Although oceans provide critical ecosystem services and support the most abundant populations on earth, the extent of damage impacting oceans and the diversity of strategies to protect them is disconcertingly, and disproportionately, understudied. While conventional modes of conservation have made strides in mitigating impacts of human activities on ocean ecosystems, those strategies alone cannot completely stem the tide of mounting threats. Biotechnology and genomic research should be harnessed and developed within conservation frameworks to foster the persistence of viable ocean ecosystems. This document distills the results of a targeted survey, the Ocean Genomics Horizon Scan, which assessed opportunities to bring novel genetic rescue tools to marine conservation. From this Horizon Scan, we have identified how novel approaches from synthetic biology and genomics can alleviate major marine threats. While ethical frameworks for biotechnological interventions are necessary for effective and responsible practice, here we primarily assessed technological and social factors directly affecting technical development and deployment of biotechnology interventions for marine conservation. Genetic insight can greatly enhance established conservation methods, but the severity of many threats may demand genomic intervention. While intervention is controversial, for many marine areas the cost of inaction is too high to allow controversy to be a barrier to conserving viable ecosystems. Here, we offer a set of recommendations for engagement and program development to deploy genetic rescue safely and responsibly.  Wildlife biodiversity and abundance are experiencing unprecedented declines, with every marine ecosystem subject to multiple anthropogenic threats . The samPelagibacter, estimated at 1028 cells, and the most abundant phototroph, Prochlorococcus, numbering 1027 cells [18 individuals [14 individuals [Human-caused biodiversity loss is significantly outpacing rates of evolution and adaptation in natural populations ,3. Under27 cells , to the ividuals , and copividuals . TherefoOver the next 30 years, the human population is expected to grow by 3 billion and per capita resource consumption is also expected to rise . Such prSince the first human genome was sequenced in 2003, medicine has been revolutionized by genomic research and its clinical application ,17,18,19Yet the integrated adaptation of genomic tools for wild ecosystems—to support biodiversity, combat disease, or create synthetic alternatives—remains nascent. Exploration of genomics as an innovative strategy to complement and support ocean conservation may provide new wholly methods for combating threats to ocean health. Advances in biotechnology and genomic research should be harnessed and developed within a wide variety of conservation frameworks to foster viable ocean ecosystems far into the future. https://ocean.reviverestore.org), which assessed the opportunities to bring novel genomic and biotechnology insight and innovation to marine conservation. The authors of this paper were part of the survey and assessment team, and undertook an extensive effort that included reviewing current research and conducting interviews with over one hundred ocean science researchers, conservation managers, biotech innovators, and ethicists. From this, we identified how novel approaches from synthetic biology and genomics might alleviate major marine threats. As an assessment of current efforts, we focus on areas where genomics is already being applied or considered, including corals, invasive species, pollution, and overexploitation. Climate change is addressed as a broader and synergistic threat that compounds and exacerbates other marine threats. Here, we distill the results of a targeted survey, titled the Ocean Genomics Horizon Scan (OGHS)  must play a role in evaluating genetic rescue tools as a solution set for ocean conservation. Interdisciplinary engagement is the only way to successfully shape the development and suitable application of a marine Genetic Rescue Toolkit. We therefore present our findings on the immediate steps that can be taken to build and apply a dynamic and effective conservation genomics toolkit for marine ecosystems, and in closing, discuss the social, ethical, and economic implications of threats to ocean biodiversity.In parallel with the Continuum of Readiness for genomic innovations in conservation, a Genetic Rescue Toolkit will necessarily span applications from “genetic insight” to “genomic intervention.” Genetic insight and genomic intervention can accomplish some of the same goals: Maintaining or augmenting genetic diversity and facilitating adaptation to disease or climate change. However, genetic insight, as defined here, primarily uses genetic information  to enhance traditional conservation practices. Genomic intervention, as defined here, on the other hand, implements genomic manipulation (engineering or precise editing) to a species of interest and extends the capabilities of genetic rescue further, to include strategies of biocontrol and de-extinction. As such, much of the technology needed for genetic insight applications is ready to deploy for conservation or requires little optimization . Most geThe following sections outline research areas in which genetic insight and genomic intervention have been demonstrated or are in development to address four major threats to ocean species. Genomics promises to play a major role in improving the traceability of traded and unreported marine wildlife. This requires adaptation of commonly used molecular forensics tools, as well as new innovations. Today, the use of eDNA for wildlife monitoring is diversifying , but itsUntil recently, a major limitation in preventing illegal catch and trade has been the lack of rapid, high throughput technologies. However, new advances that reduce cost and eliminate the need for specialized lab equipment or high-level expertise are promising. The DNA Barcode Scanner by Conservation X Labs for instance, although still in prototype stage, packages the DNA barcoding process into a portable, affordable handheld device, and produces real-time field results . This plTo enhance the efficacy of these approaches, high-resolution genomic data and expanded DNA barcoding repositories are necessary. For example, the accurate genetic discrimination of various mussel species using exceptionally small SNP panels has been demonstrated . These sGenetic insight can inform fisheries managers about the impact of fishing on stock vulnerability or the interference of bycatch with restoration efforts . A receMany cost-effective tools for genetic insight, such as the genome wide microsatellites mentioned above, are enabled by high quality reference genomes. One tool emerging from high throughput genomic tools is the enhanced ability to understand the population dynamics of fisheries through a process known as close kin mark and recapture . These tBiotechnology and synthetic molecular biology can also provide alternatives to harvested wildlife products. Several alternatives already exist; the methods by which they were developed could be replicated to alleviate harvest of many other species. These include synthetic recombinant factor C, a replacement for the blood of horseshoe crabs in endotoxin testing by the pharmaceutical industry. The adoption of rFC in drug and water testing not only has the potential to reduce horseshoe crab harvest by >90% but it is also more effective than the lysate tests derived from horseshoe crab blood .Salmo salar) has produced a faster growing farm population [While controversial, genetic engineering can reduce overharvesting of a wild product through manipulation of desirable phenotypes. For example, the integration of a pacific salmon growth gene in Atlantic salmon  are removed by divers or injected with chemicals. Targeted trapping has shown some promise for European green crab (Carcinus maenas) population control [Current control efforts of marine invasive and irruptive species primarily involve their manual killing or capture. For example, crown-of-thorns starfish  . These sMoreover, possible gene drives can be used to spread genes through a population in a way that alters the standard model of inheritance . NormallPertaining to ocean ecosystems, gene drives are currently being investigated as a biocontrol method for invasive rodents on islands . Island Genomic research has already been applied to gathering insight into adaptive variation with respect to climate change. Transcriptome analysis has been used to identify genes involved in climate adaptation in coral , and metAdaptive interventions would involve the translocation or assisted migration of individuals to match genetic variation within individuals to new environments ,66,67. ACoral reef ecosystems are experiencing more frequent and severe bleaching events by changing ocean conditions (warming) from climate change ,69. The While coral reefs will regenerate over centuries and millennia through recolonization by thermal tolerant species, the rapid collapse of entire reefs will cause the extinction of thousands of noncoral reef species that have no refuge; these species will be lost completely. It is paramount that intervention can match and overcome the pace of coral reef loss to prevent these extinctions, as well as the major economic losses that will stem from worldwide reef collapse. This pace will only be accomplished if biotechnology and genomics are integrated into intervention strategies.A major barrier to applying genetic insight and genomic intervention to coral reef rescue is the extreme difficulty in conducting laboratory research on corals because of current difficulties in cryopreserving and maintaining coral colonies ex situ. The following three sections present areas of research that could quickly overcome these technological issues. These and other projects are discussed in more detail in the Ocean Genomics Horizon Scan .Cryopreservation is a vital preventative measure for preserving threatened coral species facing extinction in the wild. Current methods involve the freezing of sperm and larvae harvested from reefs ,72. To d2, these small clonal pieces grow at exceptional rates to reconstitute larger corals [The micro-fragmentation method offers one approach to scale coral restoration efforts ,74. Whenr corals . Micro-fMultiple groups have developed husbandry methods for making/growing micro-fragments ex situ. The cryopreservation lab at the Smithsonian Institute has identified a suite of cryo-protectants that are nontoxic to micro-fragments and enable them to survive freezing (pers. comm. Mary Hagedorn). The remaining hurdle is to optimize thawing procedures so micro-fragments can grow in open water after years of storage. Once this is achieved, the entire procedure from collection, to freezing and thawing should be standardized and potentially automated. With optimization of cryopreservation techniques, the global collection of coral micro-fragments, sperm, and possibly eggs could soon begin, starting in reefs that are most threatened. Ideally, this coral ark would include 100 individuals of each species, at 10 samples from each individual. No other technology yet offers this depth and continuity for saving a species. As mentioned, coral eggs are necessary for breeding but cannot yet be cryopreserved. However, under ideal conditions and proper stimulation, corals can be induced to spawn . ScalablThe Horniman Museum’s Project Coral laboratory is pioneering this work, but scientists there are unable to provide more than one-on-one training at present. By scaling up the team’s laboratory and training capabilities, other practitioners could be trained more easily to perform the inducible spawning technique.Ongoing developments in coral stem cell technology and the clonal nature of coral will make it possible to isolate and propagate coral stems cells . Paired Stem cells derived from novel breeds could be propagated in a continuous manner in bioproduction facilities to support industrial-scale production of coral larvae for direct out planting of high-performance corals. Moreover, stem cells could be cleaved from juvenile corals, cloned, and then subpooled for genetic experimentation, such that a single coral could be independently tested for many different traits, even with a destructive assay . Stem cells with adaptive genes could themselves be transplanted directly into wild corals, in order to recolonize dying coral colonies, as a coral stem cell therapy approach. Finally, by pairing stem cells with genome engineering and gene editing , stem cThroughout this paper, the authors frame the applicability and readiness for biotechnological interventions from the perspective of technological and social factors that will influence development for effective deployment of the genetic rescue tools presented; however, the complete spectrum of factors that will shape how genetic rescue tools are used must include a treatment on bioethics and regulation. Biotechnological interventions for marine conservation will be directly impacted by a number of existing regulatory frameworks, as well as necessitating the incorporation of bioethical analyses to advance to effective and safe states. The reality of regulation is that even for simply building the genome sequence resources needed for genetic insight tools will have to navigate policies impacting every stage of work from tissue collection to database repository and reuse, including the 1972 Convention on International Trade of Endangered Species (CITES), the 1992 Convention on the Conservation of Biological Diversity (CBD), the 2014 Nagoya Protocol, and most pertinent to genomic interventions, the 2000 Cartagena Protocol . These pDifferent genomic technologies are at varying states of technological and social readiness for large-scale implementation . GeneticGenetic insight can inform conservation management in a variety of ways that are dependent on generating and facilitating access to adequate genomic resources. The exponential increase in genome assemblies and ease of resequencing individuals is establishing the foundations for genetic insight research useful to conservation. For example, cheap and rapid sequencing has enhanced our understanding of population structure for fisheries management ,84,85, hThe lack of conservation focus in genomics is evident when comparing the ratios of actual marine biodiversity A to taxoIn order to enable genetic insight to outpace the decline and loss of species, there is a need for massive investments in a strategic sequencing and biobanking initiative. Such efforts should focus on establishing baseline reference genomes across the ocean “tree of life” in proportion to taxonomic diversity, prioritized by species’ significance to ecological function, and targeted conservation applications. For example, improvement of detection and monitoring of invasives and illegal trade is an urgent priority. However, current eDNA methods can suffer from uncertainties in species identification , presenting a risk of false positives, and could have weak statistical power (leading to overconfidence when no detections are recorded) ,44. ThisSynthetic alternatives are continually being developed, yet adoption at a scale that would impact conservation outcomes are often limited by public preference and pushback from industries that alternatives threaten to replace. For example, the synthetic alternative to horseshoe crab blood, rFC, is equally effective in biomedical research, yet it has not been widely adopted due to financial incentives . On the These alternatives to wild harvest are not new; rFC was first developed in 1995 ,92 and AThere appears to be growing public readiness for plant and cellular-based alternatives; global sales of meat-alternatives have increased 8% annually since 2010 . While nin silico genomic research, (2) in vitro cellular technologies, and (3) transition from in vitro to in vivo (made possible through advanced reproductive techniques to turn in vitro cells into embryos or the in vivo gene editing of embryos or adults). Intervention tools are well established in model species for biomedical and functional genomic research , but are also advancing for agricultural species. For application to wildlife conservation, these require development and innovation, but are achievable short-term, with laboratory examples of success already established for some terrestrial species  l blight , successl blight ). The reThe use of genetic engineering for conservation is mired by concern among the public and the scientific community over unintended or negative consequences. Marine applications pose an even higher burden of proof due to the shared nature of marine ecosystems, making stakeholder engagement critical to building guidelines for standard practices. Therefore, efforts should be targeted at evaluating and mitigating risks, working with regulatory bodies, and engaging the public. For example, the technical challenges for developing genomic biocontrols of marine invasive and irruptive species are significant, but not impossible. However, genetic biocontrol is controversial and there exists concern that the unintended effects of biological control are a threat to the environment and would potentially cause nontarget species or populations to become extinct, highlighted by the current developmental shift from self-propagating gene drives to self-limiting versions . In addiThe decline of ocean species destroys more than the evolutionary potential of biodiversity; it degrades human livelihoods as well. Societies are economically reliant upon natural resources, both biotic and abiotic, which can only be provided by functional ecosystems. The Stockholm Resilience Center has identified strong links between biodiversity loss and poverty and human suffering. For example, over 35% of global food production is dependent on pollination. The loss of pollinating species could present a major food security challenge . Nearly The compounding effects of climate change stand to simultaneously reduce the value of the fisheries industry and increase the severity of invasive species problems, causing a dual front of economic loss, which will be felt more deeply in poorer coastal nations. The global community cannot be dominated by the dialogue of rich nations when considering all options to prevent biodiversity losses, a point we assert given the historic precedence of repeated health and economic harm caused by extreme biotechnology regulation in developing nations which were inspired by US and European anti-GMO lobbyists, rather than sound science or balanced discourse ,102,103.Even with powerful enhancement via genetic insight, current conservation strategies are not sufficient to address all environmental threats, and given the international complexities of oceans, no single sector, application, or method can address the multi-faceted nature of these challenges. International genetic rescue focused programs, working groups, and consortia will be necessary to achieve specific conservation goals. Augmenting traditional strategies with genetic insight and the appropriate genomic intervention, where beneficial, can ultimately produce synergistic conservation impacts. Though some may caution that genomic solutions should be a last resort, with careful and thorough development, early deployment of genomic intervention in certain situations will produce significantly higher conservation gains as opposed to postponing the intervention until no other tools can help yield success. When considering the options, there are three essential questions to ask: When should humankind intervene in a natural evolutionary process? Oceans are shared environments, and technological intervention should be developed in a manner that reflects the values and needs of the communities that depend upon and are in relationship with shared marine ecosystems. Moreover, what is the cost/benefit analysis of each solution? Equally important, what is the cost and risk of inaction? Comparing and contrasting alternative approaches may allow decision makers and the public to understand that not using genetic technologies may have equally, if not larger, negative consequences for marine ecosystems and society as a whole.With ocean resources it is nearly impossible for a nation to form conservation policies that will not impact other nations—tracking of 1648 individuals of 14 Pacific species found that per year, these 14 species alone visit and utilize resources within the jurisdictions of 86% of Pacific nations, with many individuals visiting dozens of national jurisdictions each year . Those eBuilding public trust and engaging feedback will be essential elements of conservation efforts involving genomic technologies, especially on a global scale. These processes will depend upon relationship building to earn the respect and trust of the public. Genuinely incorporating stakeholders into the decision-making process requires the design of mechanisms for input, which in turn necessitate transparency in decision-making. Responsible development of genetic rescue strategies will therefore depend upon an exploration of the underlying values that are motivating the project and shaping its design. For example, how decision makers relate to their natural environment will form the ethical justification for many of these interventions. Whether the ecosystem under consideration is perceived to have utilitarian value  versus intrinsic value  could impact the resources and care available to save that ecosystem .Weighing Risks and Benefits: Genetic rescue program developers should conduct a thorough review of both the intended outcomes and the potential benefits of applying genetic insight or genomic intervention, as well as the risks and potential consequences. Questions to consider include: Have other more established interventions failed? Do other interventions, such as the application of antibiotics or pesticides, have potentially worse environmental consequences? Is this intervention the most efficacious, lasting, and least risky solution to an environmental problem, not merely an equivalent or novel approach?Transparency: Genetic rescue program developers in collaboration with conservation managers should proactively identify and inform key stakeholders in the early stages of technology development. This includes partners from the private sector (particularly biotech firms), social science experts, public sector partners, international and research organizations, religious and ethical organizations, NGOs, and local communities. This should involve objective discussions with the public about the risks and potential impacts of proposed environmental solutions and also subjective discussions about values.Procedures: Genetic rescue program developers should conduct systematic and data-driven reviews of recommended best practices. These should include surveys of recent intentional environmental release of organisms  into natural environments and studies of the resulting long-term effects.Tests: Genetic rescue programs should first field test genomic technologies in contained environments to minimize unintended environmental consequences. These should be simple enough to be cost-effective, yet complex enough to sufficiently mimic natural ecosystems to yield useful data on the efficacy of developing technologies. Suitable test environments will be especially important for marine environments.Predictions: Genetic rescue programs should employ computational models, particularly as they grow more sophisticated with time, to predict the long-term effects on ecosystems and highlight potential failures of specific applications of genetic technologies.Measures: Genetic rescue scientists should employ standard metrics for measuring safety and efficacy at appropriate environmental scales agreed upon in collaboration with regulators, so that direct comparisons of data sets can be made, and so universal analytic tools can be developed and useful to final-stage decision making.Protocols: Genetic rescue program developers should understand the regulatory approval processes and jurisdictions that currently guide the transition of new technologies from the lab to field trials, and should be prepared to help guide new policies when needed.Public-Private Partnerships: Genetic rescue programs should collaborate with philanthropists, NGOs, agencies, and community groups in the implementation of these technologies to evaluate risk, to address controversies, and to gain critical stakeholder support. These groups can convene conversations, commission studies, identify priorities, and connect with civil society to identify safeguards.Remediation: Genetic rescue program developers should prepare for the possibility of failure. In the event of unintended consequences, will it be possible to regain control of the organism? Possibilities for this include the creation of self-limiting gene drives.Learnings: Genetic rescue program scientists and conservation managers should continuously monitor introduced organisms and the relevant environment once the technology has been deployed. Since these technologies remain new, these findings should be shared so that researchers learn of the successes as well as failures. Ultimately, transparency will allow successful intervention techniques to be more rapidly adopted globally.The following are the recommendations for stakeholder engagement and genomic program development/deployment arrived at during the Ocean Genomics Horizon Scan compilation:Innovations in genetic insight and genomic intervention technologies will diversify and strengthen the pool of strategies for ocean conservation to provide targeted responses to difficult and urgent needs ,109. ResThe goal should be to iteratively craft a set of guidelines that clearly communicate the social and ethical priorities of all affected societies, and articulate the full range of standards that genomic intervention must meet to be considered for environmental release. Such a framework would open the door to many new, powerful, and potentially less intrusive approaches to long-term environmental restoration and health. However, functional and effective guidelines need to be created through an iterative process, inclusive of bioethical, sociopolitical, and cultural considerations and values. Therefore, regulatory and governance frameworks should be developed and tested in concert with the technologies through projects and case studies. Such case studies are underway, and are building the foundation for assessments at the global level, as exemplified both by the diverse contributors of the Ocean Genomics Horizon Scan, from which the content of this paper is extracted, as well as the International Union for the Conservation of Nature’s recently published assessment of the prospects for biotechnology in conservation . Both th


Lens culinaris/Lens orientalis, Lens lamottei/Lens odemensis, Lens ervoides, and Lens nigricans. A weak correlation was observed between geographical origin and genetic relationship, except for some accessions of L. culinaris and L. ervoides. Genetic distance matrices revealed a comparable level of variation within the gene pools of L. culinaris (Nei’s coefficient 0.01468–0.71163), L. ervoides (Nei’s coefficient 0.01807–0.71877), and L. nigricans (Nei’s coefficient 0.02188–1.2219). In order to understand any genic differences at species/subspecies level, allele frequencies were calculated from a subset of 263 lentil accessions. Among all cultivated and wild lentil species, L. nigricans exhibited the greatest allelic differentiation across the genome compared to all other species/subspecies. Major differences were observed on six genomic regions with the largest being on Chromosome 1 (c. 1 Mbp). These results indicate that L. nigricans is the most distantly related to L. culinaris and additional structural variations are likely to be identified from genome sequencing studies. This would provide further insights into evolutionary relationships between cultivated and wild lentil germplasm, for germplasm improvement and introgression.Intensive breeding of cultivated lentil has resulted in a relatively narrow genetic base, which limits the options to increase crop productivity through selection. Assessment of genetic diversity in the wild gene pool of lentil, as well as characterization of useful and novel alleles/genes that can be introgressed into elite germplasm, presents new opportunities and pathways for germplasm enhancement, followed by successful crop improvement. In the current study, a lentil collection consisting of 467 wild and cultivated accessions that originated from 10 diverse geographical regions was assessed, to understand genetic relationships among different lentil species/subspecies. A total of 422,101 high-confidence SNP markers were identified against the reference lentil genome (cv. CDC Redberry). Phylogenetic analysis clustered the germplasm collection into four groups, namely,  Lens is a self-pollinating, diploid (2n = 2x = 14), cool season legume with a large genome size of c. 4 Gbp , India is still the world’s largest importer of lentil1,2.The genus c. 4 Gbp . With reears ago . Lentil ears ago . Lentil Lens for a range of agronomic and phenological traits  and secondary  gene pools was found to be higher compared to the species from tertiary (L. ervoides) and quaternary (L. nigricans) gene pools  are widely used variant calling programs to accurately and consistently identify genome variants  and cultivated species (L. culinaris – 304) were obtained from the AGG, Horsham, VIC, Australia. The geographical origins of these accessions varied widely with the majority being from Australia and the Middle East. The remaining accessions were derived from North and South America, Africa, Mediterranean, and European and Asian countries, with the number of accessions from each geographical origin varying from 2 to 240  following the manufacturer’s instructions. Using a NanoDrop UV-Visible spectrophotometer , the concentration and quality of RNA was confirmed at the wavelength ratios of A260/230 and A260/280 nm. The integrity of extracted RNA samples was evaluated using TapeStation 2200 platform with RNA ScreenTape System , following the manufacturer’s guidelines. RNA-Seq libraries were prepared using the Agilent SureSelect Strand Specific RNA Library Preparation Kit and evaluated on TapeStation 2200 platform with D1000 ScreenTape System . Each RNA-seq library was paired-end (2 × 151 bp) sequenced using Illumina Hiseq 3000 Sequencing platforms .Q ≤ 30). Reads with three consecutive unassigned nucleotides (N) were also trimmed and finally any reads shorter than 50 bp in length were removed from the final set. The remaining high-quality trimmed sequence reads were aligned to the lentil reference genome sequence of cultivar CDC Redberry (version 1.2)4 using two alignment pipelines, TopHat2 (version 2.1.1)   and STARn 2.1.1) . The numn 2.1.1) .Lens sp. – 2, L. lamottei – 1, L. odemensis – 22, L. nigricans – 24, L. ervoides – 57, L. orientalis – 57, and L. culinaris – 304), variant calling was performed separately for each species. The number of SNPs obtained from each of the mapping and variant calling methods were compared to identify the best analysis pipeline   and GATKpipeline . The STAGenetic diversity analysis was performed using the abovementioned filtered SNP data from all accessions. Genetic distances for each lentil accession were calculated using Nei’s method within the StAMPP package . A phyloL. odemensis – 22, L. nigricans – 24, L. ervoides – 57, L. orientalis – 57, and L. culinaris – 103) was chosen based on the sample numbers that belonged to each species/subspecies to capture a comparable representation from each group. Variant calling and filtering were again performed using pipelines and parameters discussed above, to generate the final output in VCF format. The allele frequencies for each SNP position were calculated using VCFtools-freq output option  file from CDC Redberry. Based on the exon–intron positions specified in the GFF file, the sequences from the corresponding genes were extracted from the CDC Redberry genome (version 1.2) fasta file using BEDtools (version 2.26.0) . The extThe total number of SNPs or variants identified for all species/subspecies was 3- to 10-fold higher when STAR aligner was used, as compared to TopHat2 , which iQ ≥ 30; 1,414,996 SNPs), and minor allele frequencies (MAF = 0.05) that resulted in a final set of 422,101 high-quality SNP markers , leaving a set of 12,522,726 SNPs. This subset of SNP markers was further filtered for percentage of missing data (50%), quality score  that was originally classified as L. lamottei actually clustered within L. odemensis. In this study, the two lentil accessions (32601 and BGRC-025688), classified as Lens sp., were clustered in L. culinaris and L. orientalis gene pools, respectively. Moreover, 22 accessions out of 467 lentil accessions were classified in clusters that did not correlate to the passport data obtained from AGG. This was more visible in L. orientalis, L. odemensis, L. ervoides, and L. nigricans clusters (L. culinaris accessions (304) with majority being originated from Australia, there was no bias observed in terms of clustering. This was further confirmed by regenerating the phylogenetic tree after removing 201 of the 304 L. culinaris accessions   .L. ervoides (ILWL042) and L. culinaris (10H063L-12HS3003) accessions with Nei’s coefficient value of 0.92295. The two L. orientalis accessions ILWL211 and ILWL069, both originating from the Former Soviet Union, exhibited the lowest genetic distance . Further analysis was performed to understand the variation in genetic distances between and within lentil species/subspecies (L. culinaris exhibited higher genetic distance to L. ervoides (0.33642–0.89817) and L. nigricans (0.31141–0.89375) compared to L. orientalis (0.12622–0.13927) and L. odemensis (0.27913–0.31805). Further analysis revealed wide genetic distance variations within the species/subspecies level .Based on genetic distance calculations, the most divergent pair was observed between bspecies . L. culiL. culinaris, some lentil accessions originating from Canada  showed wide genetic distances to some of the Australian  and Middle East Asian accessions , with Nei’s coefficient value between 0.60 and 0.70. Similar results were also observed within L. ervoides, where distant genetic relationships were observed within two Mediterranean lentil accessions ILWL042 and HOFFMAN-42 . ILWL042 also showed wide genetic distances to multiple lentil accessions originated from Middle East Asia, Russia, and Central Europe  were clustered separately from the Middle East Asian L. ervoides accessions . L. culinaris lines from Australia, North America, Asia, and Mediterranean regions were also clustered separately . In order to explore major variations at the exome levels, we focused on regions with physical distance greater than 1.0 kb  and Pisum sativum (field pea). Potential candidate genes identified are summarized in Potential candidate genes associated with variable genomic regions were identified using the GFF file from CDC Redberry genome as well as BLASTN analysis in NCBI . A totalThe current study has identified taxonomic relationships among cultivated and wild lentil gene pools using SNPs identified from transcriptome sequencing. After comparing different SNP detection pipelines, the STAR aligner-GATK-HC pipeline was chosen as the preferred method for SNP calling due to higher number of SNPs detected compared to other pipelines tested. Similar results have been reported in other studies where the efficiency of SNP and indel detection from both whole genome and exome-captured sequence data was found to be higher in GATK-HC as compared to SAMtools-mpileup . To accuLens species into four major groups, L. culinaris/L. orientalis, L. odemensis/L. lamottei, L. ervoides, and L. nigricans, which is consistent with results from L. orientalis and L. culinaris separately, which is not consistent with the results of Genetic diversity analysis clustered the six Lens accessions for future crossing strategies in order to introgress novel alleles.A weak correlation was observed between clusters and geographical origins, with some exceptions, which is in line with some previous studies . The reaL. ervoides and L. nigricans) indicate that direct hybridization between cultivated and wild lentil accessions that belong to tertiary and quaternary gene pools may be challenging. As an alternative, the use of an intermediate relative as a bridge species may be helpful in transferring alleles/genes that belong to distant gene pools. Attempts have been made in L. nigricans (quaternary gene pool) to transfer resistant genes for ascochyta blight and anthracnose diseases into L. culinaris (primary gene pool) via L. ervoides (tertiary gene pool) as a bridge species  have reported genomic rearrangements such as substitutions, insertion, and deletions  have shown better response to metal  applications, as compared to the cultivated rice (Oryza sativa). These wild species have been used for breeding purposes to enhance the metal tolerance in cultivated rice varieties. In addition, L. odemensis, L. ervoides, and L. nigricans as compared to L. culinaris  . Furtherulinaris , which iulinaris . The canL. culinaris/L. orientalis, L. odemensis/L. lamottei, L. ervoides, and L. nigricans. Wild and cultivated lentil accessions showed a weak correlation to their geographical origins. Therefore, the selection of parents for breeding purposes can be better selected by genetic distance boundaries within and between lentil species/subspecies. Allele frequency-based analysis also confirmed that L. nigricans was distantly related to L. culinaris. The identified native and exotic lentil accessions with wide genetic distances and specific agronomical traits can be used in widening the Australian lentil germplasm for breeding purposes.Assessment of genetic diversity among a global collection of wild and cultivated lentil accessions was performed using a set of genome-wide distributed SNP markers. The genetic diversity analysis revealed a clear grouping of lentil germplasm into four major clusters/gene pools, PRJNA625627, https://www.ncbi.nlm.nih.gov/sra/PRJNA625627.The datasets generated for this study can be found in NCBI SRA accession SB prepared the plant materials and performed the sequencing library preparation. RD performed the data analysis. SK, NC, and KS provided overall project leadership. RD, NC, SK, and KS all assisted in drafting the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Danggui Buxue Tang (DBT), an ancient Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix at a ratio of 5: 1, is prescribed for menopausal women. Flavonoids and its flavonoid glycosides are considered as the major active ingredients within the herbal decoction; however, their amount is not controllable during the preparation. Besides, the aglycons within DBT are believed to have better gut absorption and pharmacological efficacy.Lactobacillus plantarum. The amounts of flavonoid glucosides and its aglycones in the fermented product were analyzed by using UPLC-MS/MS. In addition, in vitro assays were employed to evaluate the efficacy of the fermented DBT in regulating the activities of α-glucosidase, α-amylase and lipase, as well as their antioxidant capacity (DPPH and T-AOC assays) and anti-glycation property .The herbal extract of DBT was fermented with L. plantarum drove a completed conversion of calycosin-7-O-β-D-glucoside and ononin to calycosin and formononetin, respectively. The chemical transformation could be probably mediated by β-glycosidase within the fermented product. Several in vitro assays corresponding to anti-diabetic functions were compared between parental DBT against its fermented product, which included the activities against α-glucosidase, α-amylase and lipase, as well as anti-oxidation and anti-glycation. The fermented DBT showed increased activities in inhibiting α-glycosidase, suppressing DPPH radical-scavenging and anti-glycation, as compared to the original herbal product.The fermentation of DBT with L. plantarum could pave a new direction for fermentation of herbal extract, as to strengthen its pharmacological properties in providing health benefits.These results suggested that DBT being fermented with the probiotic  Type 2 diabetes mellitus (T2DM) is a chronically metabolic syndrome characterized by insulin resistance and pancreatic β cell dysfunction, caused by inherited and/or environmental factors . The gloNeiwaishang Bianhuo Lun >  > by Li Dongyuan in AD 1247 in China. He described DBT should have: Astragali Radix  Bunge var. mongholicus (Bunge) Hsiao) and Angelicae Sinensis Radix  in 5 to 1 ratio. This herbal mixture has been utilized for nourishing “Qi” and enriching “Blood” for women suffering from menopausal symptoms. Pharmacologically, DBT is able to mitigate menopausal symptoms − were detected as precursor ion, then HCOOH ion and sugar moiety could be lost from the precursor ion to form the deprotonated and de-glycosylated molecule [M-H-Glc]−. The UPLC chromatogram and related MS transition spectrum were presented in Fig. The chemical characterization of DBT was reported previously . HPLC fiR2) greater than 0.99  in six replicates, representing an entire calibrating range. The RE values of intra- and inter-day accuracies were in the range of -12.8% to 11.5% and -10.8% to 11.2%, respectively  and pH value of L. plantarum being fermented with or without DBT were determined. There was no apparent growth in the first 3 h, and however an exponential growth was observed thereafter . This process leads to a loss of protein function and impaired cell functions. The glycation reaction is highly accelerated under hyperglycemia, implicating the pathogenesis of diabetics and aging. In addition, oxidative stress plays a role in forming AGEs, and thus which has been implicated in causing the progression of diabetes . In viewL. plantarum  inhibitory properties on α-glucosidase; (ii) antioxidant properties on DPPH scavenging and T-AOC; and (iii) anti-glycation capacity on various models. This fermentation approach, nevertheless, could be considered as a mean to enhance the pharmacological efficacy of TCM in general. The safety should not be a concern: because L. plantarum is a common Lactobacillus found in many fermented food products, as well as in human saliva, which has been known to show no harm to our body. Having this new direction of fermentation of Chinese herbal extract therefore can pave another method to enhance the product efficacy, in particular Lactobacillus and herbal product are commonly used as health food products on the market.Fermentation of L. plantarum. The method development of MS parameters, as well as method validation comprising parameters, e.g. specificity, linearity, sensitivity, precision and accuracy, extract efficiency, matrix effects and stability, is being described in detail at the present study. Thus, a sensitive and reliable quality control method using MRM in negative ion mode for simultaneous determination of four flavonoids in fermented DBT, i.e. calycosin-7-O-β-D-glucoside, ononin, calycosin and formononetin, has been developed.The UPLC-MS/MS analysis is a common method for simultaneous quantitation of flavonoid compounds. However, most of the developed methods are focusing on herbal or plasma samples. No reports have investigated the simultaneously quantitative analysis of the flavonoids in the fermented broth of L. plantarum, not from the herbal extract. The expression of β-D-glucosidase in the fermented product, as revealed by release of p-nitrophenol from p-nitropheny-β-D-glucopyranoside, could be markedly enhanced in DBT extract. A synergy during the fermentation of DBT with L. plantarum is being proposed here. First, DBT promoted the expression of β-D-glucosidase in fermenting L. plantarum. Second, the produced β-D-glucosidase hydrolyzed the flavonoid glycosides of DBT to aglycons. These findings suggested that an improvement in hydrolysis rate of glycoside enzymes perhaps may be ascribed to DBT inducement. In TCM preparation, the phytochemicals exhibit low oral bioavailability, in particular glycosides are possessing poor membrane permeability [The chemical transformation, i.e. cleavage of the sugar moieties, was observed in here. The enzyme β-D-glucosidase is responsible for the cleavage of β-D-glycosidic linkages, releasing glucose moieties from flavonoid glycosides . This eneability . Moreoveeability . Herein,eability .The anti-diabetic functions of fermented products should be derived from DBT. The chemical changes within DBT after fermentation could account for increased pharmacological efficacy. However, the identity of active chemicals of DBT in triggering these activities are not resolved. The increased antioxidant property could be accounted for the hydrolyzed flavonoid glycosides. Indeed, aglycons in the herbal extract were shown to have stronger antioxidative activity . AlthougThe non-enzymatic glycation, a spontaneous reaction between sugar and protein, is considered as a source of oxidative stress, as well as the primary route in forming AGEs. Increased AGE has been implicated in the pathogenesis of diabetic complication. The increased anti-glycation activity in fermented DBT could be accounted by the conversion of flavonoid glucosides to aglycons. Various phenolic compounds, inhibiting α-glucosidase, have been reported as inhibitors of glycoside hydrolase due to their binding with proteins, as such to erode the glycation reaction . The hydL. plantarum. In considering the traditionally oral intake of herbal medicine, the conversion of flavonoid glycosides in fermented DBT not only improves the absorptivity of flavonoids to gut, but which possess prominent activities against α-glucosidase, antioxidant and anti-glycation. These anti-diabetic activities could be accounted, at least partially, by the increased flavonoid aglycons in the fermented products. The result paves direction for fermentation of herbal extract, as to strengthen its pharmacological properties.To the best of our knowledge, we have reported for the first time, the conversion of calycosin-7-O-β-D-glucoside and ononin to their aglycons in DBT through a fermentation process using Additional file 1:Figure S1. Typical-HPLC fingerprints of DBT before and after fermentation. Table S1. Analytic parameters of 4 standards by UPLC-MS/MS in fermented DBT.


Genetic heterogeneity is a well-recognized feature of hepatocellular carcinoma (HCC). The coexistence of multiple genetic alterations in the same HCC nodule contributes to explain why gene-targeted therapy has largely failed. Targeting of early genetic alterations could theoretically be a more effective therapeutic strategy preventing HCC. However, the failure of most targeted therapies has raised much perplexity regarding the role of genetic alterations in driving cancer as the main paradigm. Here, we discuss the methodological and conceptual limitations of targeting genetic alterations and their products that may explain the limited success of the novel mechanism-based drugs in the treatment of HCC. In light of these limitations and despite the era of the so-called “precision medicine,” prevention and early diagnosis of conditions predisposing to HCC remain the gold standard approach to prevent the development of this type of cancer. Finally, a paradigm shift to a more systemic approach to cancer is required to find optimal therapeutic solutions to treat this disease. In the last decade, numerous studies have provided several and accurate details on the genetic alterations associated with hepatocellular carcinoma (HCC). In particular, the familial genetic alterations responsible for the development of cirrhosis and associated with HCC formation have been entirely unraveled . MoreoveAs shown in Table Based on these considerations, we can assert that the pharmacological principles of potential therapeutic agents targeting genetic alterations have been probably based on the wrong paradigm . First, The failure of these therapeutic strategies reinforces the importance of conventional therapies , which used alone or in combination, remain the only widely accepted clinical treatments for HCC –27. TherAn entirely different approach to the cure of HCC could consist of the prevention of the disease through the identification and targeting of the early genetic alterations that anticipate the neoplastic transformation. Unfortunately, this strategy seems also not to succeed in the difficulty to target/correct familial genetic alteration and oncogenic mutations. In this contest, the HBV transgenic mouse model offers the opportunity to study the process of tumor initiation and progression related to the HBV infection , 31, andAnother important aspect regarding the development of new potential therapeutic agents against HCC that should be taken into account is the role of metabolic-induced modifications acting as environmental modifiers with the ability to influence a susceptible genetic or epigenetic background . In this


F94L polymorphism of myostatingene (MSTN) linked with the gene responsible for less fat content in the carcass by means of DNAanalysis. In the article, test systems based on the polymerase chain reaction method are presented. The analysis of the Aberdeen Angus , osteopetrosis (OS),developmental duplication (DD), double muscling (M1), hypotrichosis (HY) andmaple syrup urine disease (MSUD) as well as the  Excellent productive features such as high meatyield and good organoleptic traits due to increased tenderness and marbling were registered among the animals of the Aberdeen Angus, Hereford and Belgian Bluebreeds . Also, the animals of these breeds have goodacclimatization abilities and high genetic potential . However, besides the advantages, Aberdeen Angus,Hereford and Belgian Blue cattle have a serious drawback, i.e., the appearance ofsome genetic defects .According to the American Angus Association, today in the Aberdeen Angus cattle breed there is an appearance of a number genetic anomalies, among them arthrogryposismultiplex (AM), osteopetrosis (OS), developmental duplication (DD) anddouble muscling (M1). The clinical features of these anomalies weredescribed in previous studies . The focus of our interest are the mutations caused. It should alsobe noted that AM and OS are the lethal genetic defects leading to calfdeaths, and DD and M1 are non-lethal but undesirable conditions.ISG15) gene, the 5HES4) and two first exons of the agrin gene (AGRN) .The reason for AM is the large deletion (23 347 bp) encompassing three genes: the completely ISG15 ubiquitin-like modifier (SLC4A2) (g.114437192_114439942del) .The reason for OS is the deletion of 2784 bp encompassing completely exon 2and half of exon 3 of solute carrier family 4 (anion exchanger) member 2gene (NHLRC2) (OMIA 002103-9913) .The reason for DD is the single nucleotide polymorphism (SNP)g.34618072TMSTN) . The animals carrying in theirgenotypes the mutant allele had double muscling due to the muscularfiber hyperplasia and have a meat with increased tenderness. However,they also have a high weight at birth leading to dystocia , which destroys the reproductive functions, and so this mutationhas been regarded as a genetic defect . In addition toAberdeen Angus this polymorphism has also been found in some other cattlebreeds particular in Belgian Blue cattle .The reason for M1 is an 11 bp deletion (c.821-831delTGAACACTCCA) in the thirdexon of myostatin gene (BCKDHA) located on chromosome 18 in the c.148CKRT71) gene.In the Hereford cattle breed the genetic defects of maple syrup urine disease(MSUD)  and hypotrichosis (HY) have been registered. The cause of MSUD is the SNP in the branched-chain keto aciddehydrogenase E1, alpha polypeptide gene (F94L polymorphism of the myostatin gene (MSTN), is the SNP in 2:g.6213980AF94L polymorphism the analogue of the nt821del MSTN mutation caused by M1; however, in contrast to the former, the F94L polymorphism does not influence birth weight, and due to the absence of thedystocia risk, this polymorphism could be used for the purpose of obtaining leaner meat .The gene mutations associated with agriculturally useful traits that canbeneficially affect meat productivity are also of undoubted value. One ofthem, the F94LMSTN polymorphism.The aim of our study was the analysis of the Aberdeen Angus, Hereford andBelgian Blue cattle breeds regarding the presence in the animals' genotypes of the mutant alleles associated with the appearance of the AM, OS, DD, M1, HY and MSUD genetic defects and an analysis of the frequency of the desirable 2The study was conducted in 2018–2020 at the laboratory of molecularselection basis at the L.K. Ernst Federal Science Center for Animal Husbandry.The material was animals of the Aberdeen Angus  in accordance with the manufacturer's recommendations.From the biomaterial (skin or blood) of the animals, the DNA samples were obtained by extraction by means of the “DNA-Extran-1” and “DNA-Extran-2”kits  and DD by PCR with the subsequent restrictionfragment length polymorphism analysis (RFLP) using the restrictionendonuclease The DNA samples from the animals of the Hereford cattle breed weregenotyped for the genetic defects of MSUD by a PCR-RFLP test system previously developed by Dennis et al. (1999).Testing for HY was conducted by an AS-PCR test system developed in thelaboratory of the selection molecular basis of the L.K. Ernst Federal Centre ofAnimal Husbandry.F94L gene by means of created in our lab PCR-RFLP test system (TaqI restrictionendonuclease with the sequence of recognition T/CGA).A section of the animals of the Aberdeen Angus breed  under the following conditions: initial denaturation –95 The detection of the amplification product was conducted by electrophoresis in 3 % agarose gel under 110 V for 30 min.F94L MSTN the allele After the genotyping of each genotyped population the frequency of theheterozygous genotypes (carriers) in percent was calculated by Eq. (1):33.1F94L MSTN polymorphism in the populations ofAberdeen Angus, Hereford and Belgian Blue cattle is presented in Fig. 1. The tests created allow identifying wild-type alleles and mutant ones.The working of the developed test systems for the revealing of theanimal carriers of the genetic defects of AM, OS, DD, M1 and HY and allele variants of the 3.2The screening of the Russian population of Aberdeen Angus cattle hasdemonstrated the presence of animals carrying in their genotypes the mutantalleles associated with the appearance of the AM, DD, OS and M1 genetic defects(Table 3). AMC (arthrogryposis carrier) animals were found in three of five investigatedpopulations with frequencies of 1.06 %–4.89 %. The average frequency was1.84 %. OSC (osteopetrosis carrier) animals were detected in two of three investigatedpopulations with relatively low frequencies (1.01 % and 0.67 %). Theaverage frequency of OSC animals was 0.71 %. The average frequency ofanimal carriers of the M1 genetic defect was the same as OS and consisted of 0.73 %. In only one of four populations (2.1 %) were animals M1C. The highestfrequency of carriers was for the DD genetic defect. DDC  animals weredetected in five at the six investigated populations, at 1.78 %–10.4 %depending on the population. The average figure for DDC animals was 8.87 %.3.3The genotyping of the Russian population of Hereford cattle did not find the animal carriers of the HY and MSUD genetic defects.3.4nt821del11 MSTN polymorphism was also registered in the Belgian Blue cattle breed, we also genotyped the population of this breed for this mutation. As aresult, we found that the absolute majority of the investigated animalsshowed the pattern in Fig. 2 on the electrophoresis and werecarriers of the M1 genetic defect, having in their genotypes the wild-typeallele (310 bp) and the mutant one (144 bp). This fact can be explained bythe fixation of the mutant allele due to the long-term selection for increased muscle mass in the past .Because the 3.5F94L polymorphism showed high frequencies in thetwo populations (3 and 6) of Aberdeen Angus cattle of the desirable F94L MSTN polymorphism: CC – 20.6 %; CA – 48.9 %; AA – 30.5 %.The study of the 44.1It is well known that the best method for the exact identification of themutant alleles linked with genetic disorders such as productivity traitsis DNA analysis. This provides the possibility of getting data about gene mutationsindependently of the gender, age and physiological status of the animal.Previously, DNA tests were developed which were based on the PCR strategy and directed at revealing mutant alleles associated with genetic defects in cattle genotypes in the Aberdeen Angus and Hereford cattle breeds . Until 2017, in Russia, the problem of meat cattle geneticdisorders had not been study in detail and the tools for revealing thelatent carriers of some genetic defects were absent. Only in 2019–2020,based on the data from Online Mendelian Inheritance in Animals (OMIA) and the National Center Biotechnology Information, was the test system for diagnostics of the AM, OS, DD and M1 genetic defects characteristic of the Aberdeen Angus breedand the MSUD and HY characteristic of the Hereford breed using AS-PCR and PCR-RFLPmethods created . Theseachievements are very important for the domestic meat breeding industrybecause they provide the possibility of testing meat cattle and their breeding material on Russian territory.The modern development of molecular biology gives a good possibility ofspeedy gene diagnostics by using the method of real-time PCR. Indeed, thisstrategy is more appropriate for high-throughput genotyping compared tothe end-point PCR diagnostics. However, in our opinion, it is not entirelyreasonable to refuse to use the traditional PCR methods, since any moleculargenetic laboratory should have various diagnostic tools at its disposal. Thedevelopment of test systems based on AS-PCR and PCR-RFLP methods, the mainadvantage of which is less dependence on the computer software, is theinitial stage of work on the problem, and further we do not exclude themodernization of these methods by the most modern molecular genetic techniques.4.2The screening of the investigated Aberdeen Angus cattle for the genetic defectsof AM, OS, DD and M1 mainly revealed separate animal carriers of thegenetic defects in some populations. Some populations were free ofthe mutations causing AM, OS, DD and M1. Relating to the AM, OS and M1genetic defects, the frequencies of animal carriers did not exceed 5 %.The greatest concern regards the genetic defect of developmentalduplication: in populations 4 and 5, the frequency of the occurrence of animalscarrying the defect was 10.3 % and 10.4 % (Table 3).The absence in the studied Hereford populations of animal carriers of thegenetic defects of HY and MSUD was a pleasant surprise for us and is a veryfavorable sign.It is notable that all animals of the investigated Belgian Blue cattle werecarriers of the mutant allele associated with the M1 genetic defect. Themutant allele frequency was 0.5. This fact can be in part explained by thehistory of breeding Belgian Blue cattle. At the beginning of thetwentieth century, the breed was widespread in southern Belgium and used to be adual-purpose breed called the “Mid and Upper Belgium Breed”. However, from the1950s to the 1980s, beef selection by breeders led to an unintentional fixationof the mutant allele associated, as was clarified in 1997 , with mh(muscular hypertrophy) . On the one hand, the mh allele is associated with higher meat yield and more tender meat but on the other hand with an enlarged birth weight of calves and, linked to this, calvingdifficulty, which requires a Cesarean section. This makes this trait a geneticdefect that limits the breeding of Belgian Blue cattle .4.3Despite the fact of the absence of individual carriers of genetic defects in the some investigated populations, we are sure that control of the cattle genetic defects is necessary independently of the breed due to theexistence of the risk of gene mutation spread. Previously, we have alreadyobserved on the farm the absence of animal carriers of the AM and DD geneticdefects in 2015 and the presence of these defects in 2019 (2.73 % AMC and the samefrequency for DDC) .It should be noted that state regulation of the problem by the foundation of breed associations is important. Along with the availability of data onanimal productivity, the most solid associations have data on animals thatcarry inherited abnormalities obtained through DNA analysis. The rules forthe use of such animals and their breeding material are also regulated.These measures prevent the spread of genetic defects and clinicalcases of the inherited diseases.4.4\opttitle{The \textit{F94L} MSTN genotyping displayed the existence of meatproductivity potential in the Russian populations of Aberdeen Angus andBelgian Blue cattle}The identification of genes controlling production traits infarm animal species will lead to cost-effective genetic testingof entire populations for the enhanced selection of individuals, thus providing increased product quality and production efficiency.MSTN .Sellick et al. (2007) stated that “A previously reported transversion in F94LMSTN polymorphism in the both breeds. The frequency of the F94L genotyping showed the existence ofgenetic potential for the productivity traits in the cattle of the Aberdeen Angusand Belgian Blue breeds.The result of the genotyping of the Aberdeen Angus and Belgian Bluepopulations in our study has shown the presence of the desirable 5F94L polymorphism. The use of this mutation as agenetic marker would be able to provide the possibility of obtaining leaner meatand avoiding the problems with animal health due to the absence of theinfluence on calf weight at birth and its association with dystocia.The obtained DNA analysis data have demonstrated the high genetic potentialof the Aberdeen Angus and Belgian Blue cattle due to enough high frequencies ofdesirable variants of the Taking into account the presence in the populations of Aberdeen Angus cattle of animal carriers of the AM, OS, DD and M1 genetic defects, theconstant control for congenital anomalies in Aberdeen Angus herds by means of DNA genotyping is necessary.The results showed the absence in the Hereford populations of individuals carrying the HY and MSUD genetic defects; this is a very good sign, making it possible to use this potential breed without strict limitations.The results of this research allow us to conclude that the breeding ofAberdeen Angus, Hereford and Belgian Blue cattle with proper management has good potential for improving meat cattle industry profitability.


Angiotensin-converting enzyme (ACE) inhibitors and/or in combination with calcium channel blockers (CCBs) are generally recommended as the first-line antihypertensive therapy for people with hypertension and kidney dysfunction. Evidence from large randomized controlled trials comprehensively comparing renal effects of different classes of antihypertensive drugs is lacking.We used a Mendelian randomization study to obtain unconfounded associations of genetic proxies for antihypertensives with kidney function. Specifically, we used published genetic variants in genes regulating target proteins of these drugs and then applied to a meta-analysis of the largest available genome-wide association studies of kidney function (estimated glomerular filtration rate (eGFR), urine albumin-to-creatinine ratio (UACR), and albuminuria). Inverse variance weighting was used as the main analysis and to combine estimates from different sources.Genetically predicted ACE inhibition was associated with higher eGFR  0.008, 0.11), while genetic proxies for beta-blockers were associated with lower eGFR  when meta-analyzing the UK Biobank and CKDGen. Genetic proxies for CCBs were associated with lower UACR  and lower risk of albuminuria  in CKDGen. The associations were robust to using different analysis methods and different genetic instruments.Our findings suggest the reno-protective associations of genetically proxied ACE inhibitors and CCBs, while genetic proxies for beta-blockers may be related to lower eGFR. Understanding the underlying mechanisms would be valuable, with implications for drug development and repositioning of treatments for kidney disease.The online version contains supplementary material available at 10.1186/s12916-021-01951-4. Hypertension is a leading contributor to global years of life lost because of its role in cardiovascular disease . HyperteIn these circumstances, Mendelian randomization (MR) which exploits drug target-related genetic variants to mimic drug effects provides an alternative approach. As genetic variants are randomly allocated at conception and unlikely affected by socioeconomic position or other confounders, the study design minimizes residual confounding and has been successfully applied previously to assess the cardiovascular effects of several drugs such as antihypertensives and lipid-lowering medications , 10. HerWe used an MR study to obtain unconfounded associations of genetic proxies for antihypertensives , CCBs, alpha-adrenoceptor blockers, adrenergic neuron blocking drugs, beta-adrenoceptor blockers (BBs), centrally acting antihypertensive drugs, loop diuretics, potassium-sparing diuretics (PSDs) and aldosterone antagonists, renin inhibitors, thiazides and related diuretics, and vasodilator antihypertensives) with kidney function. Specifically, we used published genetic variants in genes regulating the drug target proteins , 12, as The UK Biobank is a large, ongoing, prospective cohort study, with a median follow-up time of 11.1 years . It recrF-statistic obtained using an established approximation as previously [F-statistic > 10 were used. Details concerning these genetic variants are in Additional file r2 < 0.8, taking into account their correlations obtained from 1000 Genomes European panel using “ld_matrix.” We used Steiger filtering which enables inference of the causal direction, by calculating and comparing the variance explained by the genetic instrument in kidney function and in blood pressure [http://www.phenoscanner.medschl.cam.ac.uk) and summary statistics for creatinine from the UK Biobank, to identify whether the genetic variants (or their proxies (r2 > 0.8)) were associated with other risk factors for kidney function or disease at genome-wide significance. We conducted the analysis by drug class and also by drug target within each drug class and combined the estimates for all drug classes to obtain the overall association of genetic proxies for antihypertensive drugs with kidney function. We also assessed the association of genetic predictors of SBP, identified from a GWAS of SBP without adjustment for antihypertensive medication or BMI ) for ACE inhibitors, ARBs, CCBs, alpha-adrenoceptor blockers, adrenergic neuron blocking drugs, BBs, centrally acting antihypertensive drugs, loop diuretics, PSDs and aldosterone antagonists, renin inhibitors, thiazides and related diuretics, and vasodilator antihypertensives from published sources , 12. Speeviously ; only gepressure . To asser2 < 0.01, obtained using “ld_clump,” for both sets of genetic instruments selection. Details of these genetic instruments are in Additional file From the studies based on a GWAS meta-analysis of the UK Biobank and the International Consortium of Blood Pressure (ICBP) , 16, we The outcomes include eGFR, albuminuria, and UACR. Genetic associations with eGFR were obtained from the UK Biobank individual-level data (application #42468) and from CKDGen summary statistics. Genetic associations with UACR and albuminuria were obtained from CKDGen summary statistics . eGFR wahttps://pan.ukbb.broadinstitute.org/downloads/index.html), of European ancestry, adjusted for age, age2, sex, interaction of sex with age, age2, and 20 principal components.MR estimates were based on the SNP-specific Wald estimates, i.e., the genetic association with kidney function divided by the genetic association with the genetic proxies for antihypertensives. The MR estimates are presented in effect size of SBP, obtained from the latest Pan UK Biobank summary statistics , where applicable. The weighted median estimate is robust to invalid instruments and able to provide consistent estimation even when up to 50% of the weight is from invalid SNPs . MR-PRESAll statistical analyses were conducted using R version 4.0.1  and the R package “MendelianRandomization.”ACE)  Table  and 3. TThe associations of genetically predicted ACE inhibition and BBs with eGFR, as well as the associations of genetic proxies for CCBs with UACR and albuminuria, were replicated using another set of genetic variants based on a meta-analysis of the UK Biobank and ICBP , 16, 20 Genetic proxies for antihypertensive drugs overall had no association with eGFR  for eGFR, (b) for UACR, (c) for albuminuria. Black square refers to using genetic variants based on the study of Walker et al. [Figure S2. Associations of genetic proxies for antihypertensive drugs with eGFR by drug target in each drug class, overall antihypertensives and systolic blood pressure. SBP, systolic blood pressure. ACE, Angiotensin-converting enzyme; ARB, Angiotensin II Receptor Blocker; BBs, beta-adrenoceptor blockers; CCBs, calcium channel blockers; PSDs, potassium-sparing diuretics; SBP, systolic blood pressure. Figure S3. Associations of genetic proxies for antihypertensive drugs with UACR by drug target in each drug class, overall antihypertensives and systolic blood pressure. ACE, Angiotensin-converting enzyme; ARB, Angiotensin II Receptor Blocker; BBs, beta-adrenoceptor blockers; CCBs, calcium channel blockers; PSDs, potassium-sparing diuretics; SBP, systolic blood pressure. Figure S4. Associations of genetic proxies for antihypertensive drugs with albuminuria by drug target in each drug class, overall antihypertensives and systolic blood pressure. BBs, beta-adrenoceptor blockers; CCBs, calcium channel blockers; PSDs, potassium-sparing diuretics; SBP, systolic blood pressure.r et al.  in UK Bir et al.  and Georr et al.  in meta-


These ideotypes were optimised for current climatic conditions and based on optimal physiology, constrained by available genetic variation in target traits. A ‘genetic yield gap’ in a location was estimated as the difference between the yield potential of the optimal ideotype compared with a current, well-adapted cultivar. A large mean genetic yield potential (11–13 t ha−1) and genetic yield gap (3.5–5.2 t ha−1) were estimated under rainfed conditions in Europe. In other words, despite intensive wheat breeding efforts, current local cultivars were found to be far from their optimum, meaning that a large genetic yield gap still exists in European wheat. Heat and drought tolerance around flowering, optimal canopy structure and phenology, improved root water uptake and reduced leaf senescence under drought were identified as key traits for improvement. Closing this unexploited genetic yield gap in Europe through crop improvements and genetic adaptations could contribute towards global food security.Improving yield potential and closing the yield gap are important to achieve global food security. Europe is the largest wheat producer, delivering about 35% of wheat globally, but European wheat's yield potential from genetic improvements is as yet unknown. We estimated wheat ‘genetic yield potential’, i.e. the yield of optimal or ideal genotypes in a target environment, across major wheat growing regions in Europe by designing  •−1) and genetic yield gap (3.5–5.2 t ha−1) estimated by ideotypes for Europe.Large wheat genetic yield potential (11–13 t ha•The wheat genetic yield potential has still to be fully utilised by breeders in Europe.•Closing the unexploited genetic yield gap in Europe would contribute to global food security. It has www.yieldgap.org)  under optimal crop management (irrigated/rainfed) and the average actual yield achieved by farmers under dominant management practices . Crop yigap.org) . It shougap.org) .in silico crop ideotype design in genetic crop improvement for yield gain has been outlined by different researchers  is one of the key staple crops for global food security, providing about 20% of total dietary calories and protein  is well calibrated and validated locally and (b) has a computational framework for multidimensional optimisation of cultivar traits by utilizing the full parameter space. Designing crop ideotypes also depends on interactions between crop modellers and breeders/geneticists for identifying feasible trait combinations . In the 22.1LARS-WG 6.0)  . To acco2.2Sirius (2018) is a processed-based eco-physiological wheat simulation model with a daily timescale, which includes an optimisation framework for designing crop ideotypes and optimisation of cultivar traits for target environments . The mod2.3An ideotype was defined as a set of Sirius cultivar parameters (traits) that would deliver high yield when optimised in a target environment. A cultivar based on an ideotype and utilizing its optimal combination of trait values would deliver maximum yields for the environment in question. The wheat ideotypes were optimised for grain yield under current climate in rainfed conditions at individual study sites across Europe. Local current wheat cultivars  were used as ‘parents’ for designing site-specific wheat ideotypes. A list of the 21 Sirius cultivar parameters is presented in 2.4max). This is a key trait, influencing crop growth and final grain yield by modifying the rate of canopy expansion and the maximum achievable leaf area, which in turn change the pattern of light interception and transpiration demand  The ‘stay green’ trait (SG). This trait is important in increasing grain yield by delaying leaf senescence after anthesis and maintaining the green leaf area longer for photosynthesis to continue during grain filling . (4) Daylength response (Pp). (5) ‘Duration of grain filling’ (Gf). This trait controls the rate of phenological development and physiological maturity . This is a key trait affecting temporal patterns and total amount of water uptake (ss). This is a critical trait that controls the increase in the rate of leaf senescence due to water stress under drought  Phymaturity , 2007. Win yield . (6) ‘Rar uptake . Faster  drought . Earlier2.5At each site, both the ideotypes (iS and iT) were optimised independently for maximum grain yield in rainfed conditions under the local current climate. A universal search optimisation method, evolutionary search algorithm with self-adaptation (EASA), was used in Sirius to optimise ideotypes by utilizing the full parameter ranges in a multi-dimensional parameter space with a complex fitness function . Local w2.6https://sites.google.com/view/sirius-wheat) was used. A common medium soil-water profile (Hafren) with a total available water capacity of 177 mm was used at all sites to eliminate site-specific soil effects from the analysis. Sirius was run first for current local wheat cultivars (cv) in rainfed conditions under the current climate to assess present management optimal yields (YW). Then, Sirius was used for designing wheat ideotypes (heat and drought sensitive (iS) or tolerant (iT) around flowering) under the same current climate in rainfed conditions using the same sowing dates as for the current local cultivars. 2 level if the Rubisco specificity factor (λ) that represents the discrimination between CO2 and O2 were optimal. Thus, a 10% increased light use efficiency (LUE) was used for the wheat ideotypes. All simulations were assumed to be water-limited under rainfed conditions and optimally managed, e.g. no nutrient deficiency and no yield losses due to disease, pests or competition with weeds.Sirius version 2018 (available from 2.7iG) of wheat in water-limited condition (rainfed) under the current climate in a given location was estimated as (iW is the water-limited yield (t ha−1) of wheat ideotype optimised by Sirius in a given location, representing ‘genetic yield potential’ (W is the yield (t ha−1) of the current local well-adapted cultivars in rainfed condition under optimal management estimated by using Sirius.The ‘genetic yield gap’  of genetic yield potential as33.1W) under the current climate varied from 6 to 10 t ha−1 across Europe, with a mean yield of 7.7 t ha−1 (−1) was obtained in north-western (NW-) Europe , followed by central-western (CW-) (~8 t ha−1)  and central-eastern (CE-) (7–8 t ha−1) Europe , whereas yield was lowest (6–7 t ha−1) in south-western (SW-)  and north-eastern (NE-) Europe .The simulated management optimal grain yields of current well-adapted local wheat cultivars in rainfed condition  and iT (12.9 t ha−1) represented 45% (3.5 t ha−1) and 69% (5.2 t ha−1) greater yields, respectively, than corresponding YW. Over Europe, the mean yield of the iT ideotype was 18% (1.7 t ha−1) greater compared to iS. However, the yield difference between iT and iS ideotypes varied widely (from 3 to 55%) at individual sites as described below. Across both the ideotypes, yield potential was highest (13–15 t ha−1) in NW and CW Europe, whereas the lowest yield potential (7–11 t ha−1) was in SW Europe. Wheat yield potential was intermediate (10–13 t ha−1) in NE and CE Europe.Ys Europe . Average3.3-1; ~39–55%) of heat and drought tolerance around flowering compared with heat and drought sensitivity was obtained in SW Europe, followed by NE and CE Europe , whereas a minimum or almost no benefit was found in NW and CW Europe  (max ~77%) and stay-green trait (SG ~91%), was identified as important for high yield potentials in Europe, as was optimal phenology, in terms of anthesis time, grain filling period and physiological maturity (crop duration) (u ~73%) indicated that an improved root system would be beneficial for achieving high potential yields. In contrast, reduced leaf senescence due to water stress (Wss ~11%) would be helpful for improved photosynthesis and grain yields. Overall, a better optimal canopy architecture, phenology and reduced leaf senescence were obtained for the iT ideotype than the iS, but a greater root water uptake rate was found for iS  . Thus, turation) , Fig. 3.3.4iG) under rainfed conditions ranged between 0.6 and 6 t ha-1 across ideotypes in major wheat growing regions in Europe, representing 8–48% of wheat genetic yield potential  for the iS ideotype and 40% (5.2 t ha-1) for the iT. For the iS ideotype, the absolute yield gap was highest (4–5 t ha-1) in NW and CW Europe, whereas it was lowest (≤1 t ha-1) in SW Europe. The yield gap was intermediate (2–4 t ha-1) in NE and CE Europe. In contrast, variation in the yield gap for the iT ideotype was relatively small between sites; indeed, the yield gap was almost equal (5–6 t ha-1) at all the sites except for one site in SW Europe . The absolute yield gap as well as the percent yield gap at individual sites and over Europe as a whole were greater for the iT ideotype than iS: The mean absolute yield gap over Europe was 49% (1.7 t ha-1) greater for iT ideotype than iS, while the highest yield gap difference between the iT and iS ideotypes was in SW-Europe (3–4 t ha-1), followed by NE and CE Europe (1–3 t ha-1), whereas the difference was minimum (<1 t ha-1) in NW and CW Europe.The current wheat genetic yield gap  and genetic yield gap (YiG) under the current climate in water limited conditions across Europe by optimising ideotypes in local environmental conditions. Ideotype optimisation was based on state-of-the-art knowledge in crop physiology  as 4–13 t ha-1, with associated yield gaps of 0.2–6.9 t ha-1 across Europe, with the highest and lowest potential yield and yield gaps in NW and SW Europe, respectively, due to these regions having the most and least favourable climatic conditions  and maximum primary grain setting (carbon sink) at anthesis, and influences the climatic conditions  that prevail during grain filling . Early fh and Gf  could heh and Gf .u), is often recommended for avoidance, survival or tolerance of severe drought and heat stress (ss) linked to an overall drought tolerance during the whole crop season is an important trait for reducing leaf senescence and increasing photosynthesis and yield under water stress conditions  with minimum heat and drought stresses around flowering. This helps the better optimisation of cultivar traits, such as delayed flowering time, longer grain filling period and crop duration, resulting in greater intercepted solar radiation and yield . In contThe highest genetic yield potential and genetic yield gap were in NW and CW Europe, with intermediate genetic yield potential and genetic yield gap in CE and NE Europe, indicating that current local wheat cultivars are not yet optimised for yield  and thatA vast available genetic variation exists for different yield-improving traits in wheat germplasm  still exists in Europe. Heat and drought tolerance around flowering, optimal canopy structure and phenology, improved root water uptake and reduced leaf senescence due to water stress were identified as key traits for genetic adaptation to achieve genetic yield potentials. Closing a large unexploited genetic yield gap in Europe would contribute towards genetic yield improvement and underpin global food security.In conclusion, a large wheat genetic yield potential of 11–13 t haThe authors declare no conflicts of interest.


The gut microbiome of animals, which serves important functions but can also contain potential pathogens, is to varying degrees under host genetic control. This can generate signals of phylosymbiosis, whereby gut microbiome composition matches host phylogenetic structure. However, the genetic mechanisms that generate phylosymbiosis and the scale at which they act remain unclear. Two non‐mutually exclusive hypotheses are that phylosymbiosis is driven by immunogenetic regions such as the major histocompatibility complex (MHC) controlling microbial composition, or by spatial structuring of neutral host genetic diversity via founder effects, genetic drift, or isolation by distance. Alternatively, associations between microbes and host phylogeny may be generated by their spatial autocorrelation across landscapes, rather than the direct effects of host genetics. In this study, we collected MHC, microsatellite, and gut microbiome data from separate individuals belonging to the Galápagos mockingbird species complex, which consists of four allopatrically distributed species. We applied multiple regression with distance matrices and Bayesian inference to test for correlations between average genetic and microbiome similarity across nine islands for which all three levels of data were available. Clustering of individuals by species was strongest when measured with microsatellite markers and weakest for gut microbiome distributions, with intermediate clustering of MHC allele frequencies. We found that while correlations between island‐averaged gut microbiome composition and both microsatellite and MHC dissimilarity existed across species, these relationships were greatly weakened when accounting for geographic distance. Overall, our study finds little support for large‐scale control of gut microbiome composition by neutral or adaptive genetic regions across closely related bird phylogenies, although this does not preclude the possibility that host genetics shapes gut microbiome at the individual level. This study uses the Galápagos mockingbird species complex to test the relationship between gut microbiome composition and host genetic structure at the population level based on both MHC and microsatellite genotypes. We found no relationship between microbiome composition and genetic distance based on either MHC or microsatellite markers. This suggests that, at this geographic scale, host population genetics exerts little influence on the gut microbiome. Both host‐associated microbiomes and host genetics are subject to isolation by distance due to limited dispersal of both hosts and microbes  across four Galápagos mockingbird species to test the relative contribution of neutral and adaptive genetic markers in shaping the gut microbiome composition. The Galápagos mockingbirds comprise four generalist species distributed allopatrically across almost all islands of the Galápagos Archipelago, diverging into four genetically divergent clades approximately 500,000  years ago after a single colonization event  that allopatrically inhabit islands of the Galápagos Archipelago were sampled between 2004 and 2008 as part of one overarching study. Blood samples were collected from 2004 to 2008 and used for MHC and microsatellite genotyping; fecal samples were collected largely from different individuals  between 2006 and 2008. Sample metadata is listed in Table Four endemic mockingbird species , which was afterward removed from the final tree. We then calculated pairwise dissimilarity of allele composition between all individuals per island, also taking allele genetic similarity into account, using the distance function in phyloseq  gene. Paired‐end sequencing of the amplicons was performed with Illumina MiSeq technology over 2 × 250 cycles. The paired‐end sequencing results were processed in QIIME 2 , and one representing the island common core . We also applied 30% and 70% prevalence thresholds for the island core with no difference to the results. The island common core was applied to accentuate the differences in common gut microbes between islands, and because any effects of host genetics may act on local microbial communities. We excluded low prevalence taxa in the full microbiome dataset because they are difficult to model statistically and to limit computational time for the  Methods .2.3We conducted analyses at both the individual level (to assess variation within each genetic layer) and island level (to test for associations between microbiome composition and host genetics). We estimated the effects of species and island on dissimilarity in gut microbiota ASV distributions (full microbiome and island core), genetic dissimilarity based on MHC allele frequencies, and genetic dissimilarity based on microsatellite markers, respectively, by using PERMANOVA tests on individual distance matrices described above, presenting standardized effect sizes (SES).ecodist , there is a risk of overfitting the models, and model selection on MRMs using AIC generates spurious results  that predict mean pairwise distance of microbiome composition between islands, using the package BEDASSLE , which was developed to quantify the individual effects of geographic distance and ecological variables on population genetic structure . We found that a one‐unit increase in microsatellite genetic distance  shifted microbiome distributions by the equivalent of 45.4 km  with increased power to detect weak effects and associations between species alleles and microbial taxa, thereby helping to elucidate underlying mechanisms driving phylosymbiosis. For example, mockingbird species may share genetic elements that strongly affect the microbiome yet would not be detected here. Our results therefore do not preclude that host genetics influences gut microbe distributions in this system, yet suggest that if such control exists it is likely to be targeted towards specific microbial genera, governed by genetic regions not assessed in this study, or only detectable at larger phylogenetic scales. MHC polymorphism has been associated with the abundance of a limited number of microbial families  and adaptive (MHC) genetic regions in shaping gut microbiome diversity in Galápagos mockingbirds. Whileour study is necessarily limited by the number of mockingbird populations, our study is the first to simultaneously test the independent contributions of neutral and MHC dissimilarity on gut microbiome distributions across a well‐studied avian species complex. Our results support previous studies finding weak effects of phylogeny on bird gut microbiomes, yet go further to suggest that weak correlations between host genetics and microbiomes in allopatric populations may be a consequence of spatial autocorrelation between these factors. Future research that investigates the targeted effects of host genetics on specific microbial lineages within individuals may elucidate the relationship between avian genetics and the gut microbiome at a finer‐scale resolution.No authors have any conflicting interests in regard to this paper.Ramona Fleischer: Conceptualization (supporting); Formal analysis ; Investigation (lead); Methodology (lead); Writing‐original draft ; Writing‐review & editing . Alice Risely: Conceptualization ; Formal analysis ; Supervision ; Validation ; Writing‐original draft (lead); Writing‐review & editing . Paquita E. A. Hoeck: Data curation (lead); Investigation (supporting); Methodology (supporting); Writing‐review & editing . Lukas Fridolin Keller: Data curation (lead); Investigation (supporting); Methodology (supporting); Writing‐review & editing . Simone Sommer: Conceptualization ; Resources (lead); Supervision (lead); Writing‐review & editing .Samples were collected with the permission of Galápagos National Park Service (permit number PC‐29‐05).Fig S1‐S4Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Methods S1Click here for additional data file.