Colocasia esculenta Schott  is an excellent local cultivar of the genus polymorpha in Jiangsu Province, China.Colocasia esculenta Schott to evaluate its anti-cancerous properties.In the present study, we have performed a comparative study before and after dietary consumption with Colocasia esculenta Schott daily was conducted for 1 month. Plasma samples from the subjects before and after dietary consumption with Colocasia esculenta Schott were analyzed with proximity extension assays for the alteration of 92 proteins in relation with cancers, while blood samples were examined for physiological parameters with an automatic biochemical analyzer. Bioinformatic analyses were conducted using MalaCards and GEPIA.Forty-two healthy volunteers were recruited, and dietary consumption with 200 g of tap water cooked Colocasia esculenta Schott, circulating CYR61, ANXA1, and VIM protein levels in the subjects was found to be most significantly downregulated, while for ITGB5, EPHA2, and CEACAM1, it was upregulated. Alternation of these proteins was predicted to be associated with the development of tumors such as pancreatic adenocarcinoma and breast and prostate cancers.After taking dietary consumption with Colocasia esculenta Schott, as a healthy food, has anti-cancerous properties. Further investigation of phytochemistry in Colocasia esculenta Schott has been taken into our consideration.The present study provides evidence that  Colocasia esculenta Schott is a local cultivar of the genus polymorpha in Jingjiang City, Jiangsu Province, China.Blood samples collected from volunteers before and after the dietary intervention with Colocasia esculenta Schott are analyzed with proximity extension assays.Data demonstrate that Colocasia esculenta Schott, as a healthy food, has anti-cancerous properties.Colocasia esculenta Schott is an annual herbaceous perennial plant. This plant not only is rich in starch, vitamins, minerals, and other nutrients but also has biological properties in metabolism, including antimicrobial, anti-hepatotoxic, anti-cancerous, anti-lipid-peroxidative, anti-melanogenic activities, etc.  for the detection of nucleic acids and antibody-based methods for the detection of proteins. Proximity extension assays (PEA) are developed based on the combination of these key technologies and become the immunoassay for the detection of protein molecules via DNA ligation and amplification, offering high specificity and sensitivity –9. ThereColocasia esculenta Schott. The purpose of this study was to analyze the biological properties of Colocasia esculenta Schott from Jingjiang city, Jiangsu Province, China, in relation with oncology.In the current study, we applied the advantages of PEA to analyze the alteration of 92 proteins in relation with cancers for the healthy subjects before and after dietary intervention with A total of 42 volunteers  were recruited for this study. They were either university or postgraduate students and at the age between 20 and 35 years old. All subjects had an annual healthy examination in university hospital, and no disease in stomach, liver, and kidney was recorded. Physiological characteristics of all subjects are summarized in The current study was approved by the local ethical committee in the affiliated Zhongda Hospital, Southeast University, China, and the IBR number is 2016ZSDYLL075-P01. An informed consent was obtained from all subjects before the study began.Colocasia esculenta Schott, to be remained unchanged in their lifestyles and eating habits. Therefore, smoking and alcohol drinking was not allowed in prior to 6 weeks period and during the program. In whole study program, all participates consumed 200 g of tap water cooked Colocasia esculenta Schott  is commonly used for the evaluation of the nutritional status of adults over 18 years of age, and the formula is BMI = weight (kg)/height (m2). Body composition was analyzed using a five-factor method  and a four-pole eight-point contact electrode method.Body height, weight, waist circumference, hip circumference, heart pulse, blood pressures, body composition, and basal metabolic rate of all subjects were measured by professional nurses under the morning fasting state. Body mass index  after the program ended, physiological and biochemistry parameters of all subjects were measured, respectively. In the meanwhile, anticoagulant and non-anticoagulant blood samples (5 mL each and fasted for 12 h in advance) were collected. The anticoagulant blood samples were used for plasma extraction, while non-anticoagulant blood samples were used for serum extraction. All plasma and serum samples were stored at –80°C and then evaluated before used for analyses. The sample used for analysis was not a pool plasma.Colocasia esculenta Schott treatment were performed with the PEA protocol . Two paired antibodies (each with target antigen specificity) were coupled to unique oligonucleotides (A and B), each oligonucleotide having a combination of universal amplification primers and template location of specific detection primers. One site was used for paired annealing between oligonucleotides A and B, and another site was used for binding to molecular beacons and qPCR detection. The performance of all specific qPCR primers has been evaluated by their amplification efficiency. Totally, 92 targeted proteins for analyses were included in the PEA experiments, and all the studied proteins are listed in the Supplemental The experiments for analyses of the plasma samples before and after protocol –9. The awww.biopython.org) (https://gepia.cancer-pku.cn/) (https://www.biovista.com/vizit) based on the references from PubMed (https://www.ncbi.nlm.nih.gov/pubmed)  . MalaCarpku.cn/) . Biovist/pubmed) , 13. We,P-value less than 0.05 was considered significant.Statistical analysis was performed using the PASW (Version 22.0) and R software (Version 3.6.0). The statistical power on the size of subjects was tested prior to the experiments. Thereafter, the analyses of quantitative data were performed using the paired t-test for normally distributed parameters, while the Wilcoxon test of ranks was alternatively used for traits with non-normal distributions. OPLS-DA  analysis was performed by R statistical analysis software. All data are expressed as means or percentages with standard errors. Colocasia esculenta Schott dietary intervention. As observed in the score plots from OPLS-DA analysis, the studied 92 proteins were found to be clustered in the baseline and after Colocasia esculenta Schott dietary intervention along the tp1 direction , while five proteins were downregulated (in blue) , while integrin subunit beta 5 (ITGB5), H receptor A2 (EPHA2), and CEA cell adhesion molecule 1 (CEACAM1) were upregulated   . Further.68E–06) . Data anColocasia esculenta Schott was analyzed by using the programs of MIFTS. According to MIFTS, the hits of cancers were listed in Colocasia esculenta Schott might have antitumor effects. Further analyses with GEPIA and Biovista/vizit programs were performed and data were summarized in Supplemental Fig. 3. Several cancers, including lymphoid neoplasm diffuse large B-cell lymphoma, glioblastoma multiforme (GBM), pancreatic adenocarcinoma (PAAD), thymoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal clear cell carcinoma, testicular germ cell tumors, thyroid carcinoma, brain lower grade glioma, liver hepatocellular carcinoma, skin cutaneous melanoma, and acute myeloid leukemia (AML), were found to be associated with increased ANXA1, CYR61, and VIM expressions, while all these three protein alternations were associated with PAAD . The decreased expression of CEACAM1  and EPHA2  was found to be associated with several cancers, but ITGB5 reduction in expression was associated with acute myeloid leukemia (LAML) only .The association of all 22 alternated proteins after dietary intervention with Colocasia esculenta Schott. We have used PEA, which offers high specificity and sensitivity for protein analysis  is involved in the transduction of growth factor and hormone signaling. Several studies have demonstrated that the CYR61 expression is correlated with worse prognosis of breast, colorectal, and prostate cancers –17. ANAXColocasia esculenta Schott is included in analyses, and no patient with cancers is recruited in clinical trial. Recently, Pereira et al. have demonstrated that tarin is extracted from Colocasia esculenta Schott and exhibits recognized biocide activities against viruses and insects. Therefore, tarin, as a GNA-related lectin, may have the potential prophylactic and therapeutic actions on hematopoietic and cancer cells, as a potential immunomodulator (There is a limitation in the present study because no major chemical component in odulator .Colocasia esculenta Schott is a heathy food and has anti-cancerous biological properties. Further investigation in basic research of chemical compounds such as tarin in Colocasia esculenta Schott and clinical trial for patients with cancers has been taken into our consideration.In conclusion, the current study provides evidence that Click here for additional data file.


In this paper, an eco-efficiency analysis is conducted using the epsilon-based measure data envelopment analysis (EBM-DEA) model for Russian cities along the Northern Sea Route (NSR). The EBM-DEA model includes five input variables: population, capital, public investment, water supply, and energy supply and four output variables: gross regional product (GRP), greenhouse gas (GHG) emissions, solid waste, and water pollution. The pattern of eco-efficiency of 28 Russian cities along the NSR is empirically analyzed based on the associated real data across the years from 2010 to 2019. The empirical results obtained from the analysis show that St. Petersburg, Provideniya, Nadym, N. Urengoy, and Noyabrsk are eco-efficient throughout the 10 years. The results also indicate that the cities along the central section of the NSR are generally more eco-efficient than those along other sections, and the cities with higher level of GRPs per capita have relatively higher eco-efficiency with a few exceptions. The study provides deeper insights into the causes of disparity in eco-efficiency, and gives further implications on eco-efficiency improvement strategies. The contributions of this study lie in the fact that new variables are taken into account and new modeling techniques are employed for the assessment of the eco-efficiency of the Russian cities. The Russian Arctic covers an area of about 3 million m of land . The tot of land . In term of land . In addiFor decades, Russia has been a major producer and exporter of natural resources and its economy growth is driven by the associated exports ,7,8. TheHowever, it will be difficult for a single country to develop the NSR and the natural resources in the Russian Arctic. Its development will require extensive human resources, advanced technologies and equipment, as well as high quantities of capital investment . Hence, For a long time, the Russian authorities have prioritized resource-based economic development over environmental issues, which has led to environmental damage. As an environmentally fragile area, the Russian Arctic is at great risk due to overexploitation and the activities involved in using the NSR ,16. It iFacing the fragile ecological environment in the Russian Arctic, Russian government is taking measures to protect it and facilitate regional sustainable development. For example, the “Arctic Strategic Action Program 2009” proposes measures to prevent, eliminate, and reduce the consequences of adverse environmental impacts. In 2010, Putin launched a plan to clean up the Russian Arctic. A waste disposal practice began in 2012 and takes place every summer in the polar islands of Barents Sea and other Arctic regions . In 2012The economic development of the Russian Arctic is achieved at the cost of environmental pollution and ecological destruction. Excessive environmental protection can put a brake on economic development in some cases. Such situations drive the Russian government to balance economic development and environmental protection to ensure sustainable development of the Russian Arctic. It is hence of great significance to evaluate and analyze the relationship between the economic development and environmental protection of the Russian Arctic. Eco-efficiency can be appropriate for measuring the relationship between economic development and environmental protection ,22.In this study, data envelopment analysis (DEA) is used to evaluate the eco-efficiency of the Russian cities along the NSR (most of them are located in the Russian Arctic), as it has been widely used in the areas regarding evaluation of economic and environmental sustainability . The patThe major contributions of this study can be summarized in three aspects as follows.Comprehensive panel data for the period of 2010–2019 shown in  are collCompared with previous studies, this study introduces new variables in the evaluation of ecological total-factor energy efficiency (TFEE). It can provide a new perspective for the related stakeholders to recognize, evaluate, and analyze more comprehensively and deeply the eco-efficiency of the Russian cities along the NSR.The epsilon-based measure data envelopment analysis (EBM-DEA) model is applied to the evaluation and analysis of sustainable development of the Russian cities along the NSR. The EBM-DEA model can effectively solve the problems, which exist in radial and non-radial direction.The remainder of this paper is organized as follows. In the traditional sense, eco-efficiency is a term used to describe the quantity of economic benefits per unit of ecological energy . Higher Eco-efficiency is currently a research focus due to its theoretical value and practical significance ,29. It h2) emission are included in the associated model. Li and Hu [2, and sulfur dioxide (SO2) as outputs. Zhang et al. [2 and SO2 emissions and chemical oxygen demand (COD) in China from 2001 to 2010, they carried out an empirical analysis of regional ecological energy efficiency. Based on the SBM-DEA models, Choi et al. [2 emission and energy, potential CO2 emission reduction, and marginal cost of CO2 emission in 30 provinces of China from 2001 to 2010. Li et al. used DEA to study the eco-efficiencies of China at provincial levels and the associated driving factors [Many DEA studies have also focused on regional or international eco-efficiency. Zhou et al.  proposedi and Hu  construcg et al.  used thei et al.  analyzed factors . Lorenzo factors  extensiv factors  studied It is evident from the above studies that DEA has been widely used to evaluate eco-efficiency. In addition, DEA has been proven to be a useful and valuable tool for decision makers. Hence, DEA was used in this study to evaluate the eco-efficiency of environmental governance and economic development of Russian cities along the NSR.Compared with other methods, DEA has the following advantages. (1) It does not require the estimation of the production function in advance . (2) It The DEA model mainly evaluates the relative efficiency of DMUs. It generates the efficiency by analyzing the frontier of input and output variables. It has several variants, e.g., the Charnes–Cooper–Rhodes (CCR) model, the Banker-Charnes-Cooper (BCC) model, the SBM model, and the EBM model. The CCR model proposed by Charnes et al.  assumes The CCR and BCC model are both radial DEA models, where non-radial relaxation variables are ignored. Moreover, the SBM model fails to consider the characteristics of the radial model. Tone and Tsutsui  proposedSuppose that there are n DMUs in this study. Each DMU denoted by Under the assumption of constant returns to scale, the input-oriented CCR model is used to evaluate the technical efficiency In Equation (1), the range of Under the assumption of constant returns to scale, the input-oriented SBM model proposed by Tone  can be uIn Equation (2), The EBM model proposed by Tone and Tsutsui  has bothIn Equation (3), Proposition 1.If= 1 and= 1, the model simplifies to an input-oriented SBM model.Proposition 2.The model has a finite optimal value,.Proposition 3.is non-increasing in.Definition 1.(EBM input-efficiency). When0 is called EBM input efficiency.DMUDefinition 2.(EBM projection). Let the optimal solution to Equations (1)–(3) be (Tone and Tsutsui (2010) defined the projection of DMUas follows:EBM models are used for the eco-efficiency analysis of the 28 Russian cities along the NSR in this study. The variables used in the models include population, capital, public investment, water supply, energy supply, gross regional product (GRP), GHG emissions, solid waste, and water pollution. The first five of the abovementioned variables are used as the inputs of the EBM models, and the rest are taken as the associated outputs. The eco-efficiency scores of the cities along the NSR are generated based on the EBM model, which are exhibited in Regarding the spatial distribution of the eco-efficiency of these cities shown in , it can To look at how the eco-efficiency of the cities might be improved, it is necessary to generate the results of inputs and outputs optimization. The average annual percentage changes in the inputs and outputs for these cities are shown in In terms of population, some cities such as Onega, Vladivostok, and Olenegorsk need to reduce their populations significantly to achieve eco-efficiency according to the results of EBM-DEA models. The authorities need to develop a set of preferential policies to encourage immigration between the associated cities, so that the cities that have a real demand of population can obtain population growth and those that have excessive populations can obtain fewer population to achieve eco-efficiency. Regarding water supply, the cities such as Murmansk, Kandalaksha, Dudinka, Vanino, Nakhodka, Norilsk, and Olenegorsk need to take measures to reduce the water supply substantially to achieve eco-efficiency. With regards to energy supply, in order to be eco-efficient, 10 out of 28 cities need to cut their energy supply by more than 40%. In respect of GRP, a remarkable fact is that the GRPs of most cities need to be increased substantially , so that these cities can achieve eco-efficiency. This suggests that according to the efficiency generated by the models, most of the Russian cities along the NSR are economically inefficient, which is generally attributed to the factors such as outdated infrastructure, tiny populations, harsh natural environment, simple economic structure, and insufficient financial and technological resources. It is recommended to update these cities’ outdated infrastructure, and develop more regional infrastructure and transportation projects. The “North Latitude Passage” is one of the related key infrastructure projects. It will advance the effective development of the rich natural resources in the Russian Arctic areas . The authorities need to continue developing the communication and coastal infrastructure  along the NSR, which will ensure safety of commercial transits through the NSR. The successful functioning of the NSR will bring more development opportunities to the cities along the NSR. The logistical hubs at the end points of the NSR  can be created to serve domestic and international shipping. The Russian Arctic has great potential for economic development due to its rich natural resources and geographical significance. Its infrastructure construction and oil, gas, and mineral resource development have become the main contributors to current economic growth . It is sRegarding the excesses of negative outputs  are added to the model in this study. Regarding the policy implications, in order to achieve higher eco-efficiency, the related authorities and stakeholders should devote more resources in multiple fields to the cities along the eastern and western section of the NSR. They need to develop renewable energy or eco-efficient projects such as using wind and geothermal energy resources in these cities. It is necessary to develop more industrial sectors  that are not limited to traditional energy industries based on the regional characteristics in these cities, and extend economic functions of these cities. Investment support tools should be used to attract investment to these Russian cities. It is important to renovate obsolete infrastructures, and carry out more regional infrastructure and transportation projects. It is also suggested to promote the further construction and development of the projects about oil, gas, and mineral extraction in the Russian Arctic. Particular emphasis should be placed on the circular economy for the sustainable development of the Russian Arctic cities. The clean-up of environmental damage and establishment of specially protected natural territories and reserves are effective measures for the balance between economic development and environmental protection. More resources should be invested to the related research for the sustainable development of the Russian cities along the NSR.However, there are still unconsidered issues in this study, which can be further studied. There are many variables in the eco-efficiency model that have a carryover effect. The dynamic model will be improved to study the effect. In addition, the single process model usually ignores the conflicts between different departments within the process. The network methods will be used to conduct further research on existing models to discover the impact of conflicts between departments. Finally, the existing evaluation methods are based on the assumption of diminishing marginal productivity. It ignores that the existence of economies of scale may cause evaluation errors. Finding out how to consider the effect of economies of scale and the effect of diminishing marginal productivity within an evaluation method requires further attention.


Genetic markers are employed widely in molecular studies, and their utility depends on the degree of sequence variation, which dictates the type of application for which they are suited. Consequently, the suitability of a genetic marker for any specific application is complicated by its properties and usage across studies. To provide a yardstick for future users, in this study we assess the suitability of genetic markers for molecular systematics and species identification in helminths and provide an estimate of the cut-off genetic distances per taxonomic level.We assessed four classes of genetic markers, namely nuclear ribosomal internal transcribed spacers, nuclear rRNA, mitochondrial rRNA and mitochondrial protein-coding genes, based on certain properties that are important for species identification and molecular systematics. For molecular identification, these properties are inter-species sequence variation; length of reference sequences; easy alignment of sequences; and easy to design universal primers. For molecular systematics, the properties are: average genetic distance from order/suborder to species level; the number of monophyletic clades at the order/suborder level; length of reference sequences; easy alignment of sequences; easy to design universal primers; and absence of nucleotide substitution saturation. Estimation of the cut-off genetic distances was performed using the ‘K-means’ clustering algorithm.The nuclear rRNA genes exhibited the lowest sequence variation, whereas the mitochondrial genes exhibited relatively higher variation across the three groups of helminths. Also, the nuclear and mitochondrial rRNA genes were the best possible genetic markers for helminth molecular systematics, whereas the mitochondrial protein-coding and rRNA genes were suitable for molecular identification. We also revealed that a general gauge of genetic distances might not be adequate, using evidence from the wide range of genetic distances among nematodes.This study assessed the suitability of DNA genetic markers for application in molecular systematics and molecular identification of helminths. We provide a novel way of analyzing genetic distances to generate suitable cut-off values for each taxonomic level using the ‘K-means’ clustering algorithm. The estimated cut-off genetic distance values, together with the summary of the utility and limitations of each class of genetic markers, are useful information that can benefit researchers conducting molecular studies on helminths.The online version contains supplementary material available at 10.1186/s13071-021-04737-y. Advances in molecular biology have accelerated the use of various molecular techniques and genetic markers in the fields of molecular systematics and taxonomy. Molecular genetic markers are DNA segments of the genome that can provide molecular information enabling the differentiation of taxa , 2. The c oxidase subunit I (COI) and the NADH dehydrogenase subunit 1 (NAD1) genes and the 12S and 16S ribosomal RNA (rRNA) genes. Conversely, nDNA, particularly the nuclear rRNA genes, is more conserved than mtDNA. The highly conserved sequences make the nuclear rRNA genes a potentially helpful source of genetic markers for resolving higher taxonomic levels for organisms [Genetic markers can be designed from different DNA regions from either the nuclear or mitochondrial genomes. The utility and resolution of each genetic marker depend highly on the degree of sequence variation of the marker . Comparerganisms . Within rganisms , 10, 11.Taenia spp. for cestodes, Echinostoma and Schistosoma spp. for trematodes and Trichuris spp. for nematodes [Many studies have utilized genetic markers for molecular systematics and identification studies within the three groups of helminths . Although the three groups of helminths are collectively grouped together, due in part to their parasitic lifestyle, nematodes and platyhelminths (cestodes and trematodes) are phylogenetically far apart from each other . In moleematodes –31. In aematodes , 33.. used a likelihood ratio test to assess the fit of phylogenetic tree branch lengths to define putative species and set species boundaries for insects [Despite the many successes reported in using genetic markers in molecular studies of helminths for systematics and identification purposes, there is a caveat. Estimates to determine what constitutes ‘sufficient’ genetic variation among taxa and across taxonomic levels using genetic distances varies, depending on the genetic marker used and the taxa studied. Researchers have used genetic distance widely, at both the inter-and intra-species level, as a gauge for deciding whether specimens are conspecific. In general, a genetic difference of approximately 10% among closely related species using mitochondrial protein-coding genes is the basis for comparison to determine if the specimens are conspecific . Species insects . The Poi insects . Another insects . Given tThe aim of this study was to assess the suitability of nuclear and mitochondrial genetic markers for molecular systematics and identification purposes. The aim was achieved by comparing the properties of four classes of genetic markers  across taxonomic hierarchy levels to ascertain their suitability for molecular systematics and species identification. Secondly, we aimed to estimate a cut-off for each genetic marker using the ‘K-means’ clustering method with genetic distances. The ‘K-means’ clustering approach has been extensively used in various applications, including DNA sequences for phylogenetic analysis and identifying invasive weed species –39.Our ultimate goal was to provide a guide for researchers studying future applications of genetic markers, in terms of molecular systematics and species identification, for helminths, through our assessment of genetic markers and estimation of cut-off genetic distance values.COI, cytochrome c oxidase subunit II (COII), cytochrome B (cytb) and NAD1 genes represent the mitochondrial protein-coding genes; 12S and 16S rRNA genes represent the mitochondrial rRNA genes; 18S and 28S rRNA genes represent the nuclear rRNA genes; and the ITS1 and ITS2 regions represent the nuclear ribosomal ITS regions.Four classes of genetic markers comprising both mtDNA and nDNA were selected for study: mitochondrial protein-coding genes, mitochondrial rRNA genes, nuclear rRNA genes and nuclear ribosomal ITS regions. www.ncbi.nlm.nih.gov). In all, we used 142 sequences of the mtDNA genes from helminths of medical importance to humans and animals for our analyses: 64 sequences from nematodes, 28 from trematodes and 45 from cestodes. We also obtained close to full-length sequences from the NCBI database for the nuclear rRNA genes and nuclear ribosomal ITS regions. The number of sequences used are as follows: 18S rRNA—47 from nematodes, 33 from trematodes, 44 from cestodes; 28S rRNA—27 from nematodes, 42 from trematodes, 16 from cestodes; ITS1—32 from nematodes, 30 from trematodes, 14 from cestodes; ITS2—29 from nematodes, 29 from trematodes, 12 from cestodes. As best as possible, we selected sequences from the nuclear DNA genetic markers from the same species from which we obtained from the mtDNA genetic markers. When no sequence was available for the same species, we selected congeneric sequences. The sequences for each group of helminths and each genetic marker used in this study are listed in Additional file We obtained full-length sequences of mtDNA genes from the complete mitochondrial genomes of each helminth species contained in the National Center for Biotechnology Information (NCBI) database  and suborder in trematodes , 13–15. ce versa .P-distance as the model for the aligned sequences via MEGA 6.0 [To calculate pairwise genetic distances for each genetic marker, we first aligned sequences for each dataset using ClustalX2.1 . The aliMEGA 6.0 . The calTo obtain the number of monophyletic clades for molecular systematics and accurate phylogenetic placement for molecular identification, we conducted phylogenetic analyses using maximum likelihood (ML) and Bayesian inference (BI) algorithms. ML analysis was performed using MEGA 6.0 , with thhttps://github.com/slphy/Chan-HelminthMarkers.We applied the unsupervised ‘K-means’ clustering machine learning algorithm implemented in Wolfram Mathematica 12.1  to estimUsing the desirable properties described in the Analysis of nucleotide substitution saturation, which is an indicator of whether a genetic marker is useful for phylogenetic inferences, in the ITS sequences chosen for investigation across the taxa sampled in this study revealed that the nuclear ribosomal ITS regions were saturated Table , with Isχ2 = 1519.6, df = 9, P < 0.000001 for nematodes; χ2 = 581.7, df = 9, P < 0.000001 for trematodes; χ2 = 424.3, df = 8, P < 0.000001 for cestodes). The small genetic distance values of the nuclear rRNA genes can be a limiting factor and might render insufficient resolution for species-level identification.Comparing the mean genetic distances for each marker revealed a similar trend among the three groups of helminths. As presented in Table For the mitochondrial genes, the genetic distances were significantly higher than those of the nuclear rRNA genes. Among the mitochondrial genes, the genetic distances seen in the mitochondrial rRNA genes were comparable to those in the mitochondrial protein-coding genes.NAD1 for nematodes), with four out of six orders as monophyletic. The mitochondrial rRNA genes are more conserved than the mitochondrial protein-coding genes, and this slightly more conserved nature has led to the mitochondrial rRNA genes being used for higher-level classification of organisms [The recovery of recognized taxa as monophyletic can also indicate the resolution of the genetic marker. The highly conserved nature of the nuclear rRNA genes makes them suitable genetic markers for molecular systematics . The 18Srganisms –59. In hrganisms , 61. ChaThus, the results of our assessment of the genetic markers for their suitability for molecular systematics of helminths indicate that the nuclear ribosomal ITS regions might not be suitable for phylogenetic inferences at a higher taxa level due to nucleotide substitution saturation. In addition, the number of monophyletic clades obtained and sufficient genetic distances supported the resolution of the mitochondrial rRNA genes for molecular systematics, making them comparable to the commonly used nuclear rRNA genes.Using the four above-mentioned properties, we assessed the suitability of the genetic markers for molecular identification of nematodes, trematodes and cestodes. The results are summarized in Table χ2 = 161.7, df = 9, P < 0.000001 for nematodes; χ2 = 124.5, df = 9, P < 0.000001 for trematodes; χ2 = 129.0, df = 8, P < 0.000001 for cestodes). For the nuclear rRNA genes, the average genetic distances between species were < 0.03, suggesting low levels of sequence variation. Moreover, for the closely related taxa, sequence variation using the 18S rRNA gene was low , possibly leading to inaccurate phylogenetic placement, which is problematic in terms of species identification. Examples of this are between nematodes, such as Toxocara canis versusT. cati and Ascaris lumbricoides versusA. suum, and between trematodes, such as Opisthorchis viverrini versusClonorchis sinensis . The nuclear ribosomal ITS regions tend to be used for species identification because of their faster evolution rate, resulting in highly variable sequences between species . Moreovesinensis . HowevercytB gene for nematodes, 0.195 with the NAD1 gene for trematodes and 0.132 with the NAD1 gene for cestodes. This higher degree of sequence variation seen for the mitochondrial protein-coding genes compared to the nuclear rRNA genes is a clear illustration of their ability to resolve species-level relationships, even among closely related species. Consequently, it is not surprising that the mitochondrial protein-coding genes have been used widely for molecular identification, both at the species level and the population level, and to differentiate helminths from various host species [For the mitochondrial protein-coding genes, interspecific sequence variation was 0.026–0.036 for nematodes, 0.158–0.195 for trematodes and 0.085–0.132 for cestodes. Closely related species in the three groups of helminths could also be differentiated, with genetic distance values of up to 0.166 with the  species , 63, 64.Setaria digitata among filarial nematodes [Angiostrongylus cantonensis lineage.For the mitochondrial rRNA genes, the interspecific genetic distance values were slightly smaller than those of the mitochondrial protein-coding genes, with means of 0.015 and 0.021 for the 12S and 16S rRNA gene for nematodes, 0.133 and 0.148 for trematodes, and 0.081 and 0.080 for cestodes, respectively. However, the genetic distances were significantly higher than those for the nuclear rRNA genes, rendering the mitochondrial rRNA genes suitable for species identification. In helminths, the 12S rRNA gene has been used successfully for molecular identification, confirming the phylogenetic placement of ematodes . Moreoveematodes  showed tThus, the results of our assessment of the suitability of genetic markers for molecular identification of nematodes, trematodes and cestodes suggest that the nuclear rRNA genes might not be suitable because of low sequence variation for species discrimination. Conversely, the mtDNA genetic markers have higher sequence variation to discriminate among species and closely related species, emphasizing their suitability as markers for molecular identification.The ease of both universal primer design and sequence alignment, in addition to the availability of full-length reference sequences, represent additional advantages that could affect a genetic marker’s suitability and utility for both molecular systematics and identification Table .COI primers have also been developed and utilized for molecular-based studies [COI gene in helminths compred to other groups of organisms has led to low PCR amplification success and limited taxa for analyses [First, highly conserved sequences when using the nuclear rRNA genes, as compared to the other genetic markers, can facilitate primer design that is suitable for amplifying a broad range of taxa. Universal primers for the three helminth groups have been developed using the 18S rRNA gene, and these have been used widely in molecular systematics due to their highly conserved nature –19. Univ studies , 68. Howanalyses –44. In tanalyses , 66. SecBased on our evaluation of both molecular systematics and molecular identification in the selected helminths, the mitochondrial 12S and 16S rRNA genes show potential and could be suitable for applications in both contexts.To create a yardstick for guiding users when adopting genetic distances for helminths, we provide essential points to be considered and an alternative method of using genetic distances through the ‘K-means’ clustering algorithm.COI gene as representative genetic markers to illustrate the broad levels of genetic distances in nematodes at the same taxonomic level.A wide range of genetic distances for nematodes was observed, in contrast to trematodes and cestodes. To further investigate this observation, we selected the nuclear 18S rRNA gene, the mitochondrial 12S rRNA gene and the χ2 = 39.8, df = 6, P < 0.000001). The same pattern was observed across the three genetic markers, with Ascaris having the smallest genetic distance and Strongyloides the largest. In contrast, no significant between-genus differences were found for the trematodes and cestodes (Fig. As shown in Fig. Previous studies have used genetic distances to determine whether specimens are conspecific, and in most cases, a general genetic distance value has been used as a basis for comparison . In suchIn our study, each taxonomic level was clearly distinguishable in the three groups of helminths for the 12S and 16S rRNA genes using the ‘K-means’ clustering algorithm, as presented in Fig. This study was limited by the availability and accuracy of the sequences in the NCBI database, which restricted the number of taxa that we could compare and analyze together across the genetic markers. Inadequate sampling can affect clade arrangement as well as the number of taxa recovered as monophyletic. Also, the species complex status for some helminth species was not considered, which could further complicate species delimitation. The results of the assessment of the genetic markers and genetic distance cut-off values were restricted to the helminth taxa that we selected, and future considerations to increase the number of species sampled should be undertaken.We have assessed the suitability of four classes of genetic marker for application in molecular systematics and molecular identification of nematodes, trematodes and cestodes. By comparing various properties and genetic distances across the taxonomic hierarchy levels, we ascertained the genetic distances for each genetic marker and showed that mitochondrial rRNA genes have the potential for utilization in molecular systematics and molecular identification of helminths. We have also revealed that following a general gauge of genetic distances might not be adequate, using evidence from the wide range of genetic distances among nematodes. In addition, we have provided a novel way of analyzing genetic distances to generate suitable cut-off values per genetic marker for each taxonomic level using the ‘K-means’ clustering algorithm. A guide to the utility and limitations of each class of genetic marker for the respective applications together with the estimated cut-off values can benefit researchers conducting molecular studies on helminths. Future research perspectives can include the use of the mitochondrial rRNA genes in molecular studies and the exploration of machine learning algorithms to aid in the classification of organisms.Additional file 1: Table S1. List of NCBI sequences used for analysis.Additional file 2: Tables S2–S11. Raw genetic distances from p-distance analysis from MEGA for each genetic marker.Additional file 3: Figures S1-S3. Phylogenetic tree for each genetic marker using maximum likelihood and Bayesian Inference algorithms.Additional file 4: Table S12. General guide of the utility and limitation of each class of genetic marker for helminthes.Additional file 5: Figure S4. Violin-plot of genetic distances for a nematodes b trematodes, c cestodes between family.Additional file 6: Table S13. Estimated cut-off of each genetic marker per taxonomic level for helminths using the ‘K-means’ algorithm.Additional file 7: Figures S5-S7. Plot of the estimated cut-off of each genetic marker per taxonomic level using the ‘K-means’ algorithm.


Aedes koreicus is Korea, China, Japan, and the Russian Far East. Since 2008, this species has been recorded as an invasive species in some European countries . The invasive mosquito species Ae. koreicus is reported from the Republic of Kazakhstan for the first time. Its morphological identification was confirmed by molecular-genetic analyses of ND4 sequences using specific primers. Aedes koreicus larvae were found in an artificial water reservoir together with the larvae of Culiseta longiareolata and Culex pipiens s.l. Aedes koreicus successfully overwintered in Almaty at low winter temperatures in 2018–2019. This suggests that the Ae. koreicus acclimation capacity is greater than it has been considered until now. We assume that Ae. koreicus will spread over the west and south of the Republic of Kazakhstan and territories of Kyrgyzstan and Uzbekistan Republics bordering the Almaty region.The natural distribution range of  Aedes mosquitoes are the most abundant mosquito species, and pose a health hazard for humans worldwide as well-known potential vectors of disease pathogens [Aedes albopictus Skuse, 1894 [Ae. japonicus Theobald, 1901 [Ae. aegypti Linnaeus, 1762 [Ae. japonicus in European countries, Russia included [Ae. koreicus Edwards, 1917 [Ae. koreicus covers Korea, China, Japan, and the Russian Far East [athogens , 43. Durathogens . In partse, 1894  and Ae. ld, 1901  initiallld, 1901 . Recent us, 1762  and Ae. included , 41, 53.ds, 1917 , 50, 52.Far East , 29. ThiAedes mosquitoes in Kazakhstan comprises 38 species [Systematic Catalog of Culicidae reports only five Aedes species in Kazakhstan [Ae. gutzevichi Dubitsky et Deshevykh, 1978 [, Ae. stramineus Dubitzky, 1970 [, Ae. pulcritarsis Róndani, 1872 [, Ae. montchadskyi Dubitsky, 1968 [Ae. kasachstanicus Gutsevich, 1962 [One of the factors that contribute to the distribution of invasive species is an ever-increasing level of world travel and trade. Therefore, monitoring of the species composition of blood-sucking mosquitoes is required. Faunistic studies of the non-malaria mosquitoes in Central Asia and Kazakhstan have not been carried out for a long time. The most recent data on the species composition were published in 1970 by Dubitskii . Accordi species . Howeverzakhstan , namely,kh, 1978 , Ae. strky, 1970 , Ae. pulni, 1872 , Ae. monky, 1968 , and Ae.ch, 1962 . These cn = 12) using a GeneJet Genomic DNA purification kit (Thermo). A molecular assay based on nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) sequences was used for the identification of Ae. koreicus using multiplex PCR with the primers N4J-8502D (F) and N4N-8944D (R) [Mosquito larvae were collected in September 2018 and May 2019 in Almaty, Republic of Kazakhstan . The lar944D (R) .Ae. koreicus according to morphological characteristics were found in September 2018 at Almaty zoo. Beside Ae. koreicus, this collection of larvae also contained larvae of Culiseta longiareolata and Culex pipiens. The collection of larvae sampled from the same artificial water reservoir (a bathtub) at the zoo in spring 2019 revealed only Ae. koreicus specimens  wide paddle-shaped scales lacking a main spine. Pecten teeth evenly spaced, close to each other unlike Ae. japonicus, which has the most distal pecten teeth (one to four) detached, widely spaced, well developed, and forming a sharper corner to the siphon longitudinal axis [Aedes koreicus is well illustrated in Versteirt et al. [Ae. koreicus species using a molecular approach. All specimens of Ae. koreicus displayed a specific band of 283 bp as well as the expected 465-bp band, common for other species.In its morphology, eristics , 47. Larnal axis , 47. Thit et al. . We alsoThe probability of an invasive species to colonise a new area depends on the climate characteristics of the region, availability of suitable aquatic habitats, and presence of suitable hosts , 39, 49.Aedes koreicus is well adapted to urban settlements [Ae. koreicus larvae in a bathtub at the Almaty zoo. This might suggest that female Ae. koreicus mosquitoes can feed on both human and animal blood at the zoo. Gutsevich et al. [Ae. koreicus feed not only on humans, but also on cattle. The Italian team who discovered Ae. koreicus larvae in forest water bodies far from any settlements also inferred that females feed on animal blood [Ae. koreicus: Homo sapiens, Canis lupus  [Bos taurus [tlements , 38, 47.h et al.  assumed al blood . So far,animals) , and Boss taurus .Aedes koreicus was discovered in the region where the local mosquito species  also prefer artificial water reservoirs for their development. Based on preliminary data, we also assume that the ratio of these species changes throughout the year. Aedes koreicus was the first to appear in the collection of larvae at the beginning of the year in Almaty, while Cs. longiareolata and Cx. pipiens s.l. were found in the same water reservoir in September in addition to Ae. koreicus. The last two species also occur together with Ae. koreicus in Sochi (Russia) and in Germany [Cs. longiareolata are predators [Ae. koreicus mosquitoes display a good acclimation capacity to a moderate climate; in particular, they produce cold-tolerant eggs, and the larvae hatch from these eggs during snow melting [Ae. koreicus mosquitoes to lower temperatures allows them to start their development earlier and more massively as compared with their competitors, which hatch later.One of the major factors affecting the settlement and spread of invasive species is competition with native species , 35, 38. Germany , 40. Theredators , and the melting , 47. TheAedes koreicus occurs in countries with different climatic conditions. For the climate classification of the countries, we used the Köppen-Geiger world map, which was updated by Beck et al. [Ae. koreicus has been found. In Almaty, the climate is temperate with hot summers. The climate is classified as Dfa according to the Köppen-Geiger system. Aedes koreicus type-locality (Korea) [k et al. . Accordi (Korea)  has climAe. koreicus in Almaty since this species feels quite comfortable in both natural and artificial aquatic habitats. As for the temperature, the average temperature is higher in Korea, with temperatures below zero occurring only in January. In Almaty, the minimum temperature in January over 20 years of observation exceeds –10 °C, while temperatures below zero can be observed during 3 winter months  and in winter, it was the lowest (0.11 °C over 10 years). The temperature increased more rapidly in the western regions of Kazakhstan (from 0.24 to 0.60 °C over 10 years) and slower in the northern and northeastern regions (from 0.10 to 0.43 °C over 10 years), as well as in the mountain regions in the south (from 0.11 to 0.21 °C). The number of days with the average daily air temperature of 10 °C or higher increased by 3–5 days over 10 years and even by more than 5 days over 10 years in some southern regions [Ae. koreicus will spread over the territory of Kazakhstan westwards and southwards in the Almaty region, and it could cover the Kyzylorda, Turkestan, and Zhambyl regions and through the southwest of Aktyubinsk region to the West Kazakhstan, Atyrau, and Mangistau regions. The winter months in these regions are rather mild. The South and East of the Almaty region borders the Republics of Kyrgyzstan and Uzbekistan; most likely, Ae. koreicus as a species new to Central Asia will also spread to these territories. There are no recent faunistic studies; therefore, it can be assumed that other invasive species have already occupied their own niches in the blood-sucking mosquito fauna in the republics of Central Asia. The introduction route of Ae. koreicus is unknown, it may have occurred via international trade as Almaty is one of the largest industrial centers in Kazakhstan. The introduction of a new potential disease vector to Kazakhstan can be due to appropriate entrance points that emerged as a result of intense global trade and suitable environmental conditions. Regular monitoring of the mosquito fauna is necessary due to the development of transport routes, trade, and economic connections, which enhance the introduction of new species to the territories, where these species have not previously been recorded.Marini et al.  studied  of eggs . However winters , 50. Our018–2019 . Thus, wThe authors declare that they have no conflict of interest and they have observed all relevant ethical standards.


Pampus chinensis is a commercially important fishery species in the Indo-West Pacific region. In the present study, the genetic variation of P. chinensis among 10 sampling localities along the China coast and one from the Indonesia region was evaluated using mitochondrial DNA control region sequences. As a result, a total of 30 variable sites were detected in the 458 bp segment of the control region among 330 individuals from 11 localities, and 41 haplotypes were defined. Samples in the China coast present a high level of genetic diversity, with the values of haplotype diversity ranged from 0.674 to 0.860, and nucleotide diversity from 0.820% to 1.502%. Pairwise STF statistics showed a moderate genetic divergence (−0.027 to 0.384) among samples from different geographical locations. Median-joining network analysis revealed a similar pattern of phylogeographic structure in samples from Ningbo and Dongxing although they were far apart. Therefore, joint influences of dispersal capability, spatial distance, ocean current and geographic segregation on the formation of the present population structure in P. chinensis was proposed. The results of the present study would be helpful for the sustainable utilization and management of this species. The genetic background of fish species could facilitate the identification of appropriate management units, and a better understanding of this is, therefore, essential for resource recovery and delineation and monitoring of fishery management Ward, . GeneticPampus chinensis is a coastal teleost species belonging to the Pampus family . It has a wide distribution in the East China Sea and the South China Sea with a possible wider distribution from the Indian Ocean to eastern Indonesia. In China, it is common on the coast of Fujian, Guangdong and Guangxi province and viewed as a commercially important fish species. In recent decades, however, decline due to marine pollution and overfishing was found in this marine species . The PCR was carried out under the following conditions: initial denaturation at 94 °C for 4 min, 35 cycles of 1 min at 94 °C for denaturation, 1 min at 55 °C for annealing, and 1 min at 72 °C for extension, with a final extension at 72 °C for 10 min. After separated by a 1.5% agarose gel and purified using the DNA Gel Extraction Kit , the target PCR product was then sequenced in both directions using ABI 3730 automated DNA sequencer.Genomic DNA was isolated from muscle tissue using TIANamp Genomic DNA kit  following the introduction. Extracted DNA was checked using 1.5% agarose gel electrophoresis and then stored at −20 °C for PCR amplification. The control region partial sequence was amplified using the primer pair L15923 (5′-TTA AAG CAT CGA TCT TGT AA-3′) and H16500 (5′-GCC CTG AAA TAG GAA CCA GA-3′) , nucleotide diversity (π), and average number of pairwise nucleotide differences (k) were calculated in Arliquin v.3.01   with 30 polymorphic sites (Hd) for samples from the China coast ranged from 0.674 to 0.860, and values of nucleotide diversity (π) ranged from 0.820% to 1.502% (Hd (0.248) and π (0.305%).MtDNA control region sequences with a length of 458 base pairs (bp) were amplified and sequenced from 330 ic sites . Of the ic sites . Values o 1.502% . But, saP. chinensis populations was analyzed by AMOVA  and pairwise FST values. Results of AMOVA indicated that 76.43% of the total genetic variation occurred within the same locations, and 14.25% of the variation occurred among four different geographical groups . As for the other locations, no significant difference was found among samples from the same group. In addition, samples from Xiamen showed no significant genetic difference with those in group 2, which suggested a shallow genetic divergence among them.Genetic structure of l groups . Pairwisto 8.273 . SignifiD and Fu’s Fs tests were shown in D test and the Fu’s Fs tests showed no significant values in all localities involved. Mismatch distribution analysis was also constructed for each location  gene sequences. Results showed high level of haplotype diversity (0.540–0.828) and low level of nucleotide diversity (0.081–0.210%). And the low but significant Fst statistics suggested genetic divergence among locations from different geographical regions. In addition, Li et al.  and nucleotide diversity (1.030–1.280%) was found in samples from the China coast. We deduced that the difference of genetic diversity for two studies was due to the different rate of genetic variation between cyt b and control region sequences.i et al.  comparedP. chinensis along the China coast. Results showed high level of both haplotype diversity (0.674–0.860) and nucleotide diversity (0.820–1.502%) in China samples, which was consistent with Li et al. . Meanwhile, statistical significance of SSD indicated that samples from ND, XM, ZH, ZJ, SY and DX were at equilibrium. Hence, a historical population expansion in those populations could not be deduced based on current data. Similar results were also found in P. argenteus, a sister species of P. chinensis. Sun et al.  and the South China Sea (group 2).P. chinensis has a wide distribution in China. In the present study, genetic variation and population structure of P. chinensis along the China coast were evaluated and a combined influence of dispersal capability, spatial distribution, geographic segregation and ocean current on the formation of the present population structure in P. chinensis was assumed. The results would improve population genetic understanding and provide important implication for sustainable exploitation, fishery management and conservation of this species.Insight into the genetic diversity and population structure would facilitate the fishery management and conservation of marine fish species. As an economically important fish species, the


This collection provides a contemporary excerpt of “Cities as complex systems”. The contributions have been submitted between April and October 2020. We briefly discuss example papers addressing the themes “urban scaling”, “urban mobility”, “flows in cities”, “spatial analysis”, “information technology and cities”, and “cities in time”. After motivating the intersection of cities and complexity, we provide an introduction and additional thoughts on urban scaling. Cities represent the stage for a wide range of human activities including services, government, education, commerce, markets, finance, etc. Today they are the dominant form of living even while the process of urbanization is ongoing. However, the history of cities was not always a success story and often they are associated with traffic jams , pollutiMany of these negative characteristics can be attributed to difficulties in managing cities which in turn is due to their high level of complexity. Cities consist of interacting entities, such as people and infrastructure, and exhibit emergent properties, which appear additionally to the sum of the isolated properties . UnderstThere are various arguments for treating cities as complex systems . First, scalability as known in computer science. Bondi [Scaling is closely related to the concept of e. Bondi  describe1.15 ≈ 2.2 times the total urban GDP, i.e. 20% more (see below). This means one city generates more GDP than two cities of half the size together. Consequently, the above exercise from the daily life does not work for city systems.A typical everyday-life example could be the following. “Imagine you want to buy 2.5 kg of potatoes. One kg of potatoes costs 1.50 Euro. Then half a kg of potatoes costs 0.75 Euro and 2.5 kg cost five times as much (3.75 Euro)”. Empirical analysis has shown that for many urban measures such a linear proportionality does not hold. E.g. doubling city size leads to approximately 2S is inversely proportional to its size. Settlements and cities cover a large range of spatial and size scales. Second, many urban indicators scale non-linearly with population size. This relation is often termed “urban scaling” [One can distinguish two types of scaling among cities. First, the number of cities scales inversely with the population size. It is commonly referred to as “Zipf’s law for cities” –19 and cscaling” , 20 and scaling” –23.This collection provides a contemporary excerpt of “Cities as complex systems”. The contributions have been submitted between April and October 2020—we received over a 100 submissions and PLoS One published over 60. They can be classified into a set of themes. In the following we want to briefly discuss examples. Our choice is of course subjective and the collection includes other interesting papers.Ortman, Lobo, and Smith  take up A planned community is one whose layout exhibits a clearly recognizable plan or system. Yinger  comparesPeréz-Mendéz et al.  study reSilver, Byrne, and Adler  extend tThe idea of Mulisheva et al.  is to usXie et al.  analyze Beyond this collection, Arcaute & Ramasco  provide Cities are and have been fundamental to our civilizations for centuries. If we want to sustain urban living in view of present and future challenges, then we need to understand them. This means they need to be treated as what they are: complex systems. With a considerable number of papers, here we could only introduce a minor subset, this collection covers a range of topics around cities as complex systems. Thereby, it not only advances the field but also contributes to the establishment of the urban science discipline –33. In t


The renewed focus on cereal landraces is a response to some negative consequences of modern agriculture and conventional breeding which led to a reduction of genetic diversity. Cereal landraces are still cultivated on marginal lands due to their adaptability to unfavourable conditions, constituting an important source of genetic diversity usable in modern plant breeding to improve the adaptation to abiotic or biotic stresses, yield performance and quality traits in limiting environments. Traditional agricultural production systems have played an important role in the evolution and conservation of wide variability in gene pools within species. Today, on-farm and ex situ conservation in gene bank collections, together with data sharing among researchers and breeders, will greatly benefit cereal improvement. Many efforts are usually made to collect, organize and phenotypically and genotypically analyse cereal landrace collections, which also utilize genomic approaches. Their use in breeding programs based on genomic selection, and the discovery of beneficial untapped QTL/genes/alleles which could be introgressed into modern varieties by MAS, pyramiding or biotechnological tools, increase the potential for their better deployment and exploitation in breeding for a more sustainable agricultural production, particularly enhancing adaptation and productivity in stress-prone environments to cope with current climate changes.  The growing increase of human population and environmental stresses have led to land use changes and habitat destruction in order to produce a greater amount of food. The advent of high input and intensive monoculture systems, based on genetically similar individuals  improved for particular traits  and derived from a relatively narrow germplasm pool, resulted in a loss of potentially useful traits, reducing genetic variability that can be used by plant breeders for crop improvement. To cope with climate change and meet the needs of new varieties for marginal areas, researchers and breeders are constantly looking for new sources of genetic variability. A key component of agro-biodiversity is represented by landraces.Landraces adapt to specific agro-climatic conditions while maintaining considerable diversity between and within populations , constitLandraces have been described by Harlan  as populCereal landraces emerged in different regions of the world as a result of centuries of crop evolution in traditional agrosystems, leading to heterogeneous populations rather than a few superior genotypes ,10. FarmOver time, the ageing of rural populations and mass migration of young workers from rural areas to cities have resulted in the abandonment of traditional agricultural practices and have created serious threats for the cultivation and possible use of landraces as a source of biodiversity ,14. The This review presents an overview of the different aspects related to the conservation of the main cereal landraces and how these collections can be useful for modern breeding. Landrace collections have been extensively characterized in terms of genetic diversity and population structure and have a huge potential for the identification of genetic factors that are valuable for improving the important agronomic traits of cereal crops such as resistance to biotic and/or abiotic stresses, thus sustaining grain yield and quality in unfavourable environments.A way to defend landraces against genetic erosion and to conserve and utilize the genetic resources for the future is their conservation.Two methods are used for the conservation of cereal genetic resources: ‘in situ’ and ex situ. Both conservation methods have benefits and limitations. The in situ conservation is carried out in “the natural habitats and include the maintenance and recovery of viable populations of species in their natural surroundings” on farms, while in the ex situ conservation, “the genetic resources are outside their natural habitats”, in identified gene banks or other national or regional conservation centres .Ex situ germplasm collections could be used as base collections (by gene banks), and as working collections (by research/breeding institutions). The ex situ materials maintain the ‘genetic integrity’ of the accessions as they are not subjected to natural or farmers’ selection .On the contrary, in situ is a dynamic conservation of cereal diversity in the centre of origin and in the areas in which that cereal species has been domesticated under the action of evolutionary forces , or simply traditionally grown, developing their distinctive properties and adaptation to environmental changes ,23,24,25For a comprehensive knowledge of cereal landrace diversity, it is essential to standardize the management strategies, starting from sampling procedures and guaranteeing a correct conservation of landrace diversities in gene banks over time. This way, the identification of agronomically valuable genes and untapped alleles to be used for breeding or pre-breeding purposes is facilitated ,32.The effectiveness of the ex situ conservation of landraces is closely linked to the possibility of sharing seeds and genetic information by a large community of users  through a system based on their free and open access.http://www.ecpgr.cgiar.org/ (accessed on 30 March 2021)) is a good example of standardised procedures and compatible data documentation systems for better management, studies and exchange of genetic resources. Valuable collections of cereal landraces are held at several European and world gene banks.The European Cooperative Programme for Plant Genetic Resources ), which contains metadata and data on germplasm collections and provides the identification and analysis of cereal landraces maintained in Catalogues around the world.The FAO website maintains the WIEWS on Plant Genetic Resources (PGR) (www.genesys-pgr.org (accessed on 30 March 2021)). In eastern Europe, one of the most important cereal collections is maintained at the Research Institute of Crop Production in the Czech Republic (http://genbank.vurv.cz/genetic/resources (accessed on 30 March 2021)).On a global scale, the GENESYS portal holds the information provided by three major international project partners: the European Cooperative Programme for Plant Genetic Resources (ECPGR-EURISCO), the System-wide Genetic Resources Programme (SGRP-SINGER) of the Consultative Group on International Agricultural Research (CGIAR) and the USDA Agricultural Research Service National Genetic Resources Program, accounting for approximately 2.4 million accessions held in ex situ collections worldwide (www.ars-grin.gov (accessed on 30 March 2021)). The GRIN database contains passport data, information which describes where and when an accession was collected, donated or developed and provides information on the availability and amount of seed that can be freely distributed to scientists and farmers in the US and around the world. Gene banks are therefore provided with a global management system that is powerful, flexible, easy to-use to safeguard plant genetic resources and to encourage their use by researchers, breeders and farmer-producers [The National Plant Germplasm System (NPGS) is a public germplasm collection that maintains seed samples, representing global diversity of small grains including wheat, barley, oat, rice, rye and triticale, and various wild relatives, including Aegilops. It is part of the Agricultural Research Service (ARS), the research agency of the United States Department of Agriculture (USDA) and is responsible for collecting, conserving, characterizing, evaluating, distributing and exchanging a rich and diverse genetic resources collection containing about 500,000 accessions. In particular, the wheat genetic resources are conserved at the National Small Grains Collection (NSGC), which is part of NPGS-ARS . A partnroducers . Howeverhttps://web.archive.org/web/20120220095846/, http://www.icis.cgiar.org/icis/index.php/Main_Page (accessed on 2 April 2021)).The main issue met by the gene bank users is the scarcity of information about some landrace characteristics, especially for some minor cereal species which play an important role in overall crop diversity . Recent The CIMMYT germplasm bank contains over 170,000 wheat and 28,000 maize seed collections from across the world. These collections represent the genetic diversity of unique native varieties and wild relatives of maize and wheat and are studied and used as a source of currently untapped native diversity that could be used for plant improvement. The CIMMYT and the ICARDA wheat collections are the most important sources of landraces for wheat.Triticum turgidum ssp. durum modern varieties/advanced breeding lines, derived from worldwide breeding programs, durum wheat landraces (192), and a selection of wild and domesticated emmer wheats to maximize diversity. The TGC collection [https://wheat.pw.usda.gov/GG3/global_durum_genomic_resources (accessed on 2 April 2021)).Recently, the Expert Working Group on Durum Wheat Genomics and Breeding, in the frame of the Wheat Initiative, and the International Durum Wheat Genome Sequencing Consortium developed two large germplasm collections: The Global Durum Wheat Panel (GDP) and the Tetraploid wheat Germplasm Collection (TGC), respectively ,35. Bothllection  consistsllection  are alsoSelection activities in the frame of the ongoing breeding programs led to a reduction of the diversity in genetic materials and are considered as a bottleneck in crop evolution after the domestication process. This assumption is accompanied by the idea that many alleles useful for breeding could have been lost in the selection process. Therefore, in the last few years a growing interest has focused on analysing genetic diversity in landrace collections in cereal crops.In some cases, these studies were aimed at characterizing a limited number of important genotypes, such as those traditionally grown by farmers in particular areas. The genetic analyses, carried out on a proper number of individuals for each accession, often showed a certain degree of heterogeneity in each landrace. As an example, Mangini et al.  carried Phenotypic evaluation of landraces is important to identify sources of useful loci for traits of interest in breeding and pre-breeding programs, in relation to traits with a simple genetic basis as the resistance to diseases, but also to complex traits such as grain yield . NevertThe evolution of plant breeding with the consequent genetic erosion and the gradual shift towards a model of agriculture based on genetic uniformity results in the need to re-gain genetic variability to adapt crops to climate changes . The impSUBMERGENCE 1 (SUB1) located on chromosome 9, which codes for ethylene response factor [SUB1 locus revealed three genes: SUB1A, found in tolerant lines, and SUB1B and SUB1C, found only in intolerant lines [SUB1A has two allelic forms, SUB1A-1, associated with tolerant lines, and SUB1A-2, associated with intolerant line. Two QTLs for drought tolerance, based on leaf wilting, were recently identified on chromosomes 2H and 5H in the Chinese barley landrace ‘TX9425’ [9-cis-epoxycarotenoid dioxygenase 2 (HvNCED2), which is involved in the synthesis of abscisic acid. In another study of GWAS, two candidate genes, HvCBF10B and HvCBF10A, underlying this QTL were identified, which have regulatory function under drought condition [The identification of abiotic stress tolerant alleles in landraces of cereal crops through mapping and GWAS approaches is of great importance to improve cereal crop adaptation to stress-prone environments. Two types of studies have been carried out in this regard: on one hand, traits directly associated to tolerance to abiotic stresses have been analysed by assuming their importance in improving the agronomic performance of crops in stress-prone environments. On the other hand, landraces have been evaluated for grain yield and quality, or related traits in limited environments. For the first kind of investigation, in rice, the discovery of submergence-tolerant landrace ‘FR13A’ led to the identification of the locus e factor . The posnt lines ,77. In t‘TX9425’ , which aondition . Attemptondition  identifi+ concentration on chromosome 1 (SKC-1) [+/K+ ratio [+/Na+ homeostasis under salt stress [Saltol1, associated with Na+/K+ ratio and salinity tolerance [+ and K+ concentration and with salt injury score [+/K+ homeostasis [HvNax4, was identified on chromosome 1HL in the Algerian landrace ‘Sahara 3771’ [Rice productivity in both rain-fed and irrigated agro-ecosystems is also affected by salt stress. Rice landraces ‘Nona Bokra’ and ‘Pokkali’ are excellent sources of salt tolerance. Nona Bokra contributed a major QTL for shoot K (SKC-1) , and addK+ ratio ,83. The t stress . Pokkaliolerance  and addiry score . Furthereostasis ,87. Simieostasis , and a seostasis . Finallyra 3771’ .Bot1 gene, responsible for the high boron-toxicity tolerance of the Algerian barley landrace ‘Sahara 3771’, was identified [Bo4, which was mapped on chromosome 4AL [Another trait, potentially limiting crop production, is boron toxicity. Tolerance to toxicity is associated with the ability to maintain low boron concentrations in the shoot . The Botentified . In breasome 4AL .Cereal landraces are also important sources of beneficial alleles for grain yield and quality in low-producing environments. For this reason, collections of landraces have been assessed in mapping studies to identify genetic determinants for these traits. As grain yield is a trait with a very complex genetic basis and a strong genotype x environment interaction, in some cases traits which are strongly correlated with yield have been considered. As an example, different leaf traits were assessed in a panel of 180 Vietnamese rice landraces in controlled conditions, such as leaf dry matter percentage, which can be considered a proxy for the photosynthetic efficiency per unit leaf area, contributing to yield . GeneticPhosphate uptake 1 (Pup1), identified in the rice landrace ‘Kasalath’ and located on chromosome 12, increases phosphorus uptake and confers significant grain yield advantage in phosphorus deficient soils [Pup1 is found in landraces or cultivars adapted to drought-prone environments [Pup1 revealed the presence of a Pup1-specific protein kinase gene, named Phosphorus starvation tolerance 1 (PSTOL1), which is absent in intolerant cultivars. The overexpression of POSTL1 significantly enhances grain yield in phosphorus deficient soils, promoting early root growth, thereby enabling plants to acquire more phosphorus and other nutrients [Numerous studies have focused on the evaluation of grain yield and yield components directly. Huang et al.  identifint soils ,100. Pupronments  and it ironments . A studyutrients .For a good agronomic performance in stress-prone environments, it is important to sustain not only grain yield but also quality. A good variation has been found in landraces as an example for storage proteins in wheat grain . In the These results confirm that landraces, thanks to their long evolutionary history and adaptation to stressful environments, are ideal genetic resources to explore novel genetic variation for responses to environmental constraints. In particular, landraces are an effective source of useful alleles to sustain grain yield and quality in both favourable and limiting environments. In some cases, the loci involved in the control of yield in good conditions can still maintain a good level of production when stresses are mild . In otheMagnaporthe oryzae, from which two landraces emerged for high resistance to blast, and were therefore useful in breeding programs [Pi54 gene for broad-spectrum blast resistance, which has been cloned, and transgenic lines harboring Pi54 showed a high degree of resistance to diverse strains of blast pathogen [Plant diseases are serious constraints to the production of cereal crops. During the vegetation period, the largest infections are caused by pathogenic fungi. Powdery mildew and rusts of cereals and grasses are the most dangerous diseases of wheat and barley. Head blight caused by different fungi of the genus Fusarium also affect wheat, rye, triticale, barley and oat crops, but also maize. Genetic improvement of resistance to pathogens through breeding represents the best economical and eco-friendly alternative to minimize yield losses. Many studies aimed at identifying resistance genes/loci against various diseases are available for cereals. Most of these studies were based on the analysis of biparental populations and, more recently, GWAS has been employed. Landraces may carry new sources of resistance that can be exploited to enrich the narrow resistance spectrum currently found in adapted cultivars. Studies that have reported screening with molecular markers linked to specific resistance genes of panels including landraces grown in a particular geographic area are available, such as the collection of rice landraces and breeding lines from India evaluated for 22 genes against the fungus programs . In partpathogen .Lr67, which confers partial resistance to all three wheat rust pathogen species  and powdery mildew (Bgt), as demonstrated by using a combination of comparative genomics, mutagenesis and transformation studies, was isolated from a bread wheat landrace (PI250413) [Fhb4 and Fhb5, that were fine mapped by using NIL populations [Many studies that have focused on landraces as a good reservoir of resistance genes against rusts are available for wheat ,120,121.I250413) . Wheat lI250413) , barley I250413) , powderyI250413) ,128,129 I250413) . A recomI250413) . In partI250413) . Indeed,ulations ,133.Pm61, on chromosome 4A [PmDGM, conferring powdery mildew resistance [Pm24, Pm24b and MlHLT were identified in wheat landraces Chiyacao, Baihulu and Hulutou, respectively [Pm24 was map-based cloned, and it was found to be a rare natural allele of tandem kinase protein (TKP) with putative kinase pseudokinase domains. A 6-bp deletion at the kinase domain was considered critical for the gain of powdery mildew disease resistance [Pm3b, originating from the hexaploid wheat landrace Chul, was found by positional cloning to be a member of the coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) type of disease resistance genes [Blumeria graminis f. sp. Hordei [mlo (Mildew resistance locus o)-based resistance is considered the most reliable weapon to protect plants from infection by this fungus [mlo mutants [MlMor [Landraces from China were also considered very good sources of powdery mildew resistance. For example, the Chinese wheat landrace Xuxusanyuehuang has been found by comparative genomics analysis to possess a single recessive powdery mildew resistance gene, osome 4A , whereassistance . Resistaectively . In partsistance . Finallyce genes . Regardi. Hordei ,138,139.s fungus . Loss of mutants . Moreove mutants , whereass [MlMor ,142, stes [MlMor , barley s [MlMor  and barls [MlMor  have alsPyrenophora teres f. teres) has been carried out, resulting in 15 QTL regions, four of which had never been described in previous studies [rpg4/Rpg5 locus has been indicated to be involved in conferring resistance [Very recently, a genetic analysis of a worldwide barley collection, including 277 landraces, for resistance to net blotch disease  resistance using genome-wide association studies and genomic selection (GS) [Few genetic studies for disease resistance in maize and oat landraces are available. The well-known gene he 1970s . Very reion (GS) . Loci wiion (GS) . Finallyion (GS)  analysedIn conclusion, cereal landraces have a great potential as sources of novel disease resistance genes, and a good combination of these genes could help to alleviate diseases. Therefore, more efforts are needed to utilize genomic approaches in order to exploit genetic variability across landrace collections worldwide.The QTLs/Genes for abiotic and biotic stresses identified in cereal landraces have been summarized in Plant breeders who want to create new high-yielding varieties tend to make crosses among élite lines where they have the highest likelihood of developing new varieties. Currently, climate change is affecting grain yields worldwide, threatening food quality and security. The cereals grown in many areas worldwide, such as the Mediterranean area, are often exposed to biotic and abiotic stresses, such as high temperatures and water stress, especially during the filling kernels period. Compared to modern cultivars, landraces are often resilient to stresses and represent a valuable source of germplasm for meeting the future needs of sustainable agriculture in the context of climate change ,148,149.The wide genetic variability and high potential for adaptation to extreme climatic conditions of landraces make them important for the genetic improvement of cereals, particularly in improving agronomic traits such as yield and yield stability ,152,153.Yr51, associated to marker sun104 in wheat landrace AUS27858, is currently backcrossed into Australian and Indian wheat cultivars through marker-assisted selection, to improve resistance to yellow rust [Lr67 gene, which was introduced into wheat cultivar Thatcher to produce the near-isogenic Thatcher + Lr67 line, RL6077, confirming a resistance phenotype to a number of races of wheat rusts and powdery mildew [Pi54, a PCR-based co-dominant molecular marker targeting an InDel identified in the exonic region of the gene, co-segregating with blast resistance, has been developed and used for routine deployment in MAS in breeding programs [Improving resistance to biotic stresses is, in general, a simple task, as many resistance genes provide a complete, even if race-specific, resistance. Examples are available in different cereal species. low rust . As singy mildew ,155. Theprograms ,157.Improving tolerance to abiotic stresses is a more complex issue due to the quantitative nature of this trait. Nonetheless, landraces have also been shown to be valuable sources of beneficial alleles in this case.SUB1A-1 allele, found only in flooding tolerant lines, has been introgressed through MAS into locally adapted and widely grown rice cultivars in Asia [Saltol1 allele, associated with Na/K ratio to obtain rice cultivars highly tolerant to salt stress [Posphate uptake 1 (Pup1) from the rice landrace Kasalath [Bot1 responsible for the high boron-toxicity tolerance was introgressed by MAS into commercial barley cultivars, and the lines obtained showed lower yield than the recipient cultivars, which have been further tailored to develop lines carrying recombination events directly adjacent to Bot1 for use in barley breeding [HvFT3 gene, which contributed to an acceleration of flowering and an increase in thousand-kernel weight (TKW), and on chromosome 6H, which contributed to increasing plant height and TKW. Favourable alleles responsible for an increased ground coverage after winter, which could be exploited as an adaptive trait, were contributed only by SBCC042. South American maize landraces are known for high-altitude adaptation and tolerance to abiotic stresses [Opaque-2 mutation, which improves kernel protein quality, and was identified in a Peruvian maize landrace [Extensive studies have been done in rice, given the importance of this cereal in world food supply.  in Asia , improvi in Asia . This lot stress . Good ret stress . Other eKasalath . These lKasalath . In barlbreeding . Improvibreeding  improvedstresses . Recentlstresses ,163,164.stresses . Nonethelandrace .Despite the scientific literature on the role of cereal landraces in breeding, landrace potential has not been fully exploited in modern breeding , as indiThe use of cereal landraces as a genetic resource for breeding is a response to some negative consequences of domestication, modern agricultural practices and conventional farming, which led to the loss of genetic diversity and a reduction of yield in unfavourable environments. Most of the genetic diversity present in cereal landraces is still little known and even less used. A major part of this valuable landrace diversity is conserved in the farmers and the world’s gene bank networks and should be largely exploited for traits of interest. The information derived from gene banks, phenotypic assessment and high-throughput genetic tools is valuable for the choice of parental lines to use in crosses aimed at obtaining improved varieties, especially for enhancing the adaptation and productivity of crops in situations of global climate change, to increase the sustainability of cereal crop production in the future.


Genetic diseases in Tunisia are a real public health problem given their chronicity and the lack of knowledge concerning their prevalence and etiology, and the high rates of consanguinity. Hence, we performed systematic reviews of the literature in order to provide a more recent spectrum of these disorders and to expose the challenges that still exist to tackle these kinds of diseases. A manual textual data mining was conducted using MeSH and PubMed databases. Collected data were classified according to the CIM-10 classification and the transmission mode. The spectrum of these diseases is estimated to be 589 entities. This suggests remarkable progress through the development of biomedical health research activities and building capacities. Sixty percent of the reported disorders are autosomal recessive, which could be explained by the high prevalence of endogamous mating. Congenital malformations (29.54%) are the major disease group, followed by metabolic diseases (22%). Sixty percent of the genetic diseases have a known molecular etiology. We also reported additional cases of comorbidity that seem to be a common phenomenon in our population. We also noticed that epidemiological data are scarce. Newborn and carrier screening was only limited to pilot projects for a few genetic diseases. Collected data are being integrated into a database under construction that will be a valuable decision-making tool. This study provides the current situation of genetic diseases in Tunisia and highlights their particularities. Early detection of the disease is important to initiate critical intervention and to reduce morbidity and mortality. In Europe, a prevalence of less than 1/2000 people was specified to define a RD : “2021”[Date—Publication])).n = 970) were included in the meta-analysis and were curated manually. They were completely screened for disease name, OMIM ID, inheritance mode, associated gene(s), germinal mutation(s) (according to HGVS nomenclature), and epidemiological parameters . We also searched for the same information from the grey literature . From the selected documents reporting on genetic diseases in the Tunisian population, a recursive search for additional documents was performed using the authors’ names of referring physicians and researchers in Tunisia.The retained papers  International Classification of Disease (WHO ICD-10) in order to provide a clustering of the genetic diseases in different pathological groups. Our previous data on the genetic diseases’ spectrum published in 2011 have been compared to the updated data using the Fisher test, considering 5% as a statistical significance threshold. The data have been stored in a local database for subsequent analyses .To get knowledge about the current genetic disease screening situation in Tunisia, a selective literature search was carried out. Multiple keywords have been used to develop an advanced PubMed query string as below:  AND  AND (Tunis*))Only studies dealing with genetic disease screening among Tunisians were kept. In addition, we asked referring experts  with whom we collaborate in order to provide more information concerning screening for these disorders. Moreover, grey literature was an important resource in this part of the systematic review. Furthermore, we have defined a list of genetic diseases frequently described among Tunisians based on epidemiological data, essentially patients’ counts. The experience feedback of our collaborators was of great value in this step of analysis. We have checked the eligibility of the identified commonly-reported genetic diseases for systematic newborn and/or carrier screening in our country by responding to key questions relative to newborn screening and using the seven ACOG carrier-screening criteria mentioned in their committee opinion in 2017. These criteria are the following (a) well-defined phenotype, (b) detrimental effect on the quality of life, (c) cognitive or physical impairment, (d) surgical or medical intervention is required, (e) an early life onset, (f) availability of prenatal diagnosis, and (g) carrier frequency of 1 in 100 or greater. Evaluation of these criteria was conducted by a genetic counselor.In this study, we present the actual situation of genetic diseases in Tunisia. Compared to what has been published in 2011, 243 additional genetic conditions have been identified, with an average of approximately 23 genetic diseases reported every year. Thus, the total number of genetic diseases in Tunisia amounts to 589 clinical and/or genetic entities, in which 41.3% come from the present data mining.p-value = 9.9 × 10−4).We opted for the international medical classification (WHO ICD-10) of genetic diseases described in the Tunisian population. Therefore, such classification revealed that congenital malformations, deformations, and chromosomal abnormalities are the most commonly reported (29.54%). Endocrine, nutritional, and metabolic diseases are the next most frequent group of diseases (22%), followed by diseases of the nervous system (15.45%) . We repop-value = 5 × 10−4). Despite this difference, the whole spectrum of genetic diseases described among Tunisians (589 genetic diseases) has kept the same pattern illustrated by the high frequency of AR diseases (60%) followed by the AD conditions (24.3%) , autosomal recessive diseases (AR) are the most frequent (54.7%), followed by autosomal dominant (AD) disorders (27.16%). X-linked (XL) conditions were encountered 5% of the time. Diseases transmitted with both AR and AD  are more prevalent than in data reported before 2011. Remarkably, diseases inherited via mitochondrial mode are becoming more reported (8.2%). In order to check for an eventual change in the inheritance mode distribution between the two datasets, we have compared our current data to that of the previous spectrum. We found a significant difference  .Sixty-one percent (61 %) of the 589 genetic diseases have a known molecular etiology (358 genetic diseases), meaning that the responsible gene with at least one mutation has been identified among Tunisian patients. About 69% of these diseases are autosomal recessive, 16.2% are autosomal dominant, 7% are caused by a mutation in mitochondrial genes, 6% are X-linked, and only 0.3% are Y-linked or sporadic. Autosomal dominant or autosomal recessive diseases are encountered at 1.2% .Among these diseases, 21 conditions were of unknown molecular etiology before the last decade  . Eleven Comorbidity is defined as the expression of another disease in addition to the primary disease. This phenomenon has been reported among Tunisian families  . In our Atypical phenotypes are also reported and characterize several genetic diseases among Tunisians. By atypical clinical presentations, we mean unusual symptoms of the disease. Chanarin–Dorfman syndrome, palmoplantar keratoderma, juvenile Parkinson’s disease, and multiple endocrine neoplasia type 2A were described in Tunisian patients with unusual presentations . For epidemiological parameters, we focused on the frequency of the whole reported disease spectrum by targeting either the prevalence or the incidence. We noticed the paucity of such epidemiological data as only 26 (4.6%) genetic diseases described among Tunisians have defined prevalence and/or incidence . For somOn 1 July 2021, the second PubMed-MeSH query described above yielded 732 results, of which 26 articles have been retained for further analysis. Three articles (in French) found in Google Scholar have also been included. Ultimately 29 articles were screened. In all of these studies, the authors highlight the key role of screening for genetic diseases as a state-run health program in Tunisia. However, neonatal newborn and carrier screenings were limited to pilot experiments for only six conditions, and they have not been replicated or extended geographically to the level of the country. Systematic newborn screening for hearing impairment is exclusively conducted in the governorate of Sfax in the south . In factIn this study, we attempted to provide a comprehensive and up-to-date overview of the genetic disease spectrum described among Tunisians. Our findings showed that the number of reported genetic conditions increased from 346 to 589 in the last eleven years (2010–2021). This highlights remarkable progress in the availability of molecular diagnosis of genetic diseases in Tunisia. The development of biomedical health research activities, the building capacities in this field, and the use of high-throughput sequencing technologies facilitated by international collaborations, have played a critical role in this progress .Genetic disorders present a health burden, especially among inbred populations, including Arab countries and several African communities ,75,76. HAnother impact of the elevated rate of consanguinity and inbreeding is the co-occurrence of two or more phenotypes within the same family . ComorbiSeveral variants, either in the same gene or in related genes, were correlated with the high frequency of genetic diseases in inbred communities . In the TGFBI mutation were the severely most affected [CASR gene mutation. Their daughter had a severe hyperparathyroidism phenotype because of her homozygous state for the same mutation [Autosomal dominant diseases were found in 24.5% of reported Tunisian patients. Although intrafamilial marriages have no important effect on the prevalence of autosomal dominant diseases, significant consequences on the expression of such disorders could not be completely excluded . Indeed,affected . Allelicaffected . The parmutation .http://www.maladie-gaucher-tunisie.org/, accessed on 24 September 2021), Fanconi Anemia , and bleeding disorders [Accurate prevalence and incidence rates are crucial to developing an understanding of a disease’s natural history and population burden . Howeverisorders . Furtherisorders ,86. The isorders . Moreoveisorders . In addiisorders . Consideisorders .CFTR gene has accumulated multiple founder mutations that could be used as a target for personalized therapy [Molecular data availability on relatively frequent genetic rare diseases in inbred populations could be an ideal way to boost current research focusing on targeted therapies, including gene and mutation-specific therapies, hence leading to the implementation of precision medicine. Several diseases identified in the Tunisian population are subject to targeted therapy based on their genetic information. Bloom syndrome (BS) is a DNA-repair disease characterized by a high risk of cancer at an early age . It has  therapy . Lumacaf therapy . Gene th therapy ,97. A ge therapy . Nine Tu therapy . With muThe increased focus on genetic diseases in Tunisia over the last decades has been spurred mainly by the clinical and molecular investigations intended to improve the diagnosis. Nevertheless, genetic services are lacking and are not equally distributed across the country. Indeed, only three main medical genetic services are available: Charles Nicolle hospital in Tunis, Farhat Hached hospital in Sousse, and Habib bourguiba hospital in Sfax. A unit for congenital and inherited diseases was recently built at the “Mongi Slim hospital” in La Marsa, providing cytogenetic tests. Furthermore, breast and cervical cancer screening has been integrated into basic health care services within the framework of national cancer control plans  . In addition, the establishment of an NGS platform in Institut Pasteur de Tunis, allowed the setting up of molecular diagnosis of familial cancers, thus contributing to the better characterization and widening of the mutation spectrum of BRCA1/2 genes in the Tunisian population . The incFrom a public health perspective, experts and policymakers have opted that newborn and carrier screening should be conducted as a state-run program ,101. ThiA comprehensive genetic education and premarital genetic counseling programs can also help to reduce the burden of genetic diseases in inbred communities ,115. In Patient support groups could act as key support for rare genetic diseases. As stated earlier, hearing loss constitutes a frequent disease group in our population. In an attempt to improve the auditory health in the Tunisian population, hearing screening and awareness-raising campaigns are being organized by NGOs, such as ICHARA, in the frame of a science shop project participatory research support .Our study provides a better knowledge of the genetic diseases spectrum in Tunisia, which is useful for this country, as well as for neighboring countries, in order to improve the diagnosis, to reduce misdiagnosis, and therefore, patients will have earlier and better management. This would also help to avoid possible complications and reduce the costs of managing these diseases. Molecular and genetic data for these diseases are becoming more widely available through the development of biomedical health research activities and access to high-throughput next-generation sequencing. However, the Tunisian population still faces challenges, especially in the case of dual diagnosis and co-occurrence in the same patient or family, which delays the appropriate patient management. Taking this into account, in addition to the relatively high genetic and mutational heterogeneity, we recommend the creation of diagnostic referral centers that leverage some technologies, such as whole-exome sequencing, as a first intention tool for genetic disease elucidation in the country. These specialized structures could potentially offer accurate molecular diagnostic services at a lower cost and allow faster, efficient patient management. Furthermore, patient management should include multidisciplinary team investigators working in a well-defined care pathway. Vigilant monitoring of emerging clinical manifestations is recommended, especially in the case of genetic disorders. Furthermore, studies are needed to accurately set the epidemiological data of genetic diseases aiming to implement better and appropriate healthcare measures. Patient support groups have a significant role to play in promoting patient voice and influencing the decision-makers.