Giga-Pixel Lensfree Holographic Microscopy and Tomography Using Color Image Sensors

We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ∼350 nm lateral resolution, corresponding to a numerical aperture of ∼0.8, across a field-of-view of ∼20.5 mm². This constitutes a digital image with ∼0.7 Billion effective pixels in both amplitude and phase channels (i.e., ∼1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ±50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ∼0.35 µm×0.35 µm×∼2 µm, in x, y and z, respectively, creating an effective voxel size of ∼0.03 µm³ across a sample volume of ∼5 mm³, which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.     

In?Vivo Nonlinear Optical Imaging of Immune Responses: Tissue Injury and Infection

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5α and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ∼450 and ∼520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.     

Cuban history of CRF19 recombinant subtype of HIV-1

CRF19 is a recombinant form of HIV-1 subtypes D, A1 and G, which was first sampled in Cuba in 1999, but was already present there in 1980s. CRF19 was reported almost uniquely in Cuba, where it accounts for ∼25% of new HIV-positive patients and causes rapid progression to AIDS (∼3 years).We analyzed a large data set comprising ∼350 pol and env sequences sampled in Cuba over the last 15 years and ∼350 from Los Alamos database. This data set contained both CRF19 (∼315), and A1, D and G sequences. We performed and combined analyses for the three A1, G and D regions, using fast maximum likelihood approaches, including: (1) phylogeny reconstruction, (2) spatio-temporal analysis of the virus spread, and ancestral character reconstruction for (3) transmission mode and (4) drug resistance mutations (DRMs). We verified these results with a Bayesian approach. This allowed us to acquire new insights on the CRF19 origin and transmission patterns. We showed that CRF19 recombined between 1966 and 1977, most likely in Cuban community stationed in Congo region. We further investigated CRF19 spread on the Cuban province level, and discovered that the epidemic started in 1970s, most probably in Villa Clara, that it was at first carried by heterosexual transmissions, and then quickly spread in the 1980s within the “men having sex with men” (MSM) community, with multiple transmissions back to heterosexuals. The analysis of the transmission patterns of common DRMs found very few resistance transmission clusters.Our results show a very early introduction of CRF19 in Cuba, which could explain its local epidemiological success. Ignited by a major founder event, the epidemic then followed a similar pattern as other subtypes and CRFs in Cuba. The reason for the short time to AIDS remains to be understood and requires specific surveillance, in Cuba and elsewhere.     

Prediction of Muscle Activities from Electrocorticograms in Primary Motor Cortex of Primates

Electrocorticography (ECoG) has drawn attention as an effective recording approach for brain-machine interfaces (BMI). Previous studies have succeeded in classifying movement intention and predicting hand trajectories from ECoG. Despite such successes, however, there still remains considerable work for the realization of ECoG-based BMIs as neuroprosthetics. We developed a method to predict multiple muscle activities from ECoG measurements. We also verified that ECoG signals are effective for predicting muscle activities in time varying series when performing sequential movements. ECoG signals were band-pass filtered into separate sensorimotor rhythm bands, z-score normalized, and smoothed with a Gaussian filter. We used sparse linear regression to find the best fit between frequency bands of ECoG and electromyographic activity. The best average correlation coefficient and the normalized root-mean-square error were 0.92±0.06 and 0.06±0.10, respectively, in the flexor digitorum profundus finger muscle. The δ (1.5∼4Hz) and γ2 (50∼90Hz) bands contributed significantly more strongly than other frequency bands (P<0.001). These results demonstrate the feasibility of predicting muscle activity from ECoG signals in an online fashion.     

A Highlights from MBoC Selection: Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803–2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.     

Nanoscale Measurement of the Dielectric Constant of Supported Lipid Bilayers in Aqueous Solutions with Electrostatic Force Microscopy

We present what is, to our knowledge, the first experimental demonstration of dielectric constant measurement and quantification of supported lipid bilayers in electrolyte solutions with nanoscale spatial resolution. The dielectric constant was quantitatively reconstructed with finite element calculations by combining thickness information and local polarization forces which were measured using an electrostatic force microscope adapted to work in a liquid environment. Measurements of submicrometric dipalmitoylphosphatidylcholine lipid bilayer patches gave dielectric constants of ε_r ∼ 3, which are higher than the values typically reported for the hydrophobic part of lipid membranes (ε_r ∼ 2) and suggest a large contribution of the polar headgroup region to the dielectric response of the lipid bilayer. This work opens apparently new possibilities in the study of biomembrane electrostatics and other bioelectric phenomena.     

A Virtual Clinical Trial of FDG-PET Imaging of Breast Cancer: Effect of Variability on Response Assessment

INTRODUCTION: There is growing interest in using positron emission tomography (PET) standardized uptake values (SUVs) to assess tumor response to therapy. However, many error sources compromise the ability to detect SUV changes. We explore relationships between these errors and overall SUV variability. METHODS: We used simulations in a virtual clinical trial framework to study impacts of error sources from scanning and analysis effects on assessment of SUV changes. We varied tumor diameter, scan duration, pretherapy SUV, magnitude of change in SUV, image reconstruction filter, and SUV metric. Poisson noise was added to the raw data before image reconstruction. Variance from global sources of error, e.g., scanner calibration, was incorporated. Two thousand independent noisy sinograms per scenario were generated and reconstructed. We used SUVs to create receiver operating characteristic (ROC) curves to quantify ability to assess response. Integrating area under the ROC curve summarized ability to detect SUV changes. RESULTS: Scan duration and image reconstruction method had relatively little impact on ability to measure response. SUV_MAX is nearly as effective as SUV_MEAN, especially with increased image smoothing and despite size-matched region of interest placement. For an effective variability of 15%, we found the Positron Emission Tomography Response Criteria in Solid Tumors criteria for measuring response (±30%) similar to the European Organization for Research and Treatment of Cancer criteria (±25%). CONCLUSIONS: For typical PET variance levels, tumor response must be 30% to 40% to be reliably determined using SUVs. PET scan duration and image reconstruction method had relatively little effect.     

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.     

Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single-channel and [³H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx_a), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca²⁺ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx_a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTx_a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx_a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx_a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTx_a binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTx_a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [³H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx_a induces subconductance states in skeletal and cardiac muscle Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.     

Dissociation of Increases in PGC-1α and Its Regulators from Exercise Intensity and Muscle Activation Following Acute Exercise

Muscle activation as well as changes in peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) following high-intensity interval exercise (HIIE) were examined in young healthy men (n  = 8; age, 21.9±2.2 yrs; VO₂peak, 53.1±6.4 ml/min/kg; peak work rate, 317±23.5 watts). On each of 3 visits HIIE was performed on a cycle ergometer at a target intensity of 73, 100, or 133% of peak work rate. Muscle biopsies were taken at rest and three hours after each exercise condition. Total work was not different between conditions (∼730 kJ) while average power output (73%, 237±21; 100%, 323±26; 133%, 384±35 watts) and EMG derived muscle activation (73%, 1262±605; 100%, 2089±737; 133%, 3029±1206 total integrated EMG per interval) increased in an intensity dependent fashion. PGC-1α mRNA was elevated after all three conditions (p<0.05), with a greater increase observed following the 100% condition (∼9 fold, p<0.05) compared to both the 73 and 133% conditions (∼4 fold). When expressed relative to muscle activation, the increase in PGC-1α mRNA for the 133% condition was less than that for the 73 and 100% conditions (p<0.05). SIRT1 mRNA was also elevated after all three conditions (∼1.4 fold, p<0.05), with no difference between conditions. These findings suggest that intensity-dependent increases in PGC-1α mRNA following submaximal exercise are largely due to increases in muscle recruitment. As well, the blunted response of PGC-1α mRNA expression following supramaximal exercise may indicate that signalling mediated activation of PGC-1α may also be blunted. We also indentify that increases in PDK4, SIRT1, and RIP140 mRNA following acute exercise are dissociated from exercise intensity and muscle activation, while increases in EGR1 are augmented with supramaximal HIIE (p<0.05).     

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli,Avoiding Dangers of Runaway Overreplication

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the “natural” CRC limit of ∼8 (cells divide more slowly); the “functional” CRC limit of ∼22 (cells divide extremely slowly); and the “tolerance” CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.     

Identification of rare alleles and their carriers using compressed se(que)nsing

Identification of rare variants by resequencing is important both for detecting novel variations and for screening individuals for known disease alleles. New technologies enable low-cost resequencing of target regions, although it is still prohibitive to test more than a few individuals. We propose a novel pooling design that enables the recovery of novel or known rare alleles and their carriers in groups of individuals. The method is based on a Compressed Sensing (CS) approach, which is general, simple and efficient. CS allows the use of generic algorithmic tools for simultaneous identification of multiple variants and their carriers. We model the experimental procedure and show via computer simulations that it enables the recovery of rare alleles and their carriers in larger groups than were possible before. Our approach can also be combined with barcoding techniques to provide a feasible solution based on current resequencing costs. For example, when targeting a small enough genomic region (∼100 bp) and using only ∼10 sequencing lanes and ∼10 distinct barcodes per lane, one recovers the identity of 4 rare allele carriers out of a population of over 4000 individuals. We demonstrate the performance of our approach over several publicly available experimental data sets.     

Diversity in the Glucose Transporter-4 Gene (SLC2A4) in Humans Reflects the Action of Natural Selection along the Old-World Primates Evolution

Background

Glucose is an important source of energy for living organisms. In vertebrates it is ingested with the diet and transported into the cells by conserved mechanisms and molecules, such as the trans-membrane Glucose Transporters (GLUTs). Members of this family have tissue specific expression, biochemical properties and physiologic functions that together regulate glucose levels and distribution. GLUT4 –coded by SLC2A4 (17p13) is an insulin-sensitive transporter with a critical role in glucose homeostasis and diabetes pathogenesis, preferentially expressed in the adipose tissue, heart muscle and skeletal muscle. We tested the hypothesis that natural selection acted on SLC2A4.

Methodology/Principal Findings

We re-sequenced SLC2A4 and genotyped 104 SNPs along a ∼1 Mb region flanking this gene in 102 ethnically diverse individuals. Across the studied populations (African, European, Asian and Latin-American), all the eight common SNPs are concentrated in the N-terminal region upstream of exon 7 (∼3700 bp), while the C-terminal region downstream of intron 6 (∼2600 bp) harbors only 6 singletons, a pattern that is not compatible with neutrality for this part of the gene. Tests of neutrality based on comparative genomics suggest that: (1) episodes of natural selection (likely a selective sweep) predating the coalescent of human lineages, within the last 25 million years, account for the observed reduced diversity downstream of intron 6 and, (2) the target of natural selection may not be in the SLC2A4 coding sequence.

Conclusions

We propose that the contrast in the pattern of genetic variation between the N-terminal and C-terminal regions are signatures of the action of natural selection and thus follow-up studies should investigate the functional importance of differnet regions of the SLC2A4 gene.     

A Highlights from MBoC Selection: Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity–dependent manner.     

Adaptive Geometric Tessellation for 3D Reconstruction of Anisotropically Developing Cells in Multilayer Tissues from Sparse Volumetric Microscopy Images

The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2–4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the ‘Adaptive Quadratic Voronoi Tessellation’ (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.     

Osmotic Pressure in a Bacterial Swarm

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼30 μm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼120 μm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Whole-Genome Resequencing of Worldwide Wild and Domestic Sheep Elucidates Genetic Diversity,Introgression, and Agronomically Important Loci

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (∼16.10×) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, ∼121.2 million single nucleotide polymorphisms, ∼61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3′-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.