW287387 - MW287554). Among the haplotypes, 10 were shared among broodstocks, of which two (H1 and H2) were shared between samples from Brazilian and Argentinian fish farms. In addition, 24 unique haplotypes were detected, of which 11 were in Argentina, 9 were in Brazil, and 4 were exclusive from the base population of the breeding nucleus . The mosThe nucleotide (π) and haplotypic (Hd) diversity demonstrated moderate values in the base population of the breeding nucleus (π = 0.00265 and Hd = 0.424)  and indiFST index indicated a high genetic structure among the populations (FST = 0.13356), similarly to the ФST index . The pairwise FST matrix also revealed high genetic structure between the broodstocks  , the popodstocks , particuThe haplotype network  revealedInbreeding depression is one of the factors that most affects individuals due to the classical (phenotype-based) selection method in the breeding nucleus. The main genetic problems arising from the inappropriate use of fish in breeding programs can be reduced and even avoided using the genetic profile of the individuals and proper genetic management practices, similarly as it was performed to compose the base population of the breeding nucleus (CAUNESP) in pacu , which would reduce the problems related from narrowing the genetic base or loss of genetic potential over the various generations of selection.


Phasianus colchicus; pheasant), northern bobwhite quail , and threatened-endangered (T&E) greater prairie chicken . We first genotyped our study pheasant at 19 microsatellite DNA loci and identified three captive breeding stocks  as being significantly bottlenecked, with relatedness >1st-cousin (μR = 0.158). ‘Wild’ (non-stocked) pheasant  were also bottlenecked, significantly interrelated (μR = 0.150) and differentiated (μFST = 0.047), yet distinct from propagation stock. PHAs that encompassed significantly with larger areas also reflected greater effective population sizes . We juxtaposed these data against previously published results for prairie chicken and quail, and found population genetic structure driven by drift, habitat/climate impacts, and gender-biased selection via hunter-harvest. Each species (hunter-harvested or T&E) is independently managed, yet their composite population genetic baseline provides the quantitative criteria needed for an upland game bird MTM. Its implementation would require agricultural plots to be rehabilitated/reclaimed using a land-sharing/sparing portfolio that differs markedly from the Conservation Reserve Program (CRP), where sequestered land decreases as agricultural prices escalate. Cost-savings for an MTM would accrue by synchronizing single-species management with a dwindling hunter-harvest program, and by eliminating propagation/stocking programs. This would sustain not only native grasslands and their resident species, but also accelerate conservation at the wildlife-agroecosystem interface.Despite its imperative, biodiversity conservation is chronically underfunded, a deficiency that often forces management agencies to prioritize. Single-species recovery thus becomes a focus , whereas a more economical approach would be the transition to multi-targeted management (= MTM). This challenge is best represented in Midwestern North America where biodiversity has been impacted by 300+ years of chronic anthropogenic disturbance such that native tall-grass prairie is now supplanted by an agroecosystem. Here, we develop an MTM with a population genetic metric to collaboratively manage three Illinois upland gamebirds: common pheasant ( Anthropogenic impacts are a serious challenge for biological diversity , with maInterestingly, the interactions among anthropogenic drivers has been relatively unexplored , due larThe gradual but ongoing progression of chronic anthropogenic disturbance negatively impacts adaptive management, and in a variety of ways. For example, it seriously strains budgets , and itsIn contrast, an MTM approach  offers several positive considerations. It can reduce management conflicts when several at-risk species are incorporated, and adequately address common threats across each while simultaneously eliminating the potential for species-specific redundancy . ConservA history of chronic anthropogenic disturbance is clearly reflected in midwestern North America, with its 300+ year record of forests felled, prairies plowed, and streams sequestered for agricultural and urban purposes. The region supports an energy-intensive economy where greenhouse gas emissions exceed the national average by >20% , and botSuch enhancements, while economically positive, have also compressed regional biodiversity into novel prairie-like parcels distributed randomly across an expansive agricultural matrix. Its persistence is inexorably challenged not only by a suite of ongoing anthropogenic pressures (as above), but also by a concomitant erosion of ecosystem services . Yet thUpland birds are an historic component of North American biodiversity, well documented in both Pleistocene fossil records and the earliest regional ornithological collections . Their lChronic anthropogenic disturbance had an early initiation in North America, and subsequently became quite challenging for upland game birds, many of which are (or were) hunter-harvested . We now Ne), which reflects the loss of heterozygosity due to drift and links strongly with demographic factors such as sex ratio, population size, and lifetime fitness. Consequently, those population effects manifested through demography and environment can best be gauged by evaluating Ne.The spatial constraints of habitat fragmentation generally require management at the population genetic level, in that genetic drift  is a frNe. Importantly, these effects can be not only documented with population genetic tools, but also remediated as well   . We thenchicken)  and hunt; quail)  Fig 1)..PhasianuIn this sense, our comparative approach extends from introduced to native species, and from hunter-harvested to T&E components. This allows us to explore the capacity of population genetics as a baseline for an upland game bird MTM. Our contemporary and economical approaches were designed to resonate with stakeholders and interest groups . By doinhttps://www.orvis.com/s/upland-game-birds-of-north-america/14692) to serve as an additional upland game bird suitable for anthropogenic hunter-harvest. However, repeated serial releases by state and federal agencies were required over many years before it became a self-sustaining component of the Great Plains and an icon of hunter-harvest  and pairwise geographic distance (log10+1 transformed).An hierarchical approach was employed to: (1) evaluate genetic diversity and divergence among wild pheasant (PHAs) and propagation stocks , and (2) assess population genetic structure among PHAs. Pairwise uin v3.5 ). To gauStructure v2.3.4  would also be reversed as well.We recognize that edge habitats will not sustain large, genetically diverse populations of upland game birds, but they will provide the corridors necessary for dispersal among currently isolated fragments. Elevated connectivity and its resulting gene flow would not only counteract demographic isolation but at the same time leverage those genetic metrics already depressed, such as migration rates, bottleneck effects, and reduced Ne values for pheasant were significantly associated with larger PHAs . A potential downside would be if agriculture was intensified on neighboring plots. This, in turn, would entail greater use of agrochemical, water, and energy resources . Both require greater yields from areas already under cultivation, a process that may best be implemented at the state-level. In addition, funds now allocated for single-species conservation, and for propagation facilities that sustain a put-and-take hunter-harvest [On the positive side, the partitioning of land to protect biodiversity is recognized as a new global initiative [for example, The Half-Earth Concept  and Natu-harvest , could pS1 Fig(TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableThis includes number of sampled genotypes, alleles detected, multiplex assignment, annealing temperature, and violations of Hardy-Weinberg equilibrium (HWE) for each locus.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Disclosure of pathogenic variants to thoracic aortic dissection biobank participants was implemented. The impact and costs, including confirmatory genetic testing in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory, were evaluated.COL3A1, FBN1, LOX, PRKG1, SMAD3, and TGFBR2) were identified in 26 participants, representing 10.8% of the cohort (26/240). A second research sample was used to validate the initial findings. Mailed letters to participants disclosed that a potentially disease causing DNA alteration had been identified (neither the gene nor variant was disclosed). Participants were offered clinical genetic counseling and confirmatory genetic testing in a CLIA laboratory.We exome sequenced 240 cases with thoracic aortic dissection and 258 controls, then examined 11 aortopathy genes. Pathogenic variants in 6 aortopathy genes  and genetic counseling . The psychosocial impact was characterized by low decisional regret (11.5 ± 11.6) and distress . The average cost for 26 participants was $400, including validation and sending letters. The average cost for those who received genetic counseling and CLIA laboratory confirmation was $605.Participants were satisfied with the return of clinically significant biobank genetic results and CLIA laboratory testing; however, the process required significant time and resources. These findings illustrate the trade-offs involved for researchers considering returning research genetic results.The online version contains supplementary material available at 10.1186/s12920-021-00902-5. Genetic research studies generate individual results that may have clinical implications for participants, but these findings require confirmatory genetic testing in a Clinical Laboratory Improvement Amendments (CLIA) laboratory prior to use in clinical care , 2. The Participants report high levels of interest in receiving research genetic results that are clinically actionable –9, definWe sought to address these questions among thoracic aortic dissection biobank participants given that between 10 and 25% of patients with a thoracic aortic aneurysm or dissection are estimated to have an underlying genetic predisposition  , 13. TheParticipants were recruited to the Cardiovascular Health Improvement Project (CHIP), a longitudinal cardiovascular biobank within the Michigan Medicine Frankel Cardiovascular Center, initiated in 2013; biobank recruitment is ongoing . The priCOL3A1, FBN1, SMAD3, TGFB2, TGFBR1, TGFBR2, ACTA2, MYH11, MYLK, LOX, PRKG1), which were annotated and categorized as pathogenic, of unknown significance, or benign for aortic dissection according to the American College of Medical Genetics (ACMG) criteria [Whole exome sequencing was performed on a subset of biobank participants, including 240 patients with thoracic aortic dissection or rupture and 258 controls matched for age, sex, and ancestry with no cardiovascular conditions from the Michigan Genomics Initiative. Detailed methods on sequencing, variant calling, and quality control has been previously published . In briecriteria , 13, 16.criteria .Additional file We opted to initiate recontact via US certified mail based on survey results from 250 participants enrolled in CHIP biobank wherein 94% indicated their preference to receive clinically actionable aortic results . This prPrior to mailing letters, the cardiothoracic surgeons overseeing the participants’ clinical care were contacted via email and informed that one of their patients (identity was not disclosed) would be receiving a letter indicating that he/she likely carried an alteration in their DNA that may cause aortic disease. The cardiothoracic surgeons were also informed that genetic counseling and confirmatory genetic testing would be offered at no cost. The purpose of the email was to prepare the cardiothoracic surgeons in the event that their patient(s) would ask about the letter and/or their genetic status , and scheduled a genetic counseling appointment with the option to receive confirmatory genetic testing in a CLIA laboratory (paid by the research study). Participants were informed that the clinical genetic counseling visit and confirmatory genetic testing would be documented in EHR. Study participants were informed that the research identified a possible causative variant with the option of CLIA confirmation to validate the finding clinically and make it available for extended family. The benefits and limitations of confirmatory genetic testing were discussed with the participant, as well as the implications of genetic testing and clinical screening for themselves and their families. A detailed 3-generation family history was obtained which could be used to aid potential future cascade testing. Supportive resources both within the healthcare system and through patient support organizations were also discussed with participants. At the end of the genetic counseling visit, participants were invited to participate in a survey study about the recontact and disclosure process. Participant interested in receiving CLIA laboratory genetic testing and participating in the survey study provided separate written informed consent .They declined genetic counseling and confirmatory genetic testing in a CLIA laboratory during the phone call with the genetic counselor.They did not respond as they were deceased or lost to follow-up.Participants responded to the letter in one of the following ways:Demographics and clinical outcomes were collected from EHR. An example of the paper survey is provided , timing of genetic counseling appointment (multiple-choice), and concerns (open-ended). Information sharing was assessed by asking participants to indicate who they informed from a list of family members and other individuals (e.g. physician).We assessed the psychosocial impact of receiving genetic research results using the 12-item ‘Feelings About genomiC Testing Results’ (FACToR) Scale. The validated scale includes four subscales, with scores ranging from 0–12 for negative emotions, 0–16 for positive emotions, 0–8 for uncertainty, and 0–8 on privacy. An overall score is generated from the subscales, with higher scores being indicative of higher psychological impairment (i.e. stress) . A validTargeted sequencing using molecular inversion probes  was utilized to validate the pathogenic variants identified by the Northwest Genomic Center. Research coordinator time was tracked for letter preparation/mailing, tracking delivery of letter, and project facilitation (hourly rate of $32.21). Genetic counselor time was tracked for all phone calls and face-to-face time with participants (hourly rate of $37.50). The costs for the room charge for genetic counseling visit, phlebotomy and CLIA laboratory genetic testing, in which the gene was sequenced to validate the research genetic result (Invitae Corporation), were extracted from billing records. Mailing costs were tracked for all letters. We evaluated the total cost of the study and average cost per participant. Separate averages were also calculated for those who pursued genetic counseling and confirmatory genetic testing in a CLIA-laboratory and those who did not. The cost of whole exome sequencing and pathogenic variant annotation were not factored into the cost model as these metrics were a part of the parent research study, which preceded the recontact and disclosure process.Data analysis was restricted solely to descriptive statistics due to the small sample size. Data is presented as mean (± SD) for continuous data, n (%) for categorical data, and range for minimum and maximum response to survey items. When calculating overall scores, missingness was accounted for by averaging by the number of questions answered.COL3A1, FBN1, LOX, PRKG1, SMAD3, and TGFBR2). Additional information about the classification of these variants, including mutation types, is found in Table Twenty-six participants 26/240) were found to have pathogenic variants with twenty-four different variants in 6 genes . The vast majority of survey respondents were able to correctly identify the name of their condition (80%), gene involved (90%), risk to siblings and children of inheriting this variant (both 90%), and inheritance pattern , and similar scores were observed for length of letter, readability, resources provided, information about their genetic results, and potential implications for family members . Mean satisfaction scores were consistent with the various aspects of the genetic counseling appointment including: appointment duration , concern demonstrated , empathy demonstrated , facilitation of decision-making , and the appointment was valuable . The lowest mean item pertained to assessing the extent to which respondents felt reassured about their research genetic result .We assessed the psychological response of receiving a genetic result using the FACToR Scale. Mean scores for psychological distress immediately after the genetic counseling appointment were low . Mean subscale scores were low for negative feelings , uncertainty , and privacy concerns . Positive feelings were near the midpoint, indicating a moderate level of positive emotional responses to the genetic test result .Researchers use the term decisional regret to describe distress or remorse as a result of making a specific decision. The mean decisional regret score was 11.5 ± 11.6 . These scores indicate low levels of regret about deciding to learn their genetic test results. 90% of participants indicated that they would share this information with someone, and 60% indicated their intention to share the result with first-degree family members Table .Table 3AWe carefully enumerated the costs involved in each step of the process: (1) the cost to perform research genetic variant validation on a new DNA sample using MIPs was $4324, (2) the cost for the research coordinator to draft letters and execute study was $2370, (3) the cost for the genetic counselor which included phone calls after the letter was sent to participants, genetic counseling visit, and phone calls for results disclosure after CLIA laboratory genetic testing was $548, (4) the cost for CLIA-certified genetic testing was $2500, and (5) the cost for mailing, room charge, and phlebotomy was $663. The total cost of the process was $10,405. Table This is the first study to examine the impact and cost of returning pathogenic variants to thoracic aortic dissection biobank participants. Fifty-five percent (11/20) of pathogenic variant carriers attended genetic counseling, with 91% of those (10/11) undergoing CLIA-laboratory confirmatory genetic testing. Participants were satisfied with the disclosure process, generally understood the meaning and implications of test results, and did not experience adverse psychological effects. Through the process described here, clinically significant genetic test results from a research study were conveyed, although the process entailed time, resources, and financial costs that were beyond the budget of the parent study.The key steps involved in the recontact and disclosure process included: (1) research sample validation; (2) recontact via US certified mail disclosing a DNA alteration that is potentially causing disease; (3) genetic counselor telephoned participants; (4) genetic counseling appointment; and (5) CLIA laboratory confirmatory genetic testing. Papaz et al.  implemenAnother key aspect of our study included evaluating the impact of receiving research genetic results. Our findings suggest that participants were satisfied with the process and that they generally understood the meaning and implications of test results. Participants reported high levels of satisfaction with recontact via letter and genetic counseling. Participants reported low test-related distress and minimal regret about their decision to learn their research genetic result, which is consistent with the established literature on the psychological impact of receiving genetic test results . The stuResearch funds are typically used to support the roll out of returning genetic results to participants. We systematically analyzed the financial costs of the recontact and disclosure process to evaluate the financial undertaking. The total cost was $10,405 for all 26 participants, with an average cost of $400 per participant. The average cost of those choosing to opt-in to genetic counseling and confirmatory genetic testing was $605. Other studies, including Papaz et al.  and ChriThere are a number of study limitations, with the most notable being the low number of participants; however, thoracic aortic dissection is a rare medical complication. Nonetheless, the sample size and disease-specificity constrained the ability to examine group differences in key study outcomes and may limit the generalizability of study findings. We report that 10.8% of our TAAD cases harbored a pathogenic variant  which isAs the number of individuals who have genetic testing done on a research basis increases, the need for a proven delivery model of health-related genetic results also increases. This study demonstrates a process by which participants of a thoracic aortic dissection biobank receive results that may change their medical management with low psychological distress and high levels of understanding. The costs, time, and resources involved in disclosure of results were significant; however, knowing these ahead of time allows for planning for results disclosure during study development. Continued consideration for results disclosure following studies completing genetic testing is vital, and these results suggest a process acceptable to both researchers and participants.Additional file 1. See Supplemental Materials for details regarding (1) Table S1. Steps taken that preceded the recontact and disclosure of pathogenic variants, (2) Material 1—Cardiovascular Health Improvement Project Committees, (3) Material 2—Physician Letter, (4) Material 3—Participant Letter, and (5) Material 4—Return of Research-Level Genetic Testing Results Study.


Following chicken domestication, diversified chicken breeds were developed by both natural and artificial selection, which led to the accumulation of abundant genetic and phenotypic variations, making chickens an ideal genetic research model. To better understand the genetic structure of chicken breeds under different selection pressures, we genotyped various chicken populations with specific selection targets, including indigenous, commercial, gamecock, and wild ancestral chickens, using the 600K SNP array. We analyzed the population structure, genetic relationships, run of homozygosity (ROH), effective population number (Ne), and other genetic parameters. The wild ancestral population, red junglefowl (RJF), possessed the highest diversity, in comparison with all other domesticated populations, which was supported by linkage disequilibrium decay (LD), effective population number, and ROH analyses. The gamecock breeds, which were subjected to stronger male-biased selection for fighting-related traits, also presented higher variation than the commercial and indigenous breeds. Admixture analysis also indicated that game breed is a relatively independent branch of Chinese local breeds. Following intense selection for reproductive and productive traits, the commercial lines showed the least diversity. We also observed that the European local chickens had lower genetic variation than the Chinese local breeds, which could be attributed to the shorter history of the European breed. ROH were present in a breed specific manner and 191 ROH island were detected on four groups . These ROH islands were involved in egg production, growth and silky feathers and other traits. Moreover, we estimated the effective sex ratio of these breeds to demonstrate the change in the ratio of the two sexes. We found that commercial chickens had a greater sex imbalance between females and males. The commercial lines showed the highest female-to-male ratios. Interestingly, RJF comprised a greater proportion of males than females. Our results show the population genetics of chickens under selection pressures, and can aid in the development of better conservation strategies for different chicken breeds. Abundant phenotypic and genotypic variations make the chicken an ideal model species for genetic studies. Following domestication in Southeast Asia , many brIn the poultry industry, most local breeds are seriously threatened by the impacts of commercial lines ; only a Molecular tools provide geneticists an opportunity to estimate the genetic structure of different populations, using strategies such as genome-wide analyses, which have already been applied in many domestic animals . The popn = 56; Hongshan [HS], n = 96; Shouguang [SG], n = 109; Silkie [SK], n = 89; Tibet [TB], n = 41), one European local breed , three commercial breeds , five gamecock breeds , and the wild ancestral population . Detailed information for these samples is presented in In total, 1,254 chickens were randomly selected from 15 chicken populations, including five Chinese local breeds . Genotyping was performed using the 600K Affymetrix Axiom Chicken Genotyping Array , and included 580,841 SNPs across the entire chicken genome . Based on these data, SNPs were excluded from the analysis if the minor allele frequency and call rate were less than 0.05 and 0.90, respectively, and if the genotype frequencies deviated severely from the Hardy-Weinberg equilibrium, with a of K values ranging from 1 to 30. K-value (K = 29) had the lowest cross validation error (CV-error). The results for K = 2, K = 4, K = 16, and K = 29 are included in this report. The other K-value results and cross validation error plot of admixture are provided in Principal component analysis (PCA) was performed on all individuals using a GCTA . Two priFST , medium (0.05–0.15), high (0.15–0.25), and extreme (>0.25)  .R2 was used as a measure of LD in this study  as the abscissa, and generated the curves using R.Seven individuals were randomly selected from each population, and PopLDdecay  was usedEffective population size (Ne) is a genetic population parameter that aids the understanding of the evolutionary history of populations and the genetic mechanisms underlying complex traits. For each breed, we calculated the Ne prevalence 1,000 generations ago, using SNeP , to summSex bias is an important piece of information on demographic dynamics and social structure. The ESR refers to the proportion of females in the effective population. We calculated the ESR of the 15 populations based on a hierarchical Bayesian model, using KIMTREE (version 2.1) . To obtaτ) is measured on a diffusion time scale, and it is proportional to the time since divergence in generations (t) scaled by the effective population sizes (Ne).This method analyzes the allele frequencies along each branch, using Kimura’s time-dependent diffusion approximation for genetic drift . This coFrom the above formula, the ESR of our sample can be calculated from:We used different parameters to characterize genetic diversity, all of which were obtained from PLINK (version 1.9) . Genome-The proportion of observed heterozygosity (Ho) was estimated from the observed homozygosity (-het) as 1–(the number of observed homozygous loci/number of non-missing loci), and the expected heterozygosity (He) was calculated as the 1–(the number of expected homozygous loci/number of non-missing loci). The expected heterozygosity and observed heterozygosity estimate for all individuals within each population were averaged over all SNPs.The estimation of the inbreeding coefficient of individuals was based on the ROH. Long homozygous fragments were scanned using PLINK (version 1.9). Specific parameters were as follows: a sliding window of 50 SNP slides along the chromosome to estimate homozygosity; each sliding window allowed no more than one heterozygote, with no more than five missing SNPs, a minimum length of ROH of 100 kb, a minimum density of 1 SNP/50 kb, and a maximum gap between consecutive SNPs of 1,000 kb.To identify the genomic regions that were most commonly associated with ROH in different chicken breeds, we estimated the percentage of occurrences of SNPs in ROH by counting the number of times when the SNP was detected in those ROH. The genomic regions most often associated with ROHs were identified by selecting the top 1% SNPs observed in ROHs. Adjacent SNPs over this threshold were merged into genomic regions named ROH islands . The ROHROH) for each breed were calculated according to McQuillan .where LPopulation structures of the 15 breeds, comprising four commercial breeds, 10 Chinese indigenous breeds, and one wild breed, were analyzed using PCA, NJ tree, and ADMIXTURE. The PCA showed that the top two principal components accounted for 43.54% (PC1) and 35.06% (PC2) of the total variability . For eacK = 2, WL was separated from the other breeds. The genetic backgrounds of WL and other breeds are clearly significantly different. At K = 4, commercial breeds, except for Cornish, were identifiable from indigenous breeds. The Cornish breed contains fighting chicken blood, so it was separated later. At K = 8 and K = 11, Beijing You and Tibet were separated. Cornish, RJF and game breeds always grouped into one population, until K = 16. The optimal K-value for admixture was K = 29. All breeds exhibited independent genetic background, except for game chickens. The distinction between different populations of chicken game is blurred. The NJ tree  . At K =  NJ tree  was consFST values) pairwise estimates between 15 chicken populations are shown in FST values ranged from 0.03 to 0.27. First, the FST of commercial breeds and FST of Chinese indigenous breeds had the highest differentiation, especially Cornish. The wild breed (RJF) and FST value of Tibetan chickens was 0.08, indicating less differentiation than other breeds. This result was confirmed by PCA and NJ analyses, which supported that Tibetan chicken was adjacent to RJF. Finally, there was greater differentiation between indigenous chickens and fighting chickens (0.06–0.25), but the degree of differentiation between game chicken subgroups was lower (0.03–0.17).The genetic differentiation (The proportion of nucleotide diversity (π) and mean value of expected heterozygosity (He) and observed heterozygosity (Ho) for each population are shown in ROH) of 15 chicken breeds  exceeded 0.22, whereas Chinese indigenous breeds, game breeds, and RJF had lower inbreeding levels. Our results suggested that there were clear variations in the inbreeding of ROH arising from the selection pressure experienced by the chickens.Runs of homozygosity can reflect the degree of animal inbreeding. A long Runs of homozygosity means inbred animals with recent common ancestors, whereas a short Runs of homozygosity reflects more distant common ancestors. We summarized the runs of homozygosity and genomic inbreeding coefficient  thresholds in SNeP, are shown in The average values of effective population size over the past 1,000 generations estimated from the mean In addition, 40 individuals were randomly selected from each group to calculate contemporary Ne based on LD . The results are shown in Effective sex ratio is critical for a better understanding of social structures and demographic dynamics. We used KIMTREE  to inferK = 16, suggesting domestic breeds and wild populations have mutual introgression , and their subpopulation genetic differentiation is smaller (0.03–0.17). However, Zhangzhou Game has been mixed with foreign chicken blood  than exotic commercial breeds, which is in agreement with previous studies . For locRed junglefowl, which is considered to be the ancestor of the domesticated chicken , showed In general, there are differences in the length of ROH in the genome of chickens in various production directions. Commercial breeds had the largest Runs of homozygosity (183–280 Mb), followed by local and wild breeds . This reflects the difference in the degree of inbreeding and the population history. The higher the inbreeding level, the greater the Runs of homozygosity number and length in the genome. According to our results, WL, RIR and Cornish might experience strong inbreeding. There are several likely reasons for this: first, strict breed standards causing breeders use inbreeding to select outstanding offspring; second, the import number limit due to frequent use of a few male chickens. The utility of Runs of homozygosity to estimate inbreeding has already been proven in cattle , pigs B, and goaNECAB2, SLC38A8, and NFATC3 genes, and several potential candidate genes for silky feathers. On chromosome 3, we showed that up to 40% of Beijing You chickens had a ROH between 69.38 and 70.93 Mb, retaining the gene ASCC3 which is also a candidate gene for silky feathers . Xishuangbanna Game had the lowest LD value and inbreeding coefficient (FROH: 0.05). The other breeds followed the same trend. The effective population sizes were opposite to their LD level.We investigated LD decay and the effective population size using 15 different breeds . Our results showed that LD decreased with an increase in the physical distance between SNPs, and that indigenous breeds displayed lower LD than commercial chickens, which was consistent with the results of previous studies . PreviouWe used whole-genome SNP data to estimate effective population size. The results showed that the effective population size of all populations decreased over time, which may have been caused by a bottleneck effect, variety differentiation, or selection pressure during domestication . The wilThe proportion of females in the effective population, known as the ESR, can provide information about the reproductive potential of a population, which is essential for wildlife management of endangered species, and to better understand the underlying social structure and population dynamics . We deteIn most diversity studies, it is generally believed that the genetic diversity assessment results are more reliable when the number of individuals in the population is greater than 30. And small sample sizes are enough to assess interpopulation divergence when enough SNP markers are used . In thisIn summary, we collected in 1,254 samples representing four different selection pressures. We analyzed the population structure, genetic relationships, genomic inbreeding, ROH, effective population number and ESR using a genome-wide SNPs. Firstly, game breeds were genomically distinct from the other Chinese local chickens. They possessed their own genetic structure in group, and mutual introgression was observed among them. Then, the genetic diversity between commercial and indigenous chickens showed a highly significant different. Commercial chickens with high intense selection had the lowest diversity and local breeds had relatively high diversity. While some local breeds genetic diversity was higher than that of RJF, indicating that the diversity of red jungle fowl was declining in recent years. In addition, ROH level was consistent with estimates of the LD decay trend and effective population size. ROH islands contained many candidate genes controlling phenotypes and production performance. Moreover, there is unequal diversity between sex and autosome chromosomes, and the ESR of commercial breed was distinct from other breeds. Overall, our results provide valuable information to better understanding the genetic diversity and population structure of chicken breeds under different selection pressures.https://figshare.com/s/efe1bbbf60078f2f08aa.The datasets generated for this study can be found in FigShare The animal study was reviewed and approved by the Animal Welfare Committee of China Agricultural University.LQ and ZN conceived and designed this study. JZ performed did the analysis. CN, XLi, and JZ performed the experiments and interpreted the result data. YC, YJ, LW, XLv, and WY contributed to reagents and materials. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus subtilis response to cell envelope perturbations. A third protein, LiaF, has an inhibitory role over LiaRS in the absence of stimulus. Together, LiaF and LiaRS form a three-component system characterized by an unusual stoichiometry, a 4:1 ratio between LiaS and LiaR, the significance of which in the signal transduction mechanism of LiaRS is not entirely understood.LiaRS mediates We measured, for the first time, the kinetics of the phosphorylation-dependent processes of LiaRS, the DNA-binding affinity of LiaR, and characterized the effect of phosphorylation on LiaR oligomerization state. Our study reveals that LiaS is less proficient as a phosphatase. Consequently, unspecific phosphorylation of LiaR by acetyl phosphate may be significant in vivo. This drawback is exacerbated by the strong interaction between LiaR and its own promoter, as it can drive LiaRS into losing grip over its own control in the absence of stimuli. These intrinsic, seemingly ‘disadvantageous”, attributes of LiaRS are likely overcome by the higher concentration of LiaS over LiaR in vivo, and a pro-phosphatase role of LiaF.Overall, our study shows that despite the conservative nature of two-component systems, they are, ultimately, tailored to meet specific cell needs by modulating the dynamics of interactions among their components and the kinetics of phosphorylation-mediated processes. Two-component systems (TCS) represent a fundamental mechanism of bacterial signal transduction that allows microorganisms to perceive external signals and react appropriately with a cytoplasmic response. Typically, a two-component system (TCS) consists of a membrane bound (most of the time) histidine kinase (HK) and an intracellular soluble protein , 2. The Bacillus subtilis is involved in sensing cell envelope stress instigated by perturbation of the cytoplasmic membrane, particularly antibiotics that interfere with the Lipid II cycle of cell-wall peptidoglycan biosynthesis such as bacitracin, ramoplanin, vancomycin, and cationic antimicrobial peptides -ATP was purchased from Perkin Elmer LAS Canada Inc.  or GE Healthcare. The Proteo Extract All-in-One Trypsin Digestion Kit was purchased from EMD Bioscience. Chromosomal DNA of the Bacillus subtilis strain 168 was acquired from Cedarlane. Oligonucleotides were acquired from Sigma (Canada).Chemicals and antibiotics were purchased from Sigma  or ThermoFisher , unless otherwise stated. Chromatography media and columns were purchased from GE Healthcare . Growth media were purchased from ThermoFisher. B. subtilis liaS: Dir: 5′- CATGGATCCCGATTGGCCAGAGATCTTC-3′ and Rev.: 5′-ACGCCCG GGTTATCAATCAA TAATACTCGAATC-3′. The primers contain the restriction enzymes BamHI and SmaI , respectively. We used chromosomal DNA of B. subtilis 168 as the template for liaS amplification. The amplified gene is referred to as liaS126. The amplified gene (708 bp) was digested with the above restriction enzymes and subsequently cloned into the corresponding restriction sites of the pGEX-4 T-1 vector  to fuse liaS126 to the C-terminus of the GST protein. The resulting construct pGEX-4 T-1::liaS126 was introduced into E. coli BL21(DE3). The correctness of the liaS126 insertion into pGEX-4 T-1 was confirmed by DNA sequencing . Hereon the LiaS chimeric construct is referred to as LiaS.The following direct and reverse primers were used to amplify the nucleic acid sequence that encodes the cytoplasmic region (amino acids 126–360) of B. subtilis 168 was used as a template for the amplification of the liaR gene encoding for the full length and tagless LiaR protein, corresponding to the amino acids 1 to 211. The following primers were used for the amplification of liaR: Dir: 5′-ACGCATATGATGATTCGAGTATT ATTGAT-3′ and Rev.: 5′-ACGAAGCTTCTAATTCACGAGATGATTT-3′. The primers contained the restriction sites for NdeI and HindIII , respectively. The amplicon was digested with NdeI and HindIII and ligated into pET26b vector between the NdeI and HindIII restriction sites. The resulting construct, pET26b::liaR, was introduced into E. coli BL21(DE3). The correctness of the liaR cloning to pET26b was confirmed by DNA sequencing .Chromosomal DNA of liaRN). The mutation was carried out using Site-Directed mutagenesis kit (ThermoFisher) with the following primers Dir: 5′-AAAGTGGCGTGAAAAGTATTATCC AGGCT-3′ and Rev.: 5′-AGCCTGGATAATACTTTTCACGCCACTTT − 3′ . The pET26b::liaR vector was used as the template. The successful introduction of the stop codon was verified by DNA sequencing. The resulting pET26b::liaRN construct was used for transformation of E. coli BL21 (DE3).The Gly-131 residue of LiaR (GGA) was mutated to a stop codon (TGA) in order to clone the N-terminal domain of LiaR (liaRC) was similar to that employed for cloning of the full-length LiaR. The nucleotide sequence that encodes ED, spanning the residues 140–211 (the sequence was determined by ExPASy tools), was amplified using pET26b::liaR as the template and the following primers: Dir: 5′- ACGCATATGTCAGGTGA AAACGC-3′ and Rev.: 5′-ACGAAGCTTCTAATTCACGAGATGATTT-3′ . The subsequent construct, pET26b::liaRC, was introduced into E. coli BL21(DE3). The correctness of the liaRC cloning was verified by DNA sequencing.The cloning strategy for the C-terminal domain of LiaR (liaS and liaR was carried out using the Single-Site Directed mutagenesis kit (ThermoFisher). In the case of mutagenesis on liaS, the pGEX-4 T1::liaS126 vector was used as a template and the following primers were used as the mutagenesis primers: Dir: 5′-CCAGAGATCTTGCTGATGCGGTCAG-3′ and Rev.: 5′-CTGACCGCATCAGCAAG ATCTCTG G-3′ . In the case of mutagenesis on liaR, the pET26b::liaR vector was used as a template and the following primers were used as the mutagenesis primers: Dir: 5′-GATGTCATTTTAATGGCCCTTGTCATGG-3′ and Rev.: 5′-CC ATGACAAGGGCCATTAAAATGACATC-3′ . The successful mutations were confirmed by DNA sequencing.We carried out the mutation of the conserved phosphorylation site on LiaS, His154 (CAT) to Ala (GCT) substitution, and on LiaR, Asp54 (GAT) to Ala (GCC) substitution. The mutagenesis on liaS and its variants, and production and isolation of their respective proteins were carried out as described previously by Belcheva and Golemi-Kotra -ATP and non-radiolabeled ATP (a 1:9 ratio was maintained). Typically, LiaS (5 μM) in the phosphorylation buffer  supplemented with 10 mM CaCl2 was incubated with [γ-32P]-ATP (250 Ci) at different time intervals (at 25 °C). The reactions were quenched by addition of SDS sample buffer dye. Samples were analyzed by 12.5% SDS-PAGE. The gel was incubated on a phosphor screen for 2 h. The screen was scanned using a Typhoon Trio+ instrument. The quantification of phosphorylation was done by ImageJ (NIH). The time dependent data were fitted by a first-order rate equation using Erithacus GraFit software (version 5.0.10).The in vitro autophosphorylation of LiaS was carried out at ATP concentrations ranging from 10 μM to 250 μM (each concentration was repeated two times). Different ATP concentrations were prepared by mixing radiolabeled [γ-32P]-ATP (3000 Ci/mmol) was removed using a desalting column . Then the phosphorylated LiaS (4 μM) was added to LiaR (20 μM) in the PB buffer . The reaction mixture was incubated at 25 °C and samples of 10 μl were removed at different time intervals and quenched with SDS sample buffer. The quenched reactions were analyzed by 15% SDS-PAGE. The extent of the phosphotransfer was monitored by exposing gels to a phosphor screen  for 2 h and imaging them using a Typhoon Trio+ variable-mode imager . The gels were stained with Coomassie Blue to assess the protein amounts across the different time-point reaction mixtures. The phosphor images of the gels were analyzed by ImageJ (NIH). These experiments were repeated two times.Phosphorylation of LiaS was carried out as described above. The excess of -ATP (3000 Ci/mmol) using T4 polynucleotide kinase. The binding reactions (20 μl) were prepared in the binding buffer  supplemented with 5 mM MgCl2, 10 ng of herring sperm DNA, and 2.5% glycerol. The DNA was mixed with unphosphorylated and phosphorylated LiaR at concentrations varying from 0 to 10 μM. The reaction mixtures were incubated at 25 °C for 30 min and resolved by 9% native-PAGE. The dried gels were exposed to phosphor screens  and scanned by a Typhoon Trio+ variable-mode imager . The gel images were analyzed by ImageJ (NIH). The dissociation constant, Kd, was determined as the concentration of the protein required to shift 50% of the DNA. These experiments were repeated at least three times.The DNA-binding affinity of LiaR to the liaI spanning the nucleotide sequence from − 162 to + 31 was amplified using the primers Dir:5′-GAAAGGGAAGCAAGTGTTCATCTGTAAAG-3′ and Rev.:5′- TTCATGCAGATCCTCCTTTCGTTTT-3′. The DNase I footprinting was carried out as previously described [Pescribed .Additional file 1. Purification of LiaS and LiaR.Additional file 2. Purificaiton of LiaRC and LiaRN.Additional file 3. Autokinase activity of LiaS. Progress curve of time-dependent phosphorylation of LiaS, and SDS-PAGE analysis of LiaS and LiaSH159A phosphorylation.Additional file 4. Phosphorylation of LiaRN by acetyl phosphate.Additional file 5. CD studies on LiaR and LiaRD54A variant.Additional file 6. Phosphorylation of LiaRD54A by acetyl phosphate.Additional file 7. Oligomerization studies on LiaRN.Additional file 8. The DNA sequencing standard reactions.Additional file 9. The pull-down experiments with GST-LiaS and LiaR.


Paeonia decomposita, endemic to China, has important ornamental, medicinal, and economic value and is regarded as an endangered plant. The genetic diversity and population structure have seldom been described. A conservation management plan is not currently available.He) varied from 0.043 to 0.901 (mean 0.492) in 16 primers. Moderate genetic diversity (He = 0.405) among populations was revealed, with Danba identified as the center of genetic diversity. Mantel tests revealed a positive correlation between geographic and genetic distance among populations , demonstrating consistency with the isolation by distance model. Analysis of molecular variance (AMOVA) indicated that the principal molecular variance existed within populations (73.48%) rather than among populations (26.52%). Bayesian structure analysis and principal coordinate analysis (PCoA) supported the classification of the populations into three clusters.In the present study, 16 pairs of simple sequence repeat (SSR) primers were used to evaluate the genetic diversity and population structure. A total of 122 alleles were obtained with a mean of 7.625 alleles per locus. The expected heterozygosity (P. decomposita using SSR. Three management units were proposed as conservation measures. The results will be beneficial for the conservation and exploitation of the species, providing a theoretical basis for further research of its evolution and phylogeography.This is the first study of the genetic diversity and population structure of  Paeonia L. (Paeoniaceae) includes 32 woody and herbaceous species, mainly distributed in the northern hemisphere. Paeonia is divided into three sections: Onaepia, Moutan, and Paeonia . PCR amplification was conducted using a Bio-Rad thermal cycler (Applied Biosystems) with either of two different cycling protocols, as follows: 1. Pre-denaturation at 95 °C for 5 min, followed by 10 cycles of denaturation at 95 °C for 30 s, annealing at 62–52 °C for 30 s (1 °C drop for each cycle), and extension at 72 °C for 30 s. 2. 25 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, then extension at 72 °C for 30 s, followed by a final extension at 72 °C for 20 min. All PCR products were genotyped using capillary electrophoresis on an ABI 3730XL DNA Analyzer. The alleles of all loci were scored relative to LIZ 500, an internal product size standard, with the aid of GeneMarker Version 4.0 .SSR-PCR amplification reactions were conducted using a total volume of 10 μl consisting of 5 μl of 2 × Taq PCR MasterMix  was computed using Cervus 3.0 software [Ar) and intra-population inbreeding coefficients (Fis) calculated using FSTAT 2.9.3.2 software [PPL), number of private alleles (NP), number of observed alleles (Na), number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), fixation index (F), genetic differentiation coefficient (Fst), gene flow (Nm), Nei’s genetic distance (NGD), genetic identity (NGI), F-statistics, Hardy-Weinberg equilibrium (HWE), Mantel test, Analysis of molecular variance (AMOVA), and Principal coordinate analysis (PCoA) were computed with GenAlEx 6.5 software [K = 2–10) was performed with a run length of 1 × 105 Markov Chain Monte Carlo (MCMC) replicates after a burn-in period of 1 × 105 iterations in an admixture model with correlated allele frequency. The ΔK method [K value and the optimal number of genetic clusters on Structure Harvester V6.0 software [The polymorphic information content (software , with alsoftware . The persoftware . The popsoftware . A totalK method  was emplsoftware . An unwesoftware  on the bMonmonier’s maximum difference algorithm was used with BARRIER version 2.2  to exploAdditional file 1: Fig. S1. The distribution of ΔK over K = 1–10.Additional file 2: Table S1. Summary of Private Alleles by Population.