Current literature debates the development and implementation of three-dimensional Exoscopes in the daily routine of neurosurgical practice. This study evaluates the grade of satisfaction and intraoperative handling of the novel Aesculap Aeos Three-Dimensional Robotic Digital Microscope used in a Neurosurgical Department in the daily practice in a larger series of neurosurgical procedures. Our evaluation of this modern microscope technology focuses on three central aspects of microsurgical procedures. First, the visualization of tumor tissue and its discernment from healthy tissue, which is of utmost importance during neurooncological procedures. Furthermore, the aspect of ergonomics and fatigue during long, repetitive surgical procedures have been shown to cause work-related musculoskeletal disorders. Finally, the implementation of novel three-dimensional microscopes may be a promising tool for surgical education. Improved image quality, superior ergonomic comfort for the surgeon and increased accessibility within the surgical field have been noted as major advantages to date.Objective: Current literature debates the role of newly developed three-dimensional (3D) Exoscopes in the daily routine of neurosurgical practice. So far, only a small number of cadaver lab studies or case reports have examined the novel Aesculap Aeos Three-Dimensional Robotic Digital Microscope. This study aims to evaluate the grade of satisfaction and intraoperative handling of this novel system in neurosurgery. Methods: Nineteen neurosurgical procedures  performed over 9 weeks using the Aeos were analyzed. Ten neurosurgeons of varying levels of training were included after undergoing device instruction and training. Following every surgery, a questionnaire consisting of 43 items concerning intraoperative handling was completed. The questionnaires were analyzed using descriptive statistics. Results: No intraoperative complications occurred. Surgical satisfaction was ranked high (78.95%). In total, 84.21% evaluated surgical ergonomics as satisfactory, while 78.95% of the surgeons would like to use this system frequently. Image quality, independent working zoom function and depth of field were perceived as suboptimal by several neurosurgeons. Conclusion: The use of Aeos is feasible and safe in microsurgical procedures, and surgical satisfaction was ranked high among most neurosurgeons in our study. The system might offer advanced ergonomic conditions in comparison to conventional ocular-based microscopes.  The development and implementation of three-dimensional 3D) Exoscopes in the daily routine of neurosurgical practice is a matter of current debate ,2,3,4,5.D ExoscopSo far, the majority of reports have focused on the VITOM , the Olympus ORBEYE  and the Zeiss KINEVO  . Only a Therefore, the aim of this study was to evaluate the grade of satisfaction and the intraoperative handling of a newly developed Three-Dimensional Robotic Digital Microscope Aeos used in a Neurosurgical Department in the daily routine in a larger series of surgical procedures.In this prospective study, 19 neurosurgical procedures were performed using the Aeos Three-Dimensional Robotic Digital Microscope over a period of nine weeks in a German neurosurgical university department. A cohort of ten neurosurgeons, including the head of the department, four senior physicians, two consultants and three residents scheduled as the headsurgeon for a procedure corresponding to their level of training . The proEvery surgeon underwent a formal introduction to the Robotic Digital Microscope from the manufacturer prior to first use. During surgery, the surgeon was able to change to the conventional microscope at any step of the procedure as deemed necessary by the surgeon. Only procedures deemed suitable for exoscopic surgery were included to be performed with the Aeos.Patient age, diagnosis, surgical procedure, histopathological findings, case complexity as well as surgical time and clinical outcome were documented. In addition, a questionnaire consisting of 43 items using a 5 (to 6) -point Likert scale regarding the intraoperative satisfaction in terms of image quality, ergonomics, ease of use and physical exhaustion afterwards was completed after every operative procedure.The Aesculap Aeos Three-Dimensional Robotic Digital Microscope is a robotic-assisted 3D Heads-Up Surgery System. It consists of a camera, a 6-axis robotic arm for flexible setup options, a 3D surgical screen (16:9 wide view) with HDR imaging (Full HD (1080p HD stereoscopic image) or 4K UHD) and 3D glasses, a control screen (15.6″ display size) with a touchscreen, the base of the microscope , USB, DICOM,) and a footswitch  . The sysStatistical analysis was performed using IBM SPSS Statistics Version 26. A total of 43 questions were evaluated and presented as mean and standard deviation (mean (SD)).A total of 19 operative procedures in 19 different patients were performed fully or partially with the Aeos Three-Dimensional Robotic Digital Microscope by ten different neurosurgeons, including the head of the department, four senior physicians, two consultants and three residents. Twelve cranial procedures were performed, including three glioblastomas  , three mn = 19). No procedure was rated as low or very high in terms of the case complexity. The mean age of the patients was 59.3 years . Case complexity was rated high in 10.53%, of increased difficulty in 36.84%, medium in 42.11% and relatively low in 10.53% of the cases (The mean time in the operation theatre was 221 min (±71 min), the mean surgical time was 154 min (±61.5 min). Gross total resection (verified by MRI) of the three glioblastomas was achieved in two cases, and the third one was planned as a subtotal tumor debulking. One postoperative complication  after temporal- and amygdala resection occurred. No intraoperative complications were detected. Concerning the patients’ outcome, seven patients improved postoperatively .A total of 78.95% of the surgeons rated the surgical satisfaction as high after having performed the operation, and 26.32% evaluated the satisfaction as very high. None of the 19 surgeons reported an unsatisfactory surgical experience. A total of 68.42% of surgeons agreed that the case was suitable for operating using the Aeos microscope . Both vaA total of 84.21% of participants evaluated the surgical ergonomics as satisfactory. Overall case complexity was rated as high in two cases (10.53%) (vascular procedures), increased in seven cases (36.84%), medium for eight cases (42.11%) and relatively low in two cases (10.53%) (cerebellar metastases). When asked whether the microsurgical tasks required during the case were suitable for the Aeos microscope, 63.16% agreed or strongly agreed (36.84%). Controlling the 6-axis robotic during the procedure was easy for 52.63% of the surgeons, whereas 21.05% found the handling difficult. The hand-eye coordination was affected negatively during the digital robotic microscope utilization in two out of 19 times. The overall 3D depth perception was satisfactory in 73.68% of the cases and unsatisfactory in 21.05% in terms of image quality. The sharpness of the displayed picture on the screen was rated as satisfactory in 94.74%.A total of 78.95% assessed the depth of the field as satisfactory, while 10.53% disagreed. The luminance was found to be satisfactory in 94.74% of cases.A total of 78.95% of the surgeons in the current study would like to use the Aeos Microscope more frequently. None rated it as unnecessarily complex. Twelve participants indicated that the microscope was easy to use (63.16%), ten felt confident using it alone (52.63%), six would need the support of a technical person if using it frequently (31.58%), and only five surgeons answered that the system was cumbersome in use (26.32%). Out of the surgeons, 89.74% found that most people could learn to use the Aeos Microscope very quickly .When asked how mentally fatiguing the procedure was, the cognitive and physical demands were rated as low .Furthermore, task complexity was rated as medium and values for situational stress and distractions were rated as low. When asked about surgical satisfaction after using the new device, residents rated it as neutral to high, senior physicians as high to very high.The senior physicians, as well as the residents, evaluated the physical and mental tiredness after the operation as very low to medium. The control of the 6-axis robotic arm was evaluated as rather easy by the residents and the experienced surgeons, the surgical working zone as unblocked by the consultants and senior physicians and rather restricted by one experienced surgeon. Surgical ergonomics, in general, were highly satisfactory and valued by the senior physicians. On the other hand, the residents rated the ergonomics as neutral. In terms of the 3D depth perception, the majority of the surgeons were satisfied to very satisfied. Residents agreed on wanting the support of a technical person during periodic use of the system, but the consultants and the majority of the senior physicians disagreed in that matter.The evaluation of modern three-dimensional robotic digital microscope technology highlights three central aspects of modern microsurgical procedures.First, the visualization of tumor tissue and its discernment from healthy tissue is of utmost importance during neurooncological procedures. The introduction of 5-ALA-induced fluorescence has been a major scientific achievement in the intraoperative visualization of malignant tissue, which is independent of brain shift ,19,20,21The Three-Dimensional Robotic Digital Microscope combines the utilization of blue light and white light during the resection of high-grade malignant tumors using 5-ALA-induced fluorescence. Consequently, non-fluorescent brain tissue appears more visible next to fluorescent tumor tissue, and tumor’s margins become even more definable. Furthermore, the aspect of ergonomics and fatigue during long, repetitive surgical procedures have been shown to cause work-related musculoskeletal disorders (WMSDs).Gadjradj et al. ,23,24,25Mavrovounis et al. showed even more concerning results after having interviewed 409 members of the “European Association of Neurosurgical Societies”, the “Neurosurgery Research Listserv” and the “Latin American Federation of Neurosurgical Societies” . Out of In general, neurosurgeons have become accustomed to restricted intraoperative working conditions causing impaired ergonomics during surgery.Other drawbacks concerning working with a conventional microscope are the weight, its size, short depth-of-field (at high magnification) and the ocular dependence .The newly developed Three-Dimensional Robotic Digital Microscope, as well as other Exoscopes, now promise superior ergonomics posturing while operating.In our study, the feasibility of use in daily neurosurgical practice appears high. Both the younger as well as the more experienced surgeons agreed on the good usability of this system for most applications. The surgical satisfaction after having performed a procedure with the Aeos was high in 78.95% of all cases. This high level of contentment is comparable or even better than described in the current literature ,5,6,28. In our series, two cases of aneurysm clippings were rated as not (easy) suitable for the use of the Aeos, and the surgeon switched to the conventional microscope. These results differ from previous studies concerning the role of exoscopic surgery in the operative treatment of vascular cases ,30, illuOverall, the current state of research on the emerging field is sparse due to the novelty of the exoscopic or three-dimensional digital robotic technique. Working with the familiar conventional microscope on which every neurosurgeon is trained since the beginning of his career is still the gold standard in neurosurgical departments. As such, in our study, the experienced surgeons, in particular, described the new device, different working angles, the utilization of the 6-axis robotic arm and focusing on the screen as irritating at the beginning. The implementation of the novel Three-Dimensional Robotic Digital Microscope into the daily routine of a neurosurgical department may also be a promising tool in terms of surgical education. The lead surgeon, as well as the assistant, are able to see the whole procedure in a 3D high-resolution image on a large screen. The assistant watches all steps at exactly the same angle as the operating surgeon in an equally ergonomic position . The leaA number of limitations or disadvantages of exoscopic surgery have been noted. In general, the default settings for image quality and depth of field were perceived as suboptimal by some of the participating neurosurgeons. Blood vessels were displayed in an overly bold red, and the visualization of hemorrhagic tissue was, therefore, a subjective limitation as previously reported . This min = 10) at different educational levels, which does not allow for statistically comparable cohorts. Furthermore, learning curves and progress reports over time were not possible due to the inclusion period of nine weeks. Another limitation may be the diversity of included cases with elective cranial, spinal and peripheral nerve surgery as well as trauma surgery. One possible limitation of this study could be the number of different operating surgeons (In conclusion, the Aeos Three-Dimensional Robotic Digital Microscope seems feasible for safe utilization in a wide spectrum of microsurgical procedures in neurosurgery. Surgical satisfaction was ranked high among the majority of neurosurgeons in our study. The system might offer advanced ergonomic conditions in comparison to conventional ocular-based microscopes.


Sixty years has passed since the cracking of the genetic code by Nirenberg and Matthaei in 1961; however, the origin of the code remains one of the greatest mysteries. It is great for a cell to innovate protein synthesis manipulated by RNA, and vice versa, which is a still unsolved “chicken-and-egg” paradox. Finding a solution is of crucial importance. Yet, most biologists hold a pessimistic view that no one knows exactly how the genetic code came into being, as an exact reconstruction of the process of the construction of genetic code may never be possible, or as there is no evidence in physics or chemistry of the control of chemical reactions by a sequence of any sort or of a code between sequences, and many papers have been published with titles indicating that their subject is the origin of the genetic code, while the content actually deals only with the evolution of the genetic code.Currently, there are several prevalent hypotheses. The frozen accident hypothesis states that the allocation of codons to amino acids in a single ancestor occurred entirely by “chance” and then remained unchanged.Unfortunately, all previous hypotheses have overlooked the importance of the energetic driving force, and none of them can explain the origination of the genetic code in the context of the biochemical system (a relation of part to whole) within a confined environment. They also ignored the relation between energy transformation and informatization (process of creating information). It is impossible to understand the origin of codons based on the codons alone or even by extending the perspective to the possible relationship between codons and amino acids.only if “A” can handle two jobs  can a persistent (then heritable) connection between “B” and “C” be established  under selection. Here, “B” and “C” are polynucleotides and polypeptides, respectively, whereas “A” is hidden. Previous attention was given neither to such logical reasoning nor to the matchmaker “A.” The phenomenal chicken-and-egg relation alone cannot give us the right answer.To craft an objective self-contained model, I propose the logical argument that ATP could fuel the elongation of both polynucleotides and polypeptides without additional energy input, making it possible to establish or fix molecular relationships between strings of nucleotides in polynucleotides and amino acids in polypeptides from their numerous random combinations throughout selection for specific sequences required for cellular survival. To produce ATP efficiently, the protocell integrated various components and interactions into a sophisticated reaction system, that is, with the development of periodic division, the protocell established a set of “intrinsic” attributes such as regularity, reproducibility, and rhythmicity of interactions, subsequently innovating biochemical pathways/cycles as needed for survival or self-copying, and ultimately forming a complex molecular network. Meanwhile macromolecules became functionally differentiated, that is, some nucleotides carried amino acids (precursor of tRNA), and others built a platform for the synthesis of polypeptides (precursor of rRNA), which eventually replaced the irregular stochastic formation of polypeptides from amino acids activated by ATP. Polypeptides helped match the acceptor stem of tRNA to its anticodon, and the system developed the rule of codon-anticodon base pairing, that is, molecular recognition through stereochemical interactions , and ushered in a unified platform, rRNA, for protein synthesis according to a mRNA template. The polypeptides in turn participated not only in the construction of the transmembrane channels of hydrophilic molecules/ions but also in catalyzing the self-condensation of nucleotides. It is an astonishing imprint of the evolutionary route from RNA to DNA that RNA is still involved as a primer in the DNA replication of extant organisms. Subdivision of the genetic system  into RNA and DNA might have favored orderly management and control of information in the very small cell in which hundreds of biochemical reactions occurred simultaneously. A triplet codon was completed with cyclizing of polynucleotides and polypeptides into a feedback loop of reciprocal causation. Consequently, the biochemical system achieved a status in which mRNA was immediately destroyed after the task was completed, while the genetic information recorded by DNA began to be permanently preserved and transmitted to ensure the continuation of a species. Then the Earth magically transitioned from chaotic prebiotic chemistry to attractive self-serving biology. It was this self-servingness that ignited exponential growth of biomolecules by incessant copying of homogeneous individuals. In short, ATP fueled the informatization of polynucleotides and polypeptides into heritable and functional molecules, although ATP was not the only molecule that could do this, but was favored by protocellular selection.Here, a new hypothesis is presented to explain how ATP acted as a “matchmaker” between biopolymers , leading to the emergence of the genetic code . ATP couWe can derive inspiration from the extant organization of biochemical systems to trace back to the origin of the genetic code. The birth of the genetic code must have been driven by some forces. Randomness and selection were frequently considered to be these forces, but I contend the energetic nature, as organisms are molecular machines in which numerous biochemical reactions take place for the acquisition, conversion, and use of energy.ATP plays a central role in the energetics of biochemical systems across all extant and perhaps extinct life forms. It provides energy for metabolism through conversion of ATP/ADP/AMP, supporting the transformation of various biomolecules into each other in an exquisitely organized cell. The major metabolic pathways  are all coupled with ATP. The other four nucleotides for genetic coding are all derived from ATP and simpler life’s building blocks (LBB), as ATP provides biochemical force to these transformations. Therefore, ATP would be the most logical starting point for the origin of informatization.in vitro, as suggested by Eigen. On early Earth, fatty acids could automatically form lipid vesicles, permitting the passage of small molecules by diffusion such as CO2 and O2, but also functioning as a barrier for certain molecules and ions , leading to different concentrations on the two sides of the membrane. Such an electro-chemical gradient could initiate energy management through polypeptide channels in the protocell.Accidental encapsulation does not make a cell, and the earliest cellular life must have resulted from long sequential processes with a plethora of intermediate forms. The present biosynthesis of ATP requires a transmembrane gradient of protons, making it exceedingly unlikely for macromolecules to evolve +, causing prebiotic organosynthesis upon reaching the concentrations or energy thresholds required for polymerization. With the accumulation of large molecules and the perpetual input of small molecules, protocells likely reciprocated between cycles of enlargement and rupture, giving rise to periodic cell division.The genetic code was likely innovated through processes from energy transformation to informatization, from ATP to triplet codon, and from RNA to DNA. The scenarios started from lipid vesicles enclosing sufficient LBB. Energy drove dazzling flows of their electrons and HOccasional polymerization and self-replication of molecules occurred on early Earth, but only fluent production of ATP facilitated by coevolution of macromolecules could establish cellular memorization, informatization and structuralization/compartmentalization. ATP eventually became the energetic core of a highly dynamic system through mediating and interlocking biochemical innovations. It is only the evolutionary completions of polynucleotide and polypeptide cyclizing into a feedback loop of reciprocal causation and preservation of the genetic code from RNA to DNA that, contrary to the central dogma, marked the dawn of cellular life or the last universal common ancestor (LUCA), when Darwinian evolution began to operate.Admittedly, all hypotheses explaining the origin of the genetic code or life are speculative, as billions of years have passed since life first appeared on Earth. It is, indeed, difficult to obtain an experimentally testable model from these hypotheses, since none of them can be verified or falsified through current methods. This pervasive speculation has been the case so far and may continue for the foreseeable future. In particular, the long parallel biochemical evolution in primordial cells could be an important reason for this difficulty.In my view, a reliable hypothesis depends on its broad applicability in the interpretation of biochemical systems, as LUCA might have changed beyond recognition due to highly variable prokaryotic world. In contrast with previous hypotheses, the ATP matchmaking hypothesis provides a new logical framework, focusing on energetic force, coevolution of the genetic code with biochemical systems, and biological innovation from energy transformation to informatization. This hypothesis offers a more synthetic and mechanistic explanation than the existing dogmas that reflect separate, discrete aspects of the story. Moreover, it sheds light on the possible origins of cell division and biochemical systems, which have remained largely unknown thus far. As these are closely linked problems, understanding one requires understanding the other. Therefore, efforts to integrate multidisciplinary evidence and approaches are required to fully investigate how these biotic innovations were synergistically accomplished, with focus on energy transformation, so that we can reconstruct sequential, stepwise hierarchies of the genetic code. The present theory, by discovering the missing “matchmaker,” can yield a very plausible answer to the “DNA-Protein Paradox” that currently presents an impassable barrier to the naturalistic origin of life.


Background: The Lethrinidae (emperors) include many important food fish species. Accurate determination of species and stocks is important for fisheries management. The taxonomy of the genusLethrinus is problematic, for example with regards to the identification of the thumbprint emperorLethrinus harak. Little research has been done onL. harak diversity in the Pacific and Indian Oceans. This study aimed to evaluate the morphometric and genetic characters of the thumbprint emperor,L. harak  in the Pacific and Indian Oceans.Methods: This research was conducted in the Marine Biology Laboratory, Faculty of Marine Science and Fisheries, Hasanuddin University, and Division of Fisheries Science, University of Miyazaki. Morphometric character measurements were based on holotype character data, while genetic analysis was performed on cytochrome oxidase subunit I (COI) sequence data. Morphometric data were analysed using principal component analysis (PCA) statistical tests in MINITAB, and genetic data were analysed in MEGA 6.Results: Statistical test results based on morphometric characters revealed groupings largely representative of the Indian and Pacific Oceans. The Seychelles was separated from other Indian Ocean sites and Australian populations were closer to the Pacific than the Indian Ocean group. The genetic distance between the groups was in the low category (0.000 - 0.042). The phylogenetic topology reconstruction accorded well with the morphometric character analysis, with two mainL. harak clades representing Indian and Pacific Ocean, and Australia in the Pacific Ocean clade.Conclusions: These results indicate that the morphological character size ofL. harak from Makassar and the holotype from Saudi Arabia have changed. Genetic distance and phylogeny reconstruction are closely related to low genetic distance. One important fisheries target species, the thumbprint emperor,Lethrinus harak , is widely distributed throughout the Indo-West Pacific from the Red Sea and East Africa to Samoa, and from north Japan to northeastern Australia. This fish tends to be found living solitary or in small schools over shallow sandy areas and coral rubble, among mangroves, in lagoons and channels, and in seagrass areas inshore and adjacent to coral reefs, at depths ranging from 1m to 20m.The Lethrinidae, and in particular the genus Lethrinus, comprise many fish species of importance in commercial and subsistence fisheries around the worldp. Research on the life history, biology and genetics ofL. harak has been conducted on populations in both the Indian and Pacific Oceans. Like the majority of Lethrinid species.L. harak is a relatively long-lived fish; however, although studies on several Pacific Ocean populations have provided information on the reproductive biology ofL. harak in Japan, the Philippines and Indonesia, many aspects of the reproductive biology of this species are still unknown. Genetic studies onL. harak have reported the possible presence of cryptic species in the southwest Indian Ocean (SWIO). Genetic information indicates the possible presence of two clusters, in the Indian Ocean and the Pacific Ocean. However, to date there are no studies evaluating morphological and genetic characters ofL. harak across the Indian and Pacific Oceans.Sustainable fisheries management should be based on an understanding of fish biology and ecology, including the accurate determination of species and stocks. Species identification based on morphometric and meristic characters should be supported by molecular identification methods, as identification based solely on morphometric-meristic characters can often lead to misidentification, taxonomic ambiguity, and fluctuations in the number of recognised species. Such studies have found strong indications of separated populations with sufficient differences to warrant the designation of new species or, conversely, to indicate that currently recognised species are in fact synonyms. These characters can be related to food habits and homoplasy , as some traits can be lost or emerge depending on the environment. Such phenomena are enabled by phenotypic and genotypic plasticity and can lead to the presence of cryptic diversity) and hidden species, as well as considerable variations in colour patterns and other features between different life-stages of the same species.Combined analyses of morphometric characters and genetic variation are increasingly common. Such methods aim to evaluate the differences and similarities in the grouping of individuals and populations using measures such as similarity indices, genetic distances and cluster analysisL. harak based on morphometric and genetic characters is needed to evaluate the stocks of this fish. The genetic structure of the population can be studied based on mitochondrial DNA (mtDNA). Although mitochondria undergo rapid evolution, the cytochrome oxidase subunit I (COI) is a segment of mtDNA with low evolution (conserved region) and has been chosen as a widely used genetic marker in so-called COI barcoding. This study aimed to evaluate the relationship betweenL. harak in the Pacific Ocean and the Indian Ocean based on morphological characters and genetic diversity using a standard COI mtDNA genetic marker.Information on the population structure ofLethrinus harak in the Least Concern category (not considered at risk of extinction); this study was based on field research (surveys) with no experimental component.This research was conducted for three months  under the Enhancing International Publication Program conducted by the Directorate for Human Resource Qualifications Directorate General of Resources for Science, Technology & Higher Education Ministry of Research, Technology and Higher Education of the Republic of Indonesia in collaboration with the University of Miyazaki. This study complied with relevant ethical regulations in Japan and Indonesia. In Indonesia, the doctoral research proposal of the first author was assessed and accepted by an academic panel. This assessment included compliance with institutional ethical guidelines in vigour at Universitas Hasanuddin. A letter of acceptance was issued by the Head of the Laboratory of Fisheries Sciences, Faculty of Agriculture, Miyazaki University, permitting the research activities at the University of Miyazaki's Division of Fisheries Science Museum (MUFS). The issuance of this acceptance was based on the research proposal, including compliance with institutional ethical guidelines in vigour at Miyazaki University. The use of the samples in this study did not require specific ethical approval for the following reasons: all fish specimens used were already dead when they were acquired; there was no use of live animal specimens; specimens obtained from fish port auction houses had been captured by legal fisheries and were obtained following all applicable regulations; the IUCN Red List assessment listsL. harak were selected as paratypes at the Marine Biology Laboratory, Faculty of Marine Science and Fisheries, Universitas Hasanuddin (MSFUH), and the Division of Fisheries Science, Miyazaki University. A total of three specimens for morphometric measurements namely MSFUH000591, MSFUH0000592, MSFUH0000593, and only one specimen is taken for DNA barcoding (MSFUH000591), collected from the Makassar Strait , were obtained from the Paotere fish auction site, Makassar City, South Sulawesi, Indonesia. The samples were placed in a coolbox with ice for transport to the Marine Biology Laboratory, Universitas Hasanuddin, Makassar. The samples were cleaned under running water to remove any dirt and organic matter attached the exterior of the specimens. Each sample was placed on its right-hand side and sterilised using alcohol. A fin-clipping of around 1.8cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. Each sample was numbered and labelled according to the Marine Science and Fisheries Universitas Hasanuddin (MSFUH) catalogue number system, preserved in 10% formalin and stored in the laboratory collection. Specimens ofThe seven specimens measured in Japan formed part of the pre-existing fish collection of the University of Miyazaki Division of Fisheries Science Museum (MUFS). These specimens had been collected from Yonashiro, Uruma, and Okinawa, Japan, with catalogue and sample numbers based on the cataloguing system of Miyazaki University Department of Fisheries Science (MUFS): MUFS 43290, MUFS 4417, MUFS 6025, MUFS 8819, MUFS 12632, MUFS 2098, and MUFS 6014. One specimen had been collected from Meitsu, Nago, Miyazaki, Japan (MUFS 16284) and one from the Philippines (MUFS 6136) .Global Biodiversity Information Facility (GBIF) andThe Barcode of Life Data System (BOLD): KS66045, HQ561476, MH331781, dan JN311937. Measurements of these remotely sourced specimens were made using ImageJ version 1.52a software, except for the interorbital width character, which could not be measured from the downloaded photographs. The morphological characters were compared with reference to data from three holotypes: MNHN 9087, holotype ofL. azureus; RMNH 5758, holotype ofL. rhodopterus; and BMNH 1873.3.160, holotype ofL. bonhamensis. Measurement of morphometric characters using conventional methods by following the holotype characterL. harak included standard length, body depth, head length, pectoral length, pelvic length, orbital length, interorbital width, snout length, suborbital width, upper jaw length.The preceding samples were used in this study to represent the Pacific Ocean. The samples used to represent the Indian Ocean were downloaded from theDNA extraction specimen Makassar was carried out using DNeasy Blood and Tissue Kit  following the manufacturer’s protocols. Fin clippings (1.8cm) were taken from the pectoral fin of each specimen using surgical scissors or a surgical scalpel. Each sample was preserved in a labelled 2ml tube filled with 95% alcohol.2O.A sub-sample weighing 0.025mg was taken from each fin clipping sample and placed in a 2mL tube to which 180µL of ATL solution (tissue lysis buffer) and 20µL of proteinase K were added. The tubes were vortexed and incubated at 56°C for 1–3 hours or overnight. Then 200µL AL buffer (lysis buffer) solution and 200µL ethanol were added before vortexing and centrifuging at 8000rpm for 1 minute. The supernatant was transferred to a new tube, 500 µL AW1 (column wash buffer 1) was added, and the tube was centrifuged for 1 minute at 8000rpm. This step was repeated with AW2 (column wash buffer 2) and centrifuged at 14,000rpm for 1 minute. The column was transferred to a new 1.5mL tube and 100μL warm AE buffer was added, then centrifuged for 1 minute, incubated for 10–15 minutes, and centrifuged at 6000×g (8000rpm) for 1 minute. RNAse was added to the extracted DNA solution and incubated for 24 hours. The extracted DNA was prepared for use through dilution, at a ratio of 30µL DNA and 70µL ddHAgarose gel was prepared by mixing 1.4g of agarose 0.8% in 180mL of 1x TAE buffer solution. A colouring agent (1.5μl GelRed) was added to the gel while still warm and liquid before pouring it into the mould. Aliquots of 2μL DNA template were pipetted and mixed with 1μl of DNA loading dye solution and loaded into the wells in the agarose gel. Electrophoresis was performed at 100 volts for 90 minutes. The results were visualised using an ultraviolet transilluminator .et al.: Fish F1-5’ TCA ACC AAC CAC AAA GCA TTG GCAC 3 (forward) and Fish R1-5’ TAG ACT TCT GGG TGG CCA AGA ATCA 3 (reverse). Each reaction tube contained 5µL of PCR mix Hotstart , 3µL DNA template, 3µL ddH2O, and 0.675µL each of the forward and reverse primers. The PCR amplification procedure  comprised the following stages: initial denaturation at 95°C for 5 minutes; 35 cycles with denaturation at 94°C for 1 minute, annealing at a primer-specific temperature for 1 minute, elongation at 72°C for 1 minute; and final elongation at 72°C for 10 minutes. The amplified PCR products were stored in a freezer (-20°C) until the separation process. The annealing temperature followed a gradient of ±5°C compared to the reference temperature for the primer pair, in order to determine the optimal annealing temperature, resulting in a bright and sharp band under electrophoresis. PCR products that have been tested positive for a length of 682 bp, then proceed with the sequencing of nucleotides to obtain DNA sequences. The sequencing was carried out at the company 1st Base Asia, Singapore using BigDye® Terminator ver.3.1 cycle sequencing KIT and an automated HiSeq-X sequencing machine. The primers used for the sequencing process are the same as the primers used for the PCR amplification process.The DNA barcode analysis used a segment of mtDNA from the cytochrome b oxidase subunit 1 (COI) gene. The target sequences were amplified using the primer pair developed by Ward. The quality of the DNA sequences obtained was evaluated using the software Sequence Scanner 2.0 . The sequence data obtained from this study were complemented by additional sequences downloaded from the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDJB) databases . The forward and reverse sequence data were combined using the software Bioedit version 7.2.6.1. The sequences were trimmed and aligned using the ClustalX routine in MEGA 6. The phylogenetic topology reconstruction was implemented in MEGA 6 with the neighbour-joining method and genetic distance was analysed using the compute within groups mean distance option.Principal component analysis (PCA) was used to analyse the morphometric data, and was implemented in MINITAB version 17JASP could be used as an alternative, although we could not guarantee total equivalence between the two software packages. Genetic analyses in this study were implemented using Sequence Scanner 2.0, ClustalX 2.1 and MEGA 6.Although PCA in this research was implemented in MINITAB, the free softwareL. harak specimens , Indonesia (Makassar and Ambon), the Philippines, and Australia, are predominantly grouped in the upper quadrants of the PCA graph .L. harak.Populations from the Indian Ocean were mostly spread across the lower left quadrant, i.e. South Africa, Mozambique, and Saudi Arabia, with the Seychelles being the only population placed (very low) in the bottom right quadrant. However, the specimens from Tanzania and Madagascar grouped together with the Pacific Ocean populations in the upper right quadrant and had morphometric characters similar to the holotype ofL. harak populations in the Pacific Ocean and the Indian Ocean have a close kinship relationship , Japan, and Australia with a bootstrap value of 99%.The topology of this neighbour-joining phylogenetic reconstruction shows two that morphometric data can be extremely useful for the study of variation in form due to geographical differences. In addition, morphometric data are frequently used in taxonomy and fish description. In taxonomy, data on the holotype of a species are vital as a basis for comparison when observing other specimens. Data on the holotype can also be used to evaluate whether there are any changes in the morphometric characters of a given specimen. Such methods form the basis for establishing new fish species.This confirms the view expressed by Baur and LeuenbergerL. harak between populations experiencing similar environmental conditions. The variation of the morphological characters ofL. harak is largely determined by the level of destructive fishing and high fishing pressure. These changes can cause emperor fish spine abnormalities Conversely, environmental pressures will tend to drive adaptation through physiological and behavioural modifications. A species with the capacity to adapt through plasticity will produce a variety of phenotypes as well as genotypes in response to a given set of environmental conditions, in order to increase fitness, the ability of individuals to survive and reproduce. Matthews found adaptations in body shape, colour and fins based on the environmental conditions in which fish live, as well as adaptations in head shape associated with feeding processes.Their proximity indicates similarities in morphometric characters of. A relatively high level of geographical isolation coupled with limited geographical extent can result in significant differences in morphometric and genetic characters between stocks or populations of the same species due to a lack of gene-flow between populations. This is supported by the identification of cryptic lethrinid species with similar morphological characters by Healey, e.g.L. nebulosus andL. mahsena in the SWIO. This study found two clades, with the Seychelles separated from the other populations in the SWIO. The emergence of this phenomenon in the Seychelles could be related to high fishing pressure and habitat degradation. Wilson & Clarke report that increasing exploitation and environmental pressures can cause a decline in fish stock abundance, as well as reductions in the average size, and detrimental genetic selection which can reduce potential fecundity and mean spawning size, alter sex ratios, and interspecific balances, as well as reducing genetic diversity.Genetic effects and environmental parameters can both result in differences in growth and development processes, which will produce variations in fish body shapeet al. state that the lower the genetic distance between individuals, the closer their kinship and vice versa. According to Nei, genetic distance values can be categorised as low (0.010 - 0.099), moderate (0.1-0.99), or high (1.00-2.00). Such phylogenetic analyses can be used to construct detailed relationships between species and estimate the divergence between members of a family descended from a common ancestor.The genetic distance can indicate the level of kinship between populations. KohL. harak populations in the Pacific and Indian Oceans. Williamset al. found a genetic break between the Pacific and Indian Oceans in several invertebrate taxa. The topology of their phylogenetic reconstruction grouped northwestern Australia with the Indian Ocean for most taxa, although some taxa showed an affinity with Pacific Ocean populations, which they considered may be due to the oceanographic conditions around nine million years ago. Meanwhile, as in our study, eastern Australia populations were more closely related to Pacific Ocean populations.This pattern indicates a divergence in genetic characteristics between. As a protogynous hermaphrodite,L. harak undergoes a process of gonad differentiation associated with sex change from the female to terminal male phase. As a fish with a pelagic spawning reproductive strategy,L. harak releases pelagic eggs which drift with other plankton. The density ofL. harak eggs is similar to or slightly less than that of sea water, so that they float at or close to the sea surface. After hatching the larvae remain planktonic for a period of time, which can vary from a few hours to several months. It is therefore likely that surface currents significantly affect the distribution and genetic population structure ofL. harak.The characteristics of the water masses and oceanographic currents in the two oceans influence the distribution and population connectivity of marine organisms, and may affect their physiology and exert differential selection pressures, thus potentially influencing genetic characteristics in several ways. Surface currents are a key factor in larval dispersal. In the Pacific Ocean there are at least four currents affecting the distribution of marine organisms: the New Guinea Coastal Current (NGCC), the Halmahera Eddy (HE), the North Equatorial Coastal Current (NECC) and the North Equatorial Current (NEC). The main source of the water masses underpinning current circulation in the LLWBCs system are the South Equatorial Current (SEC) and the North Equatorial Current (NEC). A proportion of the water masses travelling westwards in the equatorial currents join the Kuroshio current flowing northward towards Japan, while some flows south in the Mindanao current or flows south to join the East Australia Current (EAC). A branch of the Mindanao current enters the Sulawesi Sea and flows through the Makassar Strait, while another branch flows east along the north coast of Sulawesi to the Halmahera Sea. These complex currents are further complicated by seasonal variations and local geography, and could enable gene-flow between several of the sites in our study.The Pacific Low Latitude Western Boundary Currents (LLWBCs) are one of the factors that can influence genetic structure in the Indian OceanL. harak from the Pacific and Indian Oceans was relatively small, unlike the large differences found by Carvalho between the Pacific and Atlantic populations ofHaemulon steindachneri. These differences were considered sufficiently large to indicate that the two putative populations ofH. steindachneri were in fact two different species, thus indicating speciation after the closing of the Isthmus of Panama. In contrast, despite the reduced connectivity between the Indian and Pacific Oceans described by Williamset al., their data indicate some continuing gene-flow between the Pacific and Indian Oceans, although oceanographic flow patterns indicate that gene-flow is more likely from the Pacific to the Indian Ocean than the reverse. Our results are consonant with such a continued  gene-flow.Despite the clear demarcation of the two clades found in our study, the genetic distances betweenLethrinus harak populations in the Pacific and Indian Oceans appear to form two major clades based on morphometric and genetic characteristics. One clade includes populations from South Africa, Mozambique, Madagascar and Saudi Arabia, while the other includes populations in Japan , Indonesia  and the Philippines. Two populations exhibit intermediate  or unique (Seychelles) morphometric characters; however, based on the genetic reconstruction topology, the Australian population grouped with the PacificL. harak clade.TheL. harak (specimen voucher MSFUH0000591) from Makassar waters on GenBank, Accession numberMT551656.Partial cytochrome C oxidase subunit I (COX1) gene mitochondrial DNA sequence of AbstractL. harak into the Pacific and Indian clusters should be emphasised clearly. The present conclusion is not representative of the major finding. The main finding of highly structured populations ofMethodJust state the software used in this study (Page 5). There is no need to include those not relevant to this study. DNA extraction protocol from museum-preserved sample is challenging and should be described . Specimens for morphometric analysis are not well defined. Most are described on Page 4 but others are under Figure 1. And apparently based on Table 1, those obtained from public databases (Page 5) for molecular analysis were also analysed for the morphometric analysis. This is rather puzzling? Did the databases provide photos (clarified ones) for measurements?   Results: Based on Figure 3, sample sizes are very low, not appropriate for DNA barcoding. At best it provides some insight into phylogeographic pattern based on COI gene which is a DNA barcoding gene, but not utilised in this context.DiscussionThe values for genetic distance should be converted to a percentage (%) which makes it 0 - 4 %. The maximum is indication of a different taxa (See Ward 2005 and other related articles). Thus, it is not correct to see all values are low (Page 9). On the whole the data is a good preliminary contribution in this field of study, region and organism investigated and is publishable after addressing these comments. Authors need to relate their findings with the factors they describe. While these factors may be relevant, there is no attempt to incorporate the observations for example Pacific and Indian Ocean are isolated, attributed to a genetic barrier.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Population  genetics and molecular ecologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Lethrinus harak  in the Pacific and Indian Oceans"Reports on "Morphological and genetic evaluation of the thumbprint emperor, Authors should include CVA or DFA, Thin plate Spline, Wireframes or Relative Warps in the method section for better interpretation of the results on morphological and shape analysis. DFA will provide more information about the discriminating variables between populations.After including analysis especially related to DFA and by including at least one visualization graph (Thin Plate or Wireframe), results of morphological study can be interpreted in a better way. Morphological results need more support by incorporating suggested analysis to reach adequate conclusion. After including the above suggestions, I believe the manuscript will be suitable for indexing. The study is important in understanding population structure of this important fish species at morphological and genetic level. However, I believe that authors should add more analytical aspects on morphological studies:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?No source data requiredIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?NoReviewer Expertise:Fish Morphometrics at various levels I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Lethrinus harak  in the Pacific and Indian Oceans" has been reviewed.  For this Version 2, there is no major or minor revision needed in this article. This is appropriate, and I approve this research article.This research article with tittle "Morphological and genetic evaluation of the thumbprint emperor, Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Goby fishI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. L. harak in the Pacific and Indian oceans based on morphometric and genetic aspects.The conclusion in abstracts is still too general. The research novelty needs to be explicitly highlighted, particularly the differences ofThe word “alcohol” was mentioned twice in the following sentences: Each sample was placed on its right-hand side and sterilised using alcohol.  A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. Please consider replacing “alcohol” with “ethanol” to avoid misinterpretation.The method described as: A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. This sentence needs to elaborate in detail. Please connect it with the different-samples explanation in the DNA extraction section.It is not clear how many samples were taken in Makassar for morphometric and molecular analysis purposes. Samples with the code MSFUH000591, MSFUH0000592, MSFUH0000593 were not specified what analysis was applied. Only the sample coded MSFUH000591 was mentioned in the results section. Likewise, in Table 1, there were stated only 3 Makassar samples with the code MSFUH000591, MSFUH915, and MSFHU917. Where were the other samples (coded MSFUH0000592 and MSFUH0000593) descriptions?In the method mentioned: One specimen had been collected from Meitsu, Nago, Miyazaki, Japan (MUFS 16284) and one from the Philippines (MUFS 6136) . However, these two samples were not shown in Figure 1. What is the purpose of writing “” at the end of the sentence?What do “preceding samples” mean in the following sentence “The preceding samples were used in this study to represent the Pacific Ocean”Two sentences seem somewhat ambiguous in measuring morphology, as follows: Measurements of these remotely sourced specimens were made using ImageJ version 1.52a software14, except for the interorbital width character, which could not be measured from the downloaded photographs. Then in the following sentence, it was stated that: The characters measured using callipers (accuracy 0.02 mm) included standard length, body depth, head length, pectoral length, pelvic length, orbital length, interorbital width, snout length, suborbital width, upper jaw length. It might be necessary to clarify which samples were measured using ImageJ and measured manually using a caliper.The second sentence's meaning in the DNA extraction sub-section is not clear: Fin clippings (2–3cm) were taken from the pectoral fin of each specimen using surgical scissors or a surgical scalpel. Each sample was preserved in a labelled 2ml tube filled with 95% ethanol. Are the specimen referred to samples from Makassar or some other areas? As in the 6th sentence in the Fish Specimen sub-section stated as: A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol.If possible, replace the subsection “Statistical Analysis” with “Data Analysis” since there were also explaining genetic analysis.In the second paragraph in the sub-section Morphological Characters, it mentioned “Indonesia ”, while in Figure 1 it was stated that: Specimens of Lethrinus harak. A. Specimen JN311937, paratype, SL 170mm, collected from Arumbai Fish Market , Ambon, Indonesia, 06 January 2016. What mentioned here was Ambon, not Maluku. The words Maluku and Ambon need to be consistent as even though Ambon is in Maluku, this will lead to confusion. Also, Figure 2 mentioned only Makassar and Indonesia, while Maluku was not mentioned. In Table 1 and Table 2, the word Maluku was not mentioned either, but in conclusion, it was written in Indonesia . Please make it consistent whether Indonesia  or Indonesia (Ambon).It needs consistency in writing. It should be written in capital letters at the beginning of each word in the stands: The Pacific low latitude western boundary currents (LLWBCs).The writing of citations is still inconsistent. Some are written with the author's name along with the serial number in the bibliography, and some are written directly without writing the author's name.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Goby fishI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for being our manuscript reviewer. We will revise your suggestions for writing our manuscript. No justification is being given for using alternative software used for genetic analysis – without guaranteeing total equivalence, two different softwares are used [page 5] Though sample details are furnished, it hasn’t been clearly mentioned anywhere as to exact number of samples subjected to DNA barcoding. However, from the report the number seems to be insufficient to interpret the results. If DNA were extracted from museum-preserved samples, the methodology used should have been specifically mentioned. The protocol is certainly different from that used for fresh/frozen samples. In the methodology it’s mentioned “Amplified PCR products with a length (in bp) consonant with the target gene segment were sent to a sequencing company  in order to obtain DNA sequence data.” Though we accept that in majority of studies DNA sequencing is outsourced, such kind of statement as given here would dilute the scientific seriousness and originality. The authors haven’t even briefly described the actual methodology of DNA sequencing as intended and executed.  In discussion the authors have described endlessly on influence of water currents and other environmental parameters in stock distribution and separation. However, the present study has no such data to substantiate those concepts. This has resulted in just copying others’ observations and presenting the same, without having any study attempted in those lines by the authors.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?NoReviewer Expertise:Aquaculture, fish genetics, molecular taxonomyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Psathyrostachys juncea is a perennial forage grass which plays an important role in soil and water conservation and ecological maintenance in cold and dry areas of temperate regions. In P. juncea, a variety of biotic and abiotic stress related genes have been used in crop improvement, indicating its agronomic, economic, forage, and breeding value. To date, there have been few studies on the genetic structure of P. juncea. Here, the genetic diversity and population structure of P. juncea were analyzed by EST-SSR molecular markers to evaluate the genetic differentiation related to tillering traits in P. juncea germplasm resources. The results showed that 400 simple sequence repeat (SSR) loci were detected in 2,020 differentially expressed tillering related genes. A total of 344 scored bands were amplified using 103 primer pairs, out of which 308 (89.53%) were polymorphic. The Nei’s gene diversity of 480 individuals was between 0.092 and 0.449, and the genetic similarity coefficient was between 0.5008 and 0.9111, with an average of 0.6618. Analysis of molecular variance analysis showed that 93% of the variance was due to differences within the population, and the remaining 7% was due to differences among populations. Psathyrostachys juncea materials were clustered into five groups based on population genetic structure, principal coordinate analysis and unweighted pair-group method with arithmetic means (UPGMA) analysis. The results were similar between clustering methods, but a few individual plants were distributed differently by the three models. The clustering results, gene diversity and genetic similarity coefficients showed that the overall genetic relationship of P. juncea individuals was relatively close. A Mantel test, UPGMA and structural analysis also showed a significant correlation between genetic relationship and geographical distribution. These results provide references for future breeding programs, genetic improvement and core germplasm collection of P. juncea. Psathyrostachys Nevski is a genus of Gramineae grasses isolated from Hordeum vulgare L. by former Soviet botanists and contains about ten species. They are native to central and northern Asia’s inland areas and their distribution has spread to East Asia, Europe, and North America due to introduction and domestication  in the Gramineae family, and it is a cross-pollinated, perennial bunch grass , perennial ryegrass (Lolium perenne L.), and alfalfa (Medicago sativa)  . In the arieties .P. juncea hinders related breeding work. Previous study of P. juncea showed that genetic variation between individuals was much greater than the variation between populations , and two accessions by the China National Medium-term Gene Bank for Forage Germplasm. The variety 549118 ‘Mengnong No. 4’ was a variety selected from the cultivar ‘Bozoisky’ which was introduced from USDA-ARS-FRRL in 1984, and ‘Mengnong No. 4’ was registered by Inner Mongolia Agricultural University in 2009. The seeds of test materials were germinated in a greenhouse in January 2018 and transplanted to the field to establish the test plot in July 2018. The information of all accessions including eco-geographical conditions are shown in A total of 21 P. juncea accessions, ranged from 14 to 30 of each. The leaves were wiped with alcohol and deionized water and 1–2 fresh leaves per plant were cut into a 5 mL cryopreservation tube, and brought to the laboratory and stored at −80°C. DNA was extracted using a plant genomic DNA extraction kit . The quality and quantity of DNA was evaluated on 1% agarose gel with a NanoDropTM2000 Spectrophotometer . After all the measurements, the DNA stock solution was diluted to 50 ng/μL for SSR marker analysis and stored at −20°C.A total of 480 individual plants were selected from 21 In order to establish EST library under the maximum genetic background, 17 accessions with significant difference in tiller number trait between individuals were selected from total 21 accessions. A group of thirty plants with dense tillers and another group of thirty plants with sparse tillers were selected from same 17 accessions . Briefly, the poly (A) mRNA was enriched via magnetic oligo (dT) beads and then broken into short fragments using an RNA Fragmentation Kit . These cleaved mRNA fragments were used as templates for first strand cDNA synthesis using random hexamer primers. Second strand cDNA was synthesized with RNase H and DNA polymerase I. The library fragments were purified with an AMPure XP system . These short fragments were ligated to sequencing adapters, and the desired fragments were separated using AMPure XP beads. Next, the purified cDNA fragments were enriched via PCR. Finally, the cDNA libraries were sequenced using Illumina HiSeq™ 2000 and paired-end reads were generated. After obtaining high-quality sequencing data, the data was assembled to obtain unigenes.1 to identify Simple Sequence Repeats. The primer pair for each SSR was designed according to differentially expressed genes and using Primer 32 with the following criteria: primer lengths of 16–23 bases (optimum 20 bp), GC content of 40–70%, annealing temperature of 50–60°C, and PCR product size of 100–500 bp. The primers were synthesized by Sangon Biotech .According to the minimum length standard , unigenes over 1 Kb were analyzed using MISAAccording to the relative expression level between the two samples, the differentially expressed genes were screened. A total of 2,020 significant differential gene expressions (DEGs) were identified, and 400 significant DEGs with SSR loci were screened. One SSR locus could synthesize three pairs of primers, and one pair of primers was selected for each locus. After removing 75 unigenes that were not suitable for designing primers, 325 pairs of primers were synthesized by Sangon Biotech . Eight individual plants from different populations were selected randomly, and used to test the amplification effect and polymorphism of 325 pairs of primers. A total of 103 primer pairs with polymorphism were selected and used in population genetic diversity and structure analysis .2, 500 μM dNTPs, 0.1 U Taq DNA polymerase, 100 mM KCl, 20 mM Tris–HCl, Tiangen Biotech, Co., Ltd, Beijing, China), 1 μL of each forward and reverse primer, 50 ng of genomic DNA and 4.5 μL of ddH2O. PCR amplification was performed using an Applied Biosystems . The PCR conditions were as follows: pre-denaturation at 94°C for 4 min followed by 33 cycles of 94°C for 30 s, 51.5–58.5°C (depending on the Tm of the primer set used) for 30 s, and 72°C for 45 s, and finally extension at 72°C for 10 min. The fragment size of PCR amplified product was detected using a QIAxcel Advanced capillary electrophoresis instrument. The 100 bp to 2.5 kb DNA Size Marker and 15 bp to 3 kb Alignment Marker were selected as standard size markers .A total of 103 selected SSR primer pairs were used in 480 individual plant DNA PCR reactions. The PCR reaction system used a 15 μL system for amplification, and reactions containing of 7.5 μL of 2 × Taq PCR MasterMix  patterns exist  and the percentage of polymorphic bands (PPB) were calculated using Excel 2013. The GeneAlEx 6.503  softwarens exist .The population genetic structure was analyzed using STRUCTURE 2.3.4  softwarede novo assembled into 100,560 unigenes by the assembling program Trinity, and the total length of the unigenes was 91,562,881 bp, with an average length of 910 bp and the N50 of unigenes was 1,739 bp. Among all the unigenes, those with length ranging from 200 to 1,000 bp accounted for 70.28%, and 29.72% of the unigenes were more than 1,000 bp  unigenes above 1 kb were identified as containing SSRs. EST-SSRs were of perfect repeat types, ranging from mononucleotide to hexanucleotide and among them, mononucleotide and trinucleotide repeats accounted for the largest proportion, at 40.30 and 36.11%, respectively and 400 EST-SSRs with compound repeats accounted for 4.3% of all sequences .In the PCR reaction, a total of 344 loci were amplified using 103 SSR primer pairs among 480 individuals, of which 308 (89.53%) exhibited polymorphic patterns. The number of polymorphic loci for each primer combination varied from 1 to 6, with an average of 2.990 loci . The amp565052 from Russia and accession 598610 from Kazakhstan was the largest (0.9050), while that of accession 502576 from Russia and accession 531828 from the United States was the smallest (0.5008).Using the amplified markers of 480 individuals as the original data, genetic similarity coefficients were obtained . The genP. juncea material indicated that the genetic similarity coefficient between the accession 598610 individual plants from Kazakhstan had the greatest variation, which ranged from 0.5145 to 0.9111, indicating that this accession had great genetic differences and rich genetic diversity, while those plants of accession 531828 from the United States had the smallest variation, indicating that the genetic variation among individual plants of this accession had relatively small genetic differences and narrow genetic background . Of the total accessions, 93% of the variance was due to differences within the accessions in the groups and the remaining 7% was due to differences among the groups. Of the individuals, 83% of the total genetic variance was due to differences within populations, while 5% was due to differences among populations and only 12% was ascribed to differences among individuals within the populations.Analysis of molecular variance (AMOVA) was implemented to evaluate variance components among groups and individuals , which sP. juncea, PCoA and cluster analysis between the populations were carried out, based on the unweighted pair-group method with arithmetic means (UPGMA) on the regional populations . Therefore, the data showed that there was evidence of significant isolation by distance  and the ordinate as ln P(K). The ln P(K) increased constantly with increasing K and there was no maximum value, so the best number of subpopulations could not be determined  > group 3 (0.4587) > group 2 (0.4442) > group 1 (0.4225) > group 5 (0.3370) ( complex . In grou(0.3370) , which s(0.3370) .P. juncea individual materials was basically in line with the results of population genetic structure analysis. Based on the UPGMA dendrogram, some individual plants from Russia were clustered with individual plants from Mongolia and Canada, while in the population structure analysis, plant materials from Russia were classified separately into group 2. The materials from Kazakhstan, Estonia, and the Former Soviet Union were clustered into one group in the UPGMA dendrogram, indicating that the genetic distance between materials from these three regions was small. In the cluster diagram, the materials from Russia were clustered into one group, and the materials from China and Mongolia were clustered into the same group. The classification of materials from Canada and the United States was extremely complex and they were clustered into different branches.The UPGMA dendrogram also showed that the accessions could be classified into five clusters . The cluP. juncea were analyzed based on the first and second principal components  ranged from 0.173 to 0.640, with an average of 0.420 in our study, which was higher than that estimated by ISSRs for the related P. huashanica (I = 0.391). This finding indicates that the selected primers can objectively reveal the genetic diversity of P. juncea germplasm resources. Overall, the 103 EST-SSR primer pairs can provide an adequate analysis of the genetic differences among P. juncea materials.The polymorphism rate among the genotypes under exploratory conditions is considered a key factor in measurement of the diversity analysis efficiency of DNA markers . A largeP. juncea in samples 1 and 2 group, the variation coefficient of nutritional tiller number was the most obvious in both samples  was greater than that of wild materials (0.658), indicating that the genetic relationship between cultivated materials was closer and that of wild materials was more distant. The same detection was reported in the analysis of genetic similarity coefficient of wild materials and cultivated varieties of alfalfa  is an index of the degree of similarity between individuals. When the genetic similarity coefficient is greater, the genetic relationship between materials is closer. The GS of 480 individual plants of  alfalfa . The genmaterial . That fiP. juncea accessions, accessions were clustered into eight subgroups for genetic structure analysis. The Shannon information index of each subpopulation ranged from 0.2615 to 0.4142, indicating a high genetic diversity level. The high genetic diversity in the P. juncea population may be related to the anemophily pollination of the species. Genetic variation within the populations (83–93%) was higher than that among populations (5–7%) in the present study. This result is consistent with the findings of previous studies of P. juncea based on AFLP analysis (77% within populations) (Populus deltoides) based on SSR   , Black cviduals) , white cviduals)  and whitviduals) . This isviduals) . Our resP. juncea gene flow (Nm) between Canada and other countries was small, and the Nm between other countries was high. Nm < 1 indicates a high level of genetic differentiation among plant populations, while Nm > 1 indicates a low level of genetic differentiation among plant populations, and P. juncea has undergone huge genetic variation and frequent exchange of genetic material with the materials of other populations. Therefore, germplasm resources with rich polymorphism and high genetic variation have been formed at the level of genetics and breeding. Germplasm collections and the introduction history of different countries may also affect gene flow and shape the new genetic structure of subpopulations.Classically, a high degree of gene flow, which could neutralize interspecific differentiation and intraspecific genetic drift, is extremely common in cross-pollinated plants, thereby contributing to low genetic diversity among individuals or populations . In our P. juncea germplasm from Eurasia to North America.The genetic structure of species is affected by the interaction of multiple factors, such as the transmission model of seeds and pollen, population demographic history, geological events, geographical or ecological barriers and divergent selection for environmental factors . Based oP. juncea materials contains hereditary substances of different individual plants, and the genetic information of all individuals was integrated by the population structure. UPGMA clustering was carried out based on the GD of P. juncea materials to cluster the closely related materials into one group, which may lead to the aggregation of materials with different genetic structure. In this study, according to the results of population genetic structure analysis, the materials with Q greater than 0.5 were assembled to five groups and the individual plants from the same region were also be clustered into different groups. In the UPGMA cluster diagram, some individual plants from Russia were clustered with individual plants from Mongolia and Canada, but in the population structure analysis Russian materials were clustered independently into group 2. The clustering may be the result of breeding and domestication, which has a great impact on the diversity structure. Selection and breeding tend to keep plants with economically valuable traits such as tillering ability. In addition, different environments can also cause genetic changes and thus affect the division of population structure. It may also be that the DNA of P. juncea individual materials from different countries and regions, it was found that the population structure of 480 P. juncea individual plants had a certain correlation with geographical distribution. The Mantel test of genetic distance and geographical distance among materials showed that there was a significant correlation . This showed an IBD model over all sampling locations that was comparable with the results of P. juncea wild germplasms using AFLP markers, and identified that the key factors inducing moderate genetic differentiation were isolation by distance (IBD). Generally, more opportunity for allelic exchange could be obtained by individuals of neighboring populations because gene flow will theoretically be obstructed by a longer geographical distance  can be found below: National Center for Biotechnology Information (NCBI) BioProject database under accession number ZL conducted the whole research and wrote the manuscript. ZG, TW, and XR helped in experiment and data analysis. LY designed the project, collected materials, and revised the manuscript. YZ helped to revised the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Consumer interest in genetic and genomic testing is growing rapidly, with more than 26 million Americans having purchased direct-to-consumer genetic testing services. Capitalizing on the increasing comfort of consumers with genetic testing outside the clinical environment, commercial vendors are expanding their customer base by marketing genetic and genomic testing services, including testing for pharmacogenomic and pathogenic variants, to employers for inclusion in workplace wellness programs. We describe the appeal of voluntary workplace genomic testing (wGT) to employers and employees, how the ethical, legal, and social implications literature has approached the issue of genetic testing in the workplace in the past, and outline the relevant legal landscape. Given that we are in the early stages of development of the wGT market, now is the time to identify the critical interests and concerns of employees and employers, so that governance can develop and evolve along with the wGT market, rather than behind it, and be based on data, rather than speculative hopes and fears. The global consumer genetic testing market is anticipated to exceed $2.5 billion by 20254. Capitalizing on the increasing comfort of consumers with genetic testing outside the clinical environment, commercial vendors are expanding their customer bases by marketing genetic and genomic testing services, including testing for pharmacogenomic and pathogenic variants, to employers for inclusion in workplace wellness programs. For the purposes of this paper, voluntary workplace genomic testing (wGT) refers to both genetic and genomic testing offered by vendors through voluntary workplace wellness programs, as the full range of testing offered through such programs has not been robustly studied to date. Workplace wellness programs commonly include features like health risk assessments, biometric testing and screening, smoking cessation programs, and gym and health-club memberships5. We describe the potential appeal of wGT to employers and employees, how the ethical, legal, and social implications (ELSI) literature has approached the issue of workplace genetic testing in the past, and outline the current relevant legal landscape in the United States (US). Given that we are in the early stages of development of the wGT market, now is the time to identify the critical interests and concerns of employees and employers, so that governance can develop and evolve along with the wGT market, rather than behind it, and be based on data and evidence, rather than speculative hopes and fears.Consumer interest in genetic and genomic testing is growing rapidly, with more than 26 million Americans having purchased direct-to-consumer (DTC) genetic testing services6. Further, many employers are self-insured or self-funded, choosing to directly finance some or all of their employee health services and benefits instead of purchasing health insurance for employees, meaning that the employer is financially responsible for the healthcare claims of their employees. According to the 2020 Employer Health Benefits Survey published by the Kaiser Family Foundation, 67% of employees across all surveyed firms were in a self-funded plan. At firms with 5000 or more employees, 94% of employees were in a self-funded plan7. Furthermore, employer-sponsored health plans cover more than 157 million people in the US8.In the US, employers often take keen interest in the health of their employees because most Americans receive their health insurance through their workplace11. DTC testing, elective return of actionable “secondary” findings  following genomic testing according to American College of Medical Genetics and Genomics (ACMG) guidelines, and expanded efforts to engage family members after a patient’s genetic diagnosis (cascade screening) are increasing the numbers of individuals receiving genetic and genomic test results14. wGT could push these numbers higher, especially among generally younger and healthier populations who may not have cause or opportunity to pursue genetic testing through a clinical care infrastructure. Those who promote wGT suggest that such testing has the potential to lower employer healthcare costs as a result of: (1) employees becoming more informed about disease risks, making lifestyle changes, increasing preventive screenings or pursuing other interventions to reduce disease risks15; and (2) employers using aggregate employee test data to add other benefits that could potentially mitigate disease risks16.Advances in genetic medicine have improved our ability to predict, prevent, and treat certain genetic conditions17; however, it is currently unknown how many companies are actually offering wGT within their workplace wellness programs, what kind of testing is being offered, and whether the programs include services beyond mere testing, such as pre- or post-test genetic counseling. Further, little is known about employers’ motivations to provide wGT or how they access and utilize the resulting aggregate data. What we do know comes largely from press releases and media reports19. For example, executives at some US companies offering wGT have described this offering as “creating goodwill” with employees and being “a differentiator” that sets them apart from competing employers20. However, we have only limited data in the US or elsewhere on employee perspectives and concerns about wGT that would enable us to evaluate such claims21.A number of commercial vendors are marketing wGT services to employers21. It is important to note that the participants in this study were employed at a biomedical research institution that specifically focuses on genomics, and their views may not be representative of the broader population of employees’ views towards wGT. Further research is needed with employees at a range of diverse organizations. Previous research on employee perspectives regarding wGT found that employees have a desire to learn about their genetic information, and want a choice regarding who controls, stores, and has access to their genetic information22. For example, in one survey of university health center employees, respondents expressed strong interest in learning about their own genetic information while also rating privacy and confidentiality protections as very important22. The participants also indicated that including this information in their employee health records and insurance files was unacceptable, as was using their identifiable information in genetic research22.In a recent survey of employees at a biomedical research institution in the US, participants provided their views on wGT after being presented with three hypothetical scenarios for accessing genetic testing: (1) in the participant’s doctor’s office; (2) a workplace setting, and (3) via DTC testing. Of the 594 respondents, 70% of respondents indicated that they would prefer genetic testing in the workplace over a doctor’s office or DTC setting, though the survey did not solicit rationale. In addition, over 60% of respondents wanted to know about relevant laws and policies in place to protect their privacy and confidentiality23. This latter stipulation underscores the privacy concerns employees have about wGT. Regardless of context, it remains the case that many people and policies, including the Genetic Information Nondiscrimination Act (GINA), regard genetic information as a unique category of health information, in that it is intrinsic, predictive, probabilistic, and has relevance for close genetic relatives24. In part due to these characteristics, policies providing consumer protection specific to genetic information enjoy broad support25.Additionally, a 2017 Wamberg Genomic Consumer Survey of 536 US consumers with employer-sponsored health insurance reported that 65% of employees would be interested in genetic testing if their employers offered easy and affordable access with strict privacy and data ownership control, allowing test result access only to the employee and their doctor28. While the terms are sometimes used interchangeably, genetic monitoring includes frequent employee testing to determine the effects of toxic substances on employee health 29, whereas genetic vulnerability screening includes testing an unselected group of employees once to identify workers with genetic susceptibilities that might make them more vulnerable to harm in the workplace 27. Since these very early experiences, concerns have been raised about the ELSI of such testing30, and these potential issues have increased with the expansion of genetic knowledge gained over the intervening decades.While wGT is a relatively new phenomenon, employer use of genetic testing and screening  has been ongoing since at least the 1990s, with an initial focus on genetic monitoring and genetic vulnerability screening to mitigate employee risk to workplace hazards26. Additionally, due to the newfound knowledge of genetic causes for some diseases, ethicists also raised the concern that this information could be used to discriminate against historically marginalized populations , and that a positive screening result could both facilitate discrimination and exacerbate stigma28. Historically, some organizations, including parts of the US military, tested individuals for sickle cell trait (SCT), for example, to restrict access to certain kinds of military service and benefits for individuals found to have the trait. However, due to conflicting evidence on the relationship between SCT and job-related risks, and concerns about genetic and racial discrimination, the testing as originally practiced was discontinued31. Furthermore, potential false positive and false negative screening results (the latter of which were more common in early genetic screening/testing) exacerbated these concerns28.A main ethical concern raised by historical workplace genetic vulnerability screening is that while employers argued that this screening could enable them to safeguard against workplace-related illness, it might also enable them to avoid legal liability for illnesses resulting from workplace hazards that could be attributed to an employee’s genetic predisposition32. Several key legal cases have demonstrated the potential for discrimination and unethical use of screening for genetic conditions in the workplace6. In Norman-Bloodsaw v. Lawrence Berkeley Labs (1998), employees sued their employer for medical testing of employee blood and urine samples without their knowledge or consent. In addition to broad syphilis testing of employees, Black employees had been tested for sickle cell carrier status, and women had been tested for pregnancy. The 9th Circuit Court of Appeals reversed summary judgment for the defendants on the grounds of the 4th Amendment’s prohibition of unreasonable searches and seizures. Additionally, the court held that Title VII of the Civil Rights Act , allowed the plaintiff’s claims that they should not be forced to undergo genetic testing by an employer during the pre-screening process33. Perhaps the most prominent case is the Equal Employment Opportunity Commission’s (EEOC) 2001 suit against the Burlington Northern Railway Company alleging violations of the ADA36. Burlington Northern had been requiring blood samples from all employees who claimed they developed work-related carpal tunnel syndrome to determine whether they had a rare genetic predisposition to this condition, a finding that might help the employer avoid worker compensation claims costs. However, the company did not tell their employees that their samples would be used for genetic testing37. This case settled for $2.2 million38 and demonstrates the financial risk employers may face for inappropriate use of genetic testing. While this is critical background for a discussion of genetic and genomic testing in the workplace, it is important to note that how we use the language of wGT has changed dramatically since this early experience.Early discussions of genetic vulnerability screening in the workplace raised the possibility that such screening could be completed as part of the pre-employment hiring process, similar to pre-employment use of other medical information and testing. The Americans with Disabilities Act (ADA), which was passed in 1990, regulates pre-employment medical testing in order to limit the potential for discrimination39 to reduce health costs and to improve employee well-being and work productivity. The Health Insurance Portability and Accountability Act (HIPAA) in 1996 and Affordable Care Act (ACA) in 2010 set rules allowing for the use of workplace wellness programs. While both these laws prohibit health plans from adjusting premiums based on health status, they have an exception for wellness programs. The laws arguably encourage wellness programs by allowing employers to incentivize employee participation. After the implementation of the ACA, some wellness programs offer incentives or impose penalties that are equal to 30% of the cost of health insurance premiums under the employer’s plan, and as high as 50% for smokers. This can translate to the gain or loss of thousands of US dollars for individuals and families alike40, raising concerns that these programs can be coercive41.In the 1970s, employers began offering workplace wellness programs promoting healthy behaviors among their employees20. However, wGT brings with it both previously identified and new ELSI concerns. In response, some ELSI researchers focusing on genetic testing in the workplace have shifted their work from genetic monitoring and vulnerability screening to employee workplace wellness programs42. Given that the US differs with respect to the structure of health insurance and healthcare as compared to other countries, many issues in this space are of unique concern in the US. Differences such as the lack of a national health system, very high healthcare costs, the fragmentation of rules of healthcare regulation, and the distribution of insurers and employers across a large number of states, shape risks, benefits, and incentives for such programs.wGT could fit well into workplace wellness programs since genetic and genomic testing may offer employees the opportunity to identify genetic risks and mitigate or prevent future disease . Indeed, a growing number of employers are offering, or considering offering, wGT as part of their wellness programs43. Studies and critiques of these programs have suggested that they do not address key employer concerns such as absenteeism44, healthcare coverage costs, or unhealthy employee behavior45, but rather serve to shift the cost of healthcare coverage from employers to employees46 by establishing rewards/penalties linked to health insurance premium levels. Additionally, there is worry that employees may forgo any concerns about the long-term risks associated with wellness programs  in order to benefit from near-term financial incentives or to avoid penalties. Many of these overall concerns raised by workplace wellness programs could also apply to wellness programs that include wGT.Beyond the narrow category of workplace wellness programs that offer genetic testing, workplace wellness programs generally inspire critique. While many employers argue that workplace wellness programs can lead to healthier employees and lower costs for employers, this is not necessarily supported by currently-available data47. Genetic testing within an employment setting itself could potentially heighten these privacy concerns if there is not adequate protection for employees’ information and employment26.One issue unique to wGT relates to genetic privacy. The public are wary of potential misuse of their genetic information—public polling in 2018 revealed that 47% of respondents who had undergone DTC genetic testing (or whose family members had done so) expressed concerns about genetic privacy48. The law also prohibits covered health insurers from setting eligibility and premium or contribution amounts for health insurance based on genetic information. It does not, however, provide protections in the contexts of disability, long-term care, or life insurance27. Notably, GINA prohibits employers and health insurers from collecting genetic information; however, there are exceptions to this rule for certain cases, including monitoring one’s genetic information for changes due to exposure of toxic substances in the workplace and for voluntary workplace wellness programs49. Current GINA regulations generally state that a program is only voluntary if there is no incentive or penalty associated with providing genetic information to the employer50. Additionally, GINA only allows employers to access aggregate genetic information  collected from employees in the wellness program. However, as is discussed further below, there have been recent efforts by regulators and Congress alike to alter this rule. While there are several other federal laws and policies that provide protections for employees in the context of voluntary workplace wellness programs, they are not specific to one’s private genetic information  on Genetic Screening in the Workplace, argues that while genetic testing of the sort being offered through wGT has the potential to inform prevention and treatment approaches by way of pharmacogenomic, susceptibility, and diagnostic testing, it must be paired with guiding ethical and legal principles, scientific validity of wGT tests, and discussion of results with a trained health professional56.While experts recognize the potential benefits of wGT  for the employers’ overall preventive health strategy57. This bill would have allowed employers to incentivize or penalize employees for their decisions to disclose genetic information in a wellness program. The allowable incentive or penalty would have been up to 30% of health coverage costs, thus mirroring current incentives allowed by the ACA for general wellness programs. Additionally, it arguably would have allowed employers to collect individual-level data about employees’ private genetic or medical information within the context of a voluntary wellness program58. This would have thus significantly diminished employees’ genetic privacy rights. Professional societies such as the American Society of Human Genetics (ASHG) and the ACMG opposed this bill that sought to exempt workplace wellness programs from GINA and the ADA60.Congress waded into the debate in 2017, with the introduction of HR 131361. For example, the proposed rules would have allowed rewards to incentivize individuals to share their genetic information, thus increasing concerns about coercion and the risks of breaches of privacy and the possibility of discrimination for employees. Of note, employers would not be required to share the type of employee health information they would be privy to or how they would use the information.Finally, the EEOC under the Trump Administration proposed rules that would have altered the protections of GINA in regard to workplace wellness programs62.However, as of July 2021, any further action on these proposed rules has been paused by the Biden Administration63 and address many of the concerns highlighted above64, they focus primarily on the vendors offering the testing and the employers contracting with them. One of these, Stakeholders Assessing Genetics with Employers (SAGE), is a 2019 federally-funded project that provides some framework and context for conducting research on wGT63 and created a checklist for employers considering wGT. The four areas for consideration are: (1) Defining wellness program goals; (2) Specific types of genetic tests to be offered to employees; (3) Legal and policy considerations to mitigate liability risks and choosing a reputable vendor; and (4) Types of evidence employers should request to ensure that their wGT goals are achieved. Ultimately, there are many questions to be answered and little published research exploring the intersection of workplace wellness programs and genetic testing65.While two existing commentaries discuss wGTGiven the expectation that wGT will become more prevalent in the future, there is an urgent need to understand its benefits and risks. There also remains a need for empirical data to address the concerns raised by wGT and potential policy safeguards. It will also be important to understand concrete issues such as whether there are differences in implementing wGT in different settings, including the academic, non-profit, and corporate settings, and within small versus large companies . Our group has undertaken an initial step to study the ELSI issues surrounding wGT in real world settings and to elicit the perspectives of a broader repertoire of stakeholders—including employees, employers, and other relevant groups—whose perspectives are necessary for a fully informed view of wGT’s risks and benefits. Our current NHGRI-funded mixed-methods study aims to address the gaps in understanding of the wGT landscape and provide much needed empirical data to inform a normative assessment of wGT.Recognizing that existing studies on wellness programs report minimal short-term benefits and do not account for wGT, our study will provide a comprehensive assessment of wGT from both the employee and employer perspectives—advancing an evidence-based approach to policy and decision making in this area of genetic testing. By undertaking a multifaceted impartial analysis, we hope to provide foundational information and insights to inform decision making for wGT, including (if appropriate) considerations for its implementation, so as to maximize its positive impact on individuals, employers, and society alike, while minimizing harm.Genetic testing offered outside the clinical setting is affordable and accessible to a large proportion of the population. US employers are beginning to offer genetic testing to their employees as part of their voluntary workplace wellness benefits, though it is unclear how widespread this practice is or will become. While this practice is in its infancy, it is important to consider the implications of wGT for clinical care, including outreach to genetic counselors, clinical geneticists, clinical assessment, follow-up, and management. Today’s wGT through commercial third-party vendors is substantially different from the historical model focused on genetic monitoring and vulnerability screening. Given this shift, there are new ELSI and policy concerns that have arisen; however, there is little to no research currently being conducted to assess these concerns. At this time, it is still unclear whether Pandora’s Box of wGT will produce benefits or risks and for whom. Our hope is that our study and the work of others will begin to address this question while there is still time to shape the outcome. If wGT becomes more widespread, as we predict, it is critical to have conceptual and empirical analyses of the risks and benefits to inform policy recommendations designed to maximize positive impact on individual health, employers, and society alike, while minimizing harm.