Little is known on maize germplasm adapted to the African highland agro-ecologies. In this study, we analyzed high-density genotyping by sequencing (GBS) data of 298 African highland adapted maize inbred lines to (i) assess the extent of genetic purity, genetic relatedness, and population structure, and (ii) identify genomic regions that have undergone selection (selective sweeps) in response to adaptation to highland environments. Nearly 91% of the pairs of inbred lines differed by 30–36% of the scored alleles, but only 32% of the pairs of the inbred lines had relative kinship coefficient <0.050, which suggests the presence of substantial redundancy in allelic composition that may be due to repeated use of fewer genetic backgrounds (source germplasm) during line development. Results from different genetic relatedness and population structure analyses revealed three different groups, which generally agrees with pedigree information and breeding history, but less so by heterotic groups and endosperm modification. We identified 944 single nucleotide polymorphic (SNP) markers that fell within 22 selective sweeps that harbored 265 protein-coding candidate genes of which some of the candidate genes had known functions. Details of the candidate genes with known functions and differences in nucleotide diversity among groups predicted based on multivariate methods have been discussed. Zea mays ssp. mays L.) is one of the top three crops globally in total production and is cultivated as a multi-purpose crop for food, feed, biofuel, and raw material for synthesis of various industrial products1. In Africa, maize is produced on a total area of nearly 37 million hectares, which is about 20% of the total maize area of the world. However, the total maize production for the continent is 70.6 million metric tons, which accounts only for 7% of the global production (http://www.fao.org). Lack of congruence between the proportion of production and the cultivated area is due to the low productivity of maize in Africa (<2.0 t ha−1) as compared to a global average of 5.6 t ha−1. In Sub-Sahara Africa (SSA), maize is the primary source of calories  and is the second most important source of protein (12 g/capita/day) only after wheat. In Ethiopia, maize is the second most popular staple crop after tef (Eragrostis tef (Zucc.) Trotter)2 with huge potential to feed over 100 million people in the country. Between 2008 and 2017, the total maize production and average grain yield in Ethiopia have increased from 3.8 to 8.1 million tons and from 2.1 to 3.7 t ha−1, respectively (http://www.fao.org).Maize , sensitive to deep planting, susceptible to inbreeding depression, slow grain drying after harvest for storage and have low harvest index. The International Maize and Wheat Improvement Center (CIMMYT) started highland maize breeding program in Mexico in the 1970s with the intention of developing high yielding and cold tolerant improved germplasm from pools and populations carrying tropical and subtropical genetic backgrounds5.Maize is broadly divided into temperate, subtropical and tropical germplasm depending on latitudinal variations and environmental characteristics4. Ecuador 573 together with Kitale Synthetic II  were used in the reciprocal recurrent selection for genetic improvement and variety development in the African highlands6. In the 1980s, CIMMYT introduced a tropical highland transition zone adapted pool (Pool 9A) to Africa. The pool was not only made available to farmers as an open-pollinated variety but also was used in various breeding programs4. The original Pool 9A was improved for maize streak virus (MSV) resistance at CIMMYT breeding hub in Harare (Zimbabwe), which was then extensively used in highland maize breeding in Africa. The initiation of Highland Maize Genepool Project in 1997 by CIMMYT, in collaboration with the National Agricultural Research Systems (NARS) in eastern Africa, further strengthened the highland maize breeding efforts in the region through introduction and improvement of highland adapted maize germplasm6.Although maize breeding for the east African highlands started in the 1950s by assembling locally available germplasm and making synthetic populations, the introduction of Ecuador 573 in 1959 to the region significantly impacted highland maize improvement13 and International Institute of Tropical Agriculture (IITA)16 using different genotyping platforms and marker density. Recently, Ertiro et al. (2017) genotyped 265 inbred lines developed by EIAR, CIMMYT, and IITA that are widely used in the mid-altitude sub-humid maize agro-ecology of Ethiopia with 220,878 SNPs. The authors reported that only 22% of the inbred lines were considered genetically pure with >95% homogeneity (genetic purity), which requires purification or further inbreeding except those lines deliberately maintained at early inbreeding level to avoid inbreeding depression. Pairwise genetic distances among the 265 inbred lines varied from 0.011 to 0.345, with only <1% of the pairs of lines differing by less than 20% of the total number of scored alleles. Finally, the different multivariate methods consistently suggested the presence of three groups, which generally agreed with pedigree information (genetic background). However, little is known about the genetic purity, variation and population structure of the maize germplasm adapted to the African highlands, which is widely used in eastern Africa. Previous genetic diversity studies conducted on highland maize inbred lines adapted to the African ecology were based on a small number of samples and low marker density21. For example, Beyene et al.  studied genetic diversity and relationships among 62 Ethiopian highland maize collections using only 20 simple sequence repeat (SSR) markers and eight amplified fragment length polymorphism (AFLP) primers. Legesse et al. (2007) assessed the genetic diversity of 35 highland inbred lines from CIMMYT-Ethiopia and 21 mid-altitude inbred lines from CIMMYT-Zimbabwe using 27 SSR markers and nine AFLP primers. Abakemal et al. (2015) studied genetic purity and patterns of relationships among 36 maize inbred lines adapted to African highland agro-ecology using 25 SSR markers.Various studies were conducted to determine the genetic diversity, relationship, population structure and heterotic grouping of maize inbred lines developed by CIMMYT24. Selection increases the frequency of a beneficial allele within a group or population and may even lead to fixation, which then increases the fitness of the individuals carrying it but reduces overall genetic diversity in specific regions that undergone selection27. Although all the highland-adapted inbred lines have undergone selection for better adaptation to the highland agro-ecology, we expect differential selection in response to target traits, including germplasm type , heterotic grouping, and abiotic and biotic stresses. Different statistical methods are available to identify genomic regions that have undergone selective sweep29. Therefore, the present study was carried out to (i) assess the genetic purity, genetic relationship and population structure of African highland adapted maize inbred lines using high-density genotyping by sequencing (GBS); (ii) identify genomic regions that have undergone selective sweeps, and examine if those selective sweeps showed greater reduction of nucleotide diversity in specific categorical variables (groups or populations) than others; and (iii) compare the extent of molecular diversity indices and genetic differentiation among different groups of highland maize germplasm.Selective sweeps leave distinct signatures in genomes, which are indicative of loci that have undergone selection6. The lines were selected for desirable agronomic performances, resistance to common leaf rust, Turcicum leaf blight, gray leaf spot, and germplasm type . The inbred lines used in our study comprised of 111 normal endosperm (non-QPM) lines derived primarily from Kitale Synthetic II , Ecuador 573, and Pool 9A. The remaining 187 samples were QPM lines that were either developed through backcross breeding30 or extracted from adapted QPM populations. CML144, CML159, and CML176 were the QPM donor parents. Heterotically, 123, 95 and 11 inbred lines belong to groups A, B and AB, respectively, while the remaining 69 inbred lines are not yet assigned.A total of 298 white-grained inbred lines from CIMMYT and Ethiopian Institute of Agricultural Research (EIAR) collaborative highland maize breeding program were used in the study  with a minor allele frequency (MAF) of ≥0.05 and a maximum missing data of 20%  and then filtered if there were SNPs with a MAF less than 0.05, which resulted in 235,019 SNPs (Dataset-2) for further statistical analyses.For each inbred line, seed samples were obtained from Ambo Research Center, Ethiopia. The detailed procedures on genomic DNA extraction, SNP genotyping using GBS34, which showed perfect positive correlation (r = 0.999). All statistical analyses except the model-based population structure were, therefore, computed on Dataset-2. The proportion of heterogeneity, relative kinship coefficients, IBS-based genetic distance matrices, and principal component analysis (PCA) were computed (from Dataset-2) using TASSEL v.5.2.51. Cluster analysis was performed on the genetic distance matrix using the neighbor-joining algorithm implemented in molecular evolutionary genetics analysis (MEGA) v.7.035. The first two principal components (PCs) from the PCA were plotted for visual examination in XLSTAT 2012  using categorical variables, which include heterotic groups, germplasm type (QPM vs. non-QPM), genetic background and group membership predicted both from population STRUCTURE and cluster analyses.We first computed identity-by-state (IBS)-based genetic distance matrices from both Dataset-1 (unimputed) and Dataset-2 (imputed) and compared the two distance matrices using Mantel test36 and ARLEQUIN v.3.5.2.237 formats using PGDSpider v.2.1.1.338. We used MEGA X36 to estimate the number of segregating sites (S), the proportion of polymorphic sites (Ps), Theta (θS), nucleotide diversity (θπ) and Tajima’s D test statistic39. Analysis of molecular variance (AMOVA)40 and FST-based pairwise genetic distance matrices41 were computed among categorical variables using ARLEQUIN v.3.5.2.237. FST values between pairs of populations or groups are indicative of the evolutionary processes that influence the structure of genetic variation with <0.05, 0.05–0.15, 0.15–0.25 and >0.25 indicating little, moderate, great and very great genetic differentiation, respectively42. To minimize the computational requirement in population structure analyses, the 235,019 SNPs in Dataset-2 were further filtered using a MAF of 0.10 and a minimum physical distance of 10-kb between adjacent markers, which resulted in 22,500 SNPs (hereafter referred as Dataset-3). Population structure was analyzed using Dataset-3 and the model-based method implemented in the software package STRUCTURE v.2.3.443 as described in our previous studies44. Inbred lines with membership probabilities >60% were assigned to the same clusters, while those with probabilities <60% in any group were assigned to a “mixed” group.HapMap format of Dataset-2 was exported to PHYLIP interleaved format using TASSEL v.5.2.51, which was then converted to both MEGA45 was used to detect selective sweeps that may have undergone selection during breeding process. For this purpose, Dataset-2 was converted into reference and alternative alleles using the variant call format (VCF) option in TASSEL v.5.2.51, which corresponds to the major and minor alleles, respectively. SweeD v.4.0.0 was run on the VCF input file as described in a previous study45 to evaluate a grid of 10,000 equidistant physical locations. The threshold score for declaring selective sweeps was set as the 99.9%, so the 0.1% with likelihood scores >3.1 were retained to represent a candidate selective sweep. The start and end of physical positions of each selective sweep region were used to search for candidate genes and their predicted functions46 at the Gramene Genome Brower (http://ensembl.gramene.org/Zea_mays/Info/Index).SweeD v.4.0.032, we increased the threshold value from 5.0% to 6.25%, which is the expected average residual heterozygosity (heterogeneity) for lines developed after four generation of inbreeding. Using this threshold criterion, only 34 of the 298 inbred lines (11.4%) were considered fixed with a heterogeneity of ≤6.25, while 57.7% and 30.8% of the inbred lines had heterogeneity varying from 6.26 to 12.50 and from 12.51 to 32.10%, respectively . Nearly 32% of the pairwise relative kinship values were close to zero (<0.05), 66% were between 0.051 and 0.500 and the remaining 3% between 0.501 to 1.98 ) and ons Fig. . The firThe neighbor-joining (NJ) tree constructed from the genetic distance matrix grouped 296 of the 298 inbred lines into three major groups as the model-based STRUCTURE and five sub-groups; two inbred lines were not assigned into any of the sub-groups Fig. . Nearly To get insight on the patterns of relationship among the 298 inbred lines, we constructed various phylogenetic trees Fig.  and alsoFST values between groups were compared  or great (0.15–0.25) genetic differentiation.Results from the partitioning of the molecular variance by different categorical variables revealed that differences in heterotic groups (A vs. B) and germplasm type (QPM vs. non-QPM) accounted for 12.0% and 8.1% of the genetic variation, respectively, which both fell under moderate genetic differentiation. On the other hand, the differentiation among groups based on genetic background (pedigree information), groups predicted based on cluster analysis and the model-based population structure accounted for 18.8–21.6% and 25.3–29.6% of the total molecular variation, respectively  that fell within the 22 selective sweeps with the genome-wide SNPs (Dataset-2) showed reduction in both Group-2 and Group-3 than Group-1. A less obvious reduction were noted among inbred lines belonging to the different heterotic groups, germplasm type and genetic backgrounds  were found to be genetically homogeneous  during line development, which is less common both at CIMMYT and IITA. In addition, the high level of genetic heterogeneity within EIAR maize inbred lines could also be due to human errors  during line development and/or line maintenance. If such types of errors occur, the sib-mating method is more prone to introducing new sources of genetic variability that in turn reduces genetic purity than selfing of individual plants. Because of the extensive collaboration between CIMMYT-Ethiopia and EIAR, including sharing nurseries, most of the inbred lines analyzed in the present study could have resulted from a combination of sib-mating and selfing.Maintenance of genetic purity in inbred lines by minimizing residual heterozygosity (heterogeneity) is important for quality seed production49, which are suitable for developing open-pollinated varieties (OPVs) but may not be suitable for extracting genetically pure inbred lines. Third, some of the inbred lines were deliberately extracted from early generation (such as S3) lines and maintained by sib-mating to avoid severe inbreeding depression upon continuous self-pollination4. Although such approach is useful to attain higher seed yield per unit area, which in turn decreases the cost of seed production and increases access to seed by small-scale farmers50, it would be very challenging in terms of line maintenance. However, the third case is a less likely scenario as there are multiple lines with heterogeneity greater than 12.5%, which is the expected average heterogeneity among lines extracted at S3 generation.Second, most of the source germplasm used for developing the inbred lines in the current study were composites, pools, and synthetics51. As a result, maize breeders are using fixed lines in their new pedigree starts up and advance each generation through selfing than sib-mating. One of the immediate solutions for improving genetic purity of the inbred lines used in the present study may be to purify seed stocks of those lines with higher than expected heterogeneity by rouging off-types in seed maintenance and production plots, but such method requires enormous efforts and incurs additional costs. The long-term solution is to use doubled haploid (DH) technology in developing genetically pure doubled haploid (DH) lines that can be derived in a short period of time54. In partnership with the Kenya Agriculture and Livestock Research Organization (KALRO), a state-of-the-art maize DH facility for Africa has been established in 2013 by CIMMYT at Kiboko station, Kenya, which is annually producing nearly 70,000 DH lines from African-adapted maize source germplasm.Currently, there is more demand in developing uniform hybrids using genetically pure parental lines, especially doubled haploid lines, as this has several advantages, including better heterosis, simplicity in parental line maintenance and implementing quality control during hybrid seed production32. The 32% pairs of highland maize inbred lines with kinship coefficients close to zero was six-fold greater than the 5% reported in maize inbred lines originated from CIMMYT ESA breeding programs7, but nearly half of the values reported for maize inbred lines adapted to the mid-altitude ecologies of Ethiopia32, the global maize collection10, inbred lines from INERA and IITA14, inbred lines from CIMMYT and IITA9 and CIMMYT maize inbred lines8. On the other hand, nearly 91% of the pairs of 298 inbred lines differed by 30–36% of the scored alleles  as compared with just 10% of the pairs that differed by ≤30% of the scored alleles . Several previous studies reported the lack of consistencies between heterotic classification based on genotype data and combining ability or pedigree information in tropical maize germplasm32. The broad genetic base of the germplasm, lack of clear information on origin and heterotic background, inadequate pedigree information, short breeding history, and use of variable testers and testcross evaluation for assigning lines to heterotic groups have been frequently cited as possible reason for disagreement between markers-based heterotic grouping and combining ability and pedigree-based heterotic assignment.The high genetic differences among most pairs of inbred lines agrees with pedigree information and breeding history, as have been reported in other studiesglycosyl transferases in family 61 protein that mediate arabinofuranosyl transfer onto xylan in grasses56, which plays an essential structural role in cell walls of all plants and valuable components of human and animal nutrition due to its major dietary fiber composition in cereals58. One of the selective sweeps on chromosome 1 (1:106060208-110288743) consisted of 45 candidate genes, including (i) the WRKY-transcription factors that play crucial roles in plant growth and development, defense regulation and response to different biotic and abiotic stresses60; (ii) roothairless6 (rth6), which is one of the genes that control root hairs formation and facilitates nutrient uptake and optimal development62; (iii) JUMONJI-transcription factor 14 (JMJ14) and CCAAT-binding transcription factor that control flowering time65. The selective sweep on chromosome 5 (5:184394549-184699627) harbors the maize red aleurone1 (pr1) that encodes a CYP450-dependent flavonoid 3′-hydroxylase, which is required for the biosynthesis of purple and red anthocyanin pigments. Anthocyanins accumulate in maize pericarps, cob glumes, and silks66 and believed to have a protective role in plants against extreme temperatures. The pr1 locus has also been extensively used as a phenotypic marker in determining kernel aleurone color by hydroxylation of anthocyanin compounds67. Different studies have implicated members of the bZIP family of transcription factors (proteins that bind to the G-box) as mediators of abscisic acid dependent gene expression69 of which bZIP transcription factor 1 (bzip1) is located in one of the selective sweep regions (3:215786813-216005903) identified in the present study. Abscisic acid plays a central role in plants abiotic stress resistance by regulating a large number of stress-responsive genes to confer abiotic stress tolerance in plants70. The candidate genes mentioned above could be potentially influencing traits of adaptation to highland agro-ecologies in Africa, and the observed selective sweeps might be due to positive natural selection or deliberate selection during the development of inbred lines or source populations.As shown in the Manhattan plot in Fig. 72; (b) glutathione transferases that catalyze the conjugation of glutathione to xenobiotic compounds in the detoxification process73; (c) G2-like transcription factors that play a central role in regulating chloroplast development, which contain the green pigment chlorophyll and are responsible for the light-powered reactions of photosynthesis ; and (d) basic leucine zipper (bZIP) gene family that play important roles in multiple biological processes, such as light signaling, seed maturation, flower development as well as abiotic and biotic stress responses74. The four selective sweep regions identified on chromosome 7 consisted of multiple candidate genes, including Kinesin-related proteins (KRPs) that play central roles in the transport of various vesicles and organelles in eukaryotic cells75; gibberellin 2-oxidases (GA2oxs) that regulates plant growth by inactivating endogenous bioactive Gibberellins76; the maternally expressed gene (Meg) family, which encodes cysteine-rich proteins (CRPs)77 that are involved in both cell-signaling and antimicrobial processes79; the Endosperm5 (o5) showed moderate correlation (R2 = 0.66) with Opaque2 (o2) and affect different aspects of storage protein synthesis in maize80; the cellulose synthase (CesA) gene family that are primary determinant of wall formation, stalk strength and improve harvest index81; carbonic anhydrase (CA) that catalyzes the reversible hydration of CO2 into bicarbonate82, and implicated in photosynthesis83, stomatal conductance and guard cell movement in C3 plants84, and providing bicarbonate to the initial carboxylating enzyme phosphoenolpyruvate carboxylase in C4 plants85; the basic helix-loop-helix (bHLH) transcription factors that play key roles in diverse biological processes, including seed germination, shade avoidance response, flowering time regulation, stress responses and anthocyanins synthesis88; pentatricopeptide repeat (PPR) proteins that have been implicated in RNA editing, RNA processing, translation, photosynthesis, respiration and kernel development90; the maize D-cyclin gene asceapen1 (asc1) that plays a role in leaf and shoot development91 and regulates progression through the G1 phase of the cell cycle92.Multiple candidate genes of known function have been identified on chromosome 6, which include (a) elongation factors that are highly correlated with total lysine content of the endospermAP2/EREBP (APETALA2/ethylene responsive element-binding protein) transcription factors that are involved in many different pathways, including drought and high salt concentration93, low temperature94, diseases96 and the control of flowering97; receptor for activated C kinase that plays a role in plant response to fungal phytopathogens98, affect different signal transduction pathways and multiple developmental processes in plants99; MYB transcription factors that are involved in controlling responses to biotic and abiotic stresses, development, differentiation, metabolism, hormone signal transduction101; and AP kinase kinase kinase 18 (MAPKKK18) that controls plant growth by adjusting the timing of senescence via its protein kinase activity102.On chromosome 8, the selective sweeps consisted of several 4 or later generations, which suggests the need for revising the line development strategy, including line finishing and use of genetically pure parental lines for line development ; generating reference genotype data as one of the requirements for releasing lines; implementing quality assurance (QA) and quality control (QC) genotyping methods to regularly check genetic purity of key inbred lines during line maintenance; and more frequent use of DH technology in developing breeding lines. The germplasm used in the current study showed clear population structure, primarily by pedigree information and breeding history, and less so by heterotic groups and germplasm type. There was a high level of genetic difference among most pairs of inbred lines although they have a large proportion of alleles in common, which is expected when a limited number of parental lines are used for line development. We identified 944 SNPs that fell within 22 selective sweep regions, which harbored 265 annotated genes whose functions provide clues on the adaptation of the tropical maize to the African highlands. Molecular diversity indices computed across multiple categorical variables using SNPs that fell within the selective sweeps showed a two-fold reduction on polymorphic sites and nucleotide diversity in two of the three groups predicted based on the model-based STRUCTURE as compared to the genome-wide SNPs. Such thorough analyses on the genotypic data depict a significant contribution of this study to the available knowledge on selective sweeps in maize. Results from this study provide valuable information for further improvement of highland maize breeding programs in Africa, including the need for revising the line development strategy, diversifying parental lines for developing new inbred lines, and verifying genetic purity of newly fixed inbred lines using QC genotyping.Most of the 298 inbred lines adapted to the African highland ecology showed high level of genetic heterogeneity than expected for lines extracted from SSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary Information


This study aimed to evaluate the different serological techniques for early diagnosis of acute concurrent infections with linguatulosis in the definitive host (dogs) and an intermediate host (goats). This evaluation compared between the gold standard (GS) test , sandwich enzyme-linked immunosorbent assay (S-ELISA), and indirect ELISA.Linguatula serrata nymphs and adults, respectively, besides the collection of blood samples from the examined animals for serologic testing.Fifty goats and fifty dogs were examined for the presence of In goats; GS, S-ELISA, and indirect ELISA showed positivity in 32 (64%), 28 (56%), and 39 (78%) samples, respectively. In dogs; GS, S-ELISA, and indirect ELISA showed positivity in 25 (50%), 25 (50%), and 30 (60%) samples, respectively. S-ELISA displayed significant higher agreement with the GS test (≥0.83) than indirect ELISA (≤0.67) in both hosts. Infection with linguatulosis showed significant relation with the age of goats and dogs and the sex of goats (p<0.05).S-ELISA displayed more sensitivity and specificity for the detection of concurrent infections with linguatulosis in both dogs and goats than indirect ELISA, which could detect the prior infections. Similarly, these assays could be used for diagnosis of concurrent infections with linguatulosis in human, especially the chronic ones. Linguatula serrata is a cosmopolitan worldwide aberrant endoparasite of the class Pentastomida  test) and different serologic techniques (sandwich ELISA [S-ELISA] and indirect ELISA).This study aimed for evaluation of the potential use of All study procedures were approved by the Institutional Animal Care and Use of Ethical Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt.L. serrata nymphs, according to Attia et al. [Fifty slaughtered goats from butcher shops were inspected from March 2018 to August 2018. Blood samples for serum were collected from each animal, and MLNs were obtained and grossly examined by naked eyes for detection of a et al. , and plaL. serrata adult parasites from its nasal cavity. All were dogs examined for the presence of L. serrata eggs in feces and nasal swabs, according to Attia et al. [Blood, fecal, and nasal swabs were collected from 50 dogs from different governorates from March 2018 to August 2018. A positive control case was recorded in the hospital of Faculty of Veterinary Medicine, Cairo University; which sneezed et al. [et al. [The collected nymphs and adults were washed in saline solution, relaxed, flattened, fixed, stained, mounted, and identified, according to Attia et al.  and Raza [et al. .et al. [The nymphs and adults were washed several times with saline solution. Antigens were prepared from 500 nymphs and ten adults recovered from dogs. Antigens were preserved in phosphate-buffered saline (PBS) (pH 7.2) and prepared . The proet al. .Rattus norvegicus albino) about 150 g in weight were raised for preparation of hyperimmune sera versus L. serrata nymphs and adults according to Innocenti et al. [L. serrata nymphs; and two immunized against L. serrata adults) in conventional rat cages. The rats were supplied with balanced rats’ pellets and water ad libitum. The immunized rats were subcutaneously injected with 1 mg protein of antigens, which mixed in 1 ml mineral oil (1st dose). Then, two subsequent intramuscular injections of 0.5 mg protein antigens mixed in the same volume of mineral oil were injected each 2 weeks interval. Rats were slaughtered for collection of blood for sera, 2 weeks after the last dose. The collected hyperimmune rats’ sera were stored at −20°C until used.Six male rats  were obtained from an ELISA reader  at 450 nm. The sera were considered to be positive when the absorbance values were as or more than the cut off value equal to a double fold of the mean negative sera; the ELISA was done, according to Kouam et al. [The checkerboard titration for determination of the lowest antigen concentration and sera dilution was done, according to Harlow and Lane . A ninetm et al. .et al. [et al. [Checkerboard titration was done according to Harlow and Lane  to obtaiet al. , and Dix [et al. .The cutoff point for a positive result was taken as 2× mean of OD of negative control sera . To evalStatistical analysis of agreement between ELISA, S-ELISA, and GS tests was performed using the Kappa agreement test. The Landis and Koch scale  was usedL. serrata as recorded by Attia et al. [The nymphs, adults, and its eggs identified for a et al.   showed positivity in 32 samples (64%). The indirect ELISA test of goats’ sera showed seropositivity in 39 samples (78%) while the S-ELISA showed seropositivity in 28 samples (56%) .The indirect ELISA test of goats’ sera showed a sensitivity of 100%, and specificity of 61.11%, with an accuracy of 86% while the S- ELISA test showed sensitivity of 87.5%, and specificity of 100%, with an accuracy of 92% .For the agreement analysis, the Kappa values obtained were significantly substantial between ELISA and GS tests  and between S-ELISA and GS , while it was significantly moderate between ELISA and S-ELISA  .Using the Pearson’s test for correlation, a significant positive correlation was proved between the number of nymphs detected by GS test and the optical densities obtained from indirect ELISA and S-ELISA tests  .The examination of feces in dogs (GS) showed positivity in 25 samples (50%). The indirect ELISA test of dogs’ sera showed seropositivity in 30 samples (60%), while the S-ELISA showed seropositivity in 25 samples (50%) .The indirect ELISA test of dogs’ sera showed sensitivity of 84% and specificity of 64%, with an accuracy of 74%, while the S-ELISA test showed sensitivity of 96% and specificity of 96%, with an accuracy of 96% .For the agreement analysis, the Kappa value obtained was significantly moderate between ELISA and GS tests , and between ELISA and S-ELISA , while it was significantly almost perfect between S-ELISA and GS  .L. serrata infection was related to the sex of the animal and was significantly higher in female goats than in males (p=0.006). However, in dogs, the frequency of infection was not statistically related to the sex of the animal (p>0.05)  , Table-5L. serrata infection was related to the age of the animal and was significantly higher in goats aged 3 years or more (p=0.000). In addition, in dogs, it was statistically related to the age of the animal. Dogs of 1 year old and 5 years old showed the highest frequencies of infection (p=0.000), , .In goats, the GS test revealed 64% positivity, while S-ELISA revealed 56% positivity in comparison to indirect ELISA which revealed 78% positivity; this is due to that S-ELISA could detect the concurrent infection, while indirect ELISA could detect the antibody levels of the previous infection from 6 months ago. Positive S-ELISA results were higher than GS, which may be due to the presence of infection in different organs other than MLNs.From these comparisons, it was revealed that the sensitivity of the two tests was similar in goats while high in S-ELISA in dogs. The specificity of S-ELISA was high in dogs and goats.This sensitive S-ELISA improves and simplifies the detection of linguatulosis in animals.Pentastomiasis has a zoonotic potential to human, which is difficult to be diagnosed or might be misdiagnosed with other chronic diseases such as malignant tumors, cysticercosis, and lung diseases .L. serrata), and we used two animals; the definitive host (dogs) and an intermediate host (goats) that represented the most grazing animal around the dogs, to be a good example for human testing.Advanced technique as polymerase chain reaction test was previously used for identification but had not been evaluated for diagnosis of linguatulosis . For thiL. serrata antigens was presented a reliable, non-invasive diagnostic tool for detecting active infections. The sensitivity and specificity of any immunoassay are dependent on the antigen capture efficiency of an antibody which is subsequently governed by the nature of antibody. This led to the different assay sensitivities of the commercially available kits using different types of antibodies which could be monoclonal or polyclonal at the capture stage in ELISA.Using the crude extract of Considering linguatulosis importance in human and animals, it seems necessary to do more investigations in both domestic and wild carnivores and herbivores. Only a few reports described the seroprevalence and evaluated the indirect ELISA for 121 sheep sera and determined its sensitivity, specificity, PPV, NPV, and accuracy of the somatic antigens, which were 90.4%, 75.0%, 84.6%, 83.7%, and 84.2%, and those for excretory-secretory antigen were 89.0%, 93.8%, 95.6%, 84.9%, and 90.9%, respectively . HoweverOther serological tests were used as counter immunoelectrophoresis used for diagnosis of linguatulosis in sheep , but thaIn this study, we differentiated between two assays to distinguish the sensitivity and specificity of each test. S-ELISA showed higher sensitivity and specificity than indirect ELISA, so, it can be applied in the field studies or laboratories for diagnosing of linguatulosis because it detected the concurrent circulating antigens. S-ELISA could be considered as a new serological technique for the diagnosis of linguatulosis in goats and dogs. Moreover, S-ELISA could be used in the diagnosis of linguatulosis in human with halzoun or visceral form.MMA, EI, and NMKS conceived and designed the experiments. MMA and NMKS performed the experiments. EI analyzed and interpreted the data. All authors participated in draft and revision of the manuscript. All author have read and approved the final manuscript.


Oryza rufipogon and O. nivara in Sri Lanka were studied using 33 microsatellite markers. A total of 315 individuals of 11 natural populations collected from the wet, intermediate, and dry zones of the country were used in the study. We found a moderate to high level of genetic diversity at the population level, with the polymorphic loci (P) ranging from 60.6% to 100% (average 81.8%) and the expected heterozygosity (HE) varying from 0.294 to 0.481 (average 0.369). A significant genetic differentiation between species and strong genetic structure within species were also observed. Based on species distribution modeling, we detected the dynamics of the preferred habitats for the two species in Sri Lanka and demonstrated that both O. rufipogon and O. nivara populations have expanded substantially since the last internal glacial. In addition, we showed that the geographical distribution of the two species corresponded to the climate zones and identified a few of key environmental variables that contribute to the distribution of the two species, implying the potential mechanism for ecological adaptation of these two species in Sri Lanka. These studies provided important insights into the population genetics and evolution of these wild species in Sri Lanka and are of great significance to the in situ conservation and utilization of these wild resources in genetic improvement of rice.Genetic diversity and population genetic structure of the wild rice species  The detailed information of these populations is provided in Supporting Information Table A total of 11 wild rice populations including five 2.2https://gramene.org). Detailed information of the primer pairs is given in Table 2O), 7 µg 2× Taq PCR MasterMix , 1 µl each of the forward and reverse primer (10 µM), and 1 µg template DNA. The PCR cycle profile was 94°C, 3 min; 35 cycles of 94°C, 30 s, 55°C, 30 s, and 72°C, 1 min; and 72°C, 10 min for the final extension.The gDNA was extracted from 20 mg of silica gel‐dried leaves, using the Plant genomic DNA kit (Biomed DL114‐01) following by the CTAB protocol  were diluted in 8 µl of ultrapure water and scoured in 100% alcohol for 15 min. The diluted DNA was dried and mixed with highly deionized formamide (Applied Biosystems) and then submitted for fragment analysis by capillary electrophoresis using an Applied Biosystems 3130×l DNA analyzer (Applied Biosystems). The DNA fragment size analysis and allele calling were performed using GeneScan and GeneMapper software (Applied Biosystems), followed by the manual allele binning. The matrix standard kit was used to generate the “multicomponent matrix” when analyzing the FAM‐, TAMRA‐, and HEX‐labeled DNA fragments with the ABI3130 series systems. Data Collection Software (Applied Biosystems) used the multi component matrix for the instrument to analyze automatically for three different colored fluorescent dye‐labeled samples in a single capillary. In addition, reference lanes were further verified by polyacrylamide gel electrophoresis. In the case of non‐amplification, the PCR was repeated to exclude technical failure and a null allele was recorded if both PCRs failed.2.3A), the mean observed heterozygosity (HO), the percentage of polymorphic loci (P), and the unbiased expected heterozygosity (HE). All the estimates of genetic diversity were calculated using GenAIEx 6.5.01 software  through the dudi.pca function of “adegenet” R package . To estimate the relative contributions of the environmental variables on the species distribution, we performed multiple relative importance tests including the percentage contribution, permutation test, and jackknife test. To predict species occurrence, environmental layers of 19 biologically meaningful climate variables (BIO1‐19) were downloaded from the WorldClim database  under present day (1950–2000), the last glacial maximum  and the last interglacial  conditions. In addition, we added 107 O. nivara and 38 O. rufipogon records collected from Sri Lanka from field investigations. We defined the study extent according to the commonly accepted range of the two species, between 5.550N and 9.51ON, 79.42OE and 81.53OE. All the 145 localities of our own collection sites were used to build the models using the default parameters for convergence threshold (10–5) and number of iterations (500). To insure the consistency of the model predictions, 75% of the localities were used to train the model and 25% were used to test it.A species distribution model (SDM) was generated for 3p > 0.05), suggestive of an absence of linkage disequilibrium between loci. All 33 loci displayed polymorphism among the 11 populations with a total of 172 alleles identified. The most variable locus (RM426) had 30 alleles, while the RM457 showed only three alleles across the populations, with an average of 11.66 alleles per population  and the lowest  diversity, respectively. It is evident that O. rufipogon populations maintain higher level of genetic diversity than those of O. nivara populations  occurred between two major groups  were sampled from the intermediate zone  and further divided to populations  is the most important variable for determining the distribution of both species. In addition, the mean temperature of coldest quarter (BIO11) and the mean diurnal range (BIO2) were important for the distribution of O. rufipogon, while the mean temperature of driest quarter (BIO9) and the mean temperature of wettest quarter (BIO8) were critical for the distribution of O. nivara, indicating the differences in habitat preference of the two species.Finally, the SDMs under present, LGM and LIG with all 19 bioclimatic variables showed that the estimated current distribution patterns of the two species were highly consistent with their actual distribution areas at present in Sri Lanka Figure , that is4O. nivara and O. rufipogon given that thousands of SSR markers across rice genome have been well characterized  are featured as co‐dominant, highly polymorphic, abundant, and randomly distributed markers in the genomes and have been successfully used for a variety of studies in rice biology  across Asia Pacific and found that the average HE values for the species was 0.70 and 0.67 for O. rufipogon and O. nivara, respectively, with the lowest for the Nepalese O. nivara (0.39) and the highest for the Southeast Asian O. rufipogon (0.66). In a recent study on 418 O. rufipogon and O. nivara accessions collected across the entire Indian peninsula, Singh et al. (HE = 0.63 (O. rufipogon) and 0.64 (O. nivara), respectively.To date, many investigations on genetic diversity of  and Liu  found a a et al.  studied o et al.  investigh et al.  reportedO. rufipogon and 0.33 for O. nivara) or at the species level (0.62 for O. rufipogon and 0.58 for O. nivara) were relatively high given the small area of the island. This result indicates that the wild rice populations in Sri Lank may serve as an important genetic reservoir for rice improvement and deserve effective conservation. Consistent with previous studies  provide a unique system to investigate adaptive divergence and ecological speciation in plant species. Together, our study on O. nivara and O. rufipogon populations in Sri Lanka not only provide important insights into the population genetics and evolution history of these wild species, but also is of great significance for in situ conservation and management of these genetic resources.One interesting and challenging question is whether the Sri Lankan nd Kumar  reportednd Kumar . In addiNone declared.S. G., D. R., and S. S. conceived the ideas. D. R. and Q‐L. M. collected the data. S. S., A. T., and A. M. analyzed the data; and S. G., A. T., A. M., B. M., and S. S. wrote the paper.Data available from the Dryad Digital Repository. Click here for additional data file.


AbstractSebastiscusmarmoratus is an ovoviviparous fish widely distributed in the northwestern Pacific. To examine the gene flow and test larval dispersal strategy of S.marmoratus in Chinese and Japanese coastal waters, 421 specimens were collected from 22 localities across its natural distribution. A 458 base-pair fragment of the mitochondrial DNA (mtDNA) control region was sequenced to examine genetic diversity and population structure. One-hundred-six variable sites defined 166 haplotypes. The populations of S.marmoratus showed high haplotype diversity with a range from 0.8587 to 0.9996, indicating a high level of intrapopulation genetic diversity. Low non-significant genetic differentiation was estimated among populations except those of Hyogo, Behai, and Niiigata, which showed significant genetic differences from the other populations. The demographic history examined by neutrality tests, mismatch distribution analysis, and Bayesian skyline analysis suggested a sudden population expansion dating to the late Pleistocene. Recent population expansion in the last glacial period, wide dispersal of larvae by coastal currents, and the homogeneity of the environment may have important influences on the population genetic pattern. Knowledge of genetic diversity and genetic structure will be crucial to establish appropriate fishery management of S.marmoratus. Sebastiscusmarmoratus , valued for its high nutritional value and palatability  control region has been shown to be particularly effective in detecting population genetic structure and diversity, owing to its high polymorphism, maternal inheritance, high mutation rate, and nonrecombinant DNA .The mitochondrial DNA  Pacific shapes intra-species diversity of the fish fauna.The goals of this study were to estimate genetic diversity, to characterize genetic structure, and to reconstruct the evolutionary relationships of  decline . ElucidaS.marmoratus from 22 locations in coastal China and Japan, 10–24 specimens per site  were carried out in 25 μL of reaction mixture containing 10–100 ng template DNA, 0.1 µL (5 U/µL) Taq DNA polymerase , 1.5 µL (10 pmol/µL) of each forward and reverse primer, and 2 µL (200 µmol/L) deoxy-ribonucleoside triphosphate (dNTP). The PCR amplification was conducted in a Biometra thermal cycler under the following conditions: 2 min initial denaturation at 95 °C; 40 cycles of 60 s at 94 °C for denaturation, 45 s at 52 °C for annealing, and 60 s at 72 °C for extension; and a final extension at 72 °C for 8 min. The PCR product was purified with a Gel Extraction Mini Kit . The purified product was used as the template DNA for cycle sequencing reactions performed using BigDye Terminator Cycle Sequencing Kit , and bi-directional sequencing was conducted on an Applied Biosystems Instrument Prism 3730 automatic sequencer  with both forward and reverse primers. The primers used for sequencing were the same as those used for PCR amplification.Polymerase chain reactions , nucleotide diversity (π), mean number of pairwise differences (k), and number of polymorphic sites were calculated by ARLEQUIN v. 3.5  using default settings and were manually corrected. The genetic diversity indices of N v. 3.5 .NJ) tree of the haplotypes was rooted with the out-group Sebastesschlegelii .Nucleotide sequence evolution models were evaluated using likelihood-ratio tests implemented by Modeltest v. 3.06 . The neihlegelii  using MEhlegelii  to reconndex Fst , and theN v. 3.5 . The P vrrection . The FstAMOVA) of different gene pools , and τ  (SSD) and Harpending’s raggedness index (HRI) were used to test goodness-of-fit of the observed unimodal mismatch distribution to that expected under the sudden expansion model. The time since population expansion was estimated using the equation τ = 2μt, where μ is the mutation rate for the entire DNA sequence under study, and t is the time since expansion. We used the sequence divergence rate of 5%-10%/MY  . Historial time) . Unimodaal time) . The sum%-10%/MY  for the Bayesian skyline analyses, implemented in BEAST v. 1.7.4 , were pemtDNA control region was amplified, and 106 polymorphic sites were detected, including 89 transitions and 17 transversions. A total of 166 haplotypes were identified based on the sequence variation in 421 individuals from 22 locations. Among these, 84 haplotypes were shared. The most common haplotypes, Hap4 and Hap5, were both shared by 40 individuals. Haplotype sequences were deposited in GenBank under accession numbers KY703229–KY703394.A 458 bp segment of the π) and haplotype diversity (h) for the locations are shown in Table π was 0.0220±0.0110 with highest in Kochi (0.0250±0.0132) and the lowest in Hyogo (0.0098±0.0056). The mean value of h was 0.9560±0.0035 with the highest in Awaji (0.9996±0.0243) and the lowest in Hyogo (0.8587±0.0337).The estimated nucleotide diversity  rooted with the outgroup ies Fig. . The relies Fig. . The MSTFst values . Some pairwise Fst estimates were negative, indicating that within-population variation was greater than that between populations. The global AMOVA showed about 13.59% of the genetic variation to be among populations. Other grouping methods also indicated most genetic variation to be within populations, rather than among groups and populations.Genetic differentiation among the 22 locations was evaluated based on es Table , Fig. 5 es Table . In geneD  and Fs test  results were negative, indicating departure from selective neutrality  and divergence rate of 5–10% per site per Myr (1/θ0θ) was 56.84  of S.marmoratus in the northwestern Pacific Ocean, its nucleotide diversity was low (π = 0.0220±0.0110). The high mutation rate of the D-loop region may be a factor in this phenomenon (Glyptocephalusstelleri (Trachurusjaponicus (Circusspilonotus (Inbreeding depression and other genetic problems impacted by human behavior can be monitored by assessing genetic diversity under natural conditions . The adaenomenon . Haplotyenomenon . The retenomenon . The phestelleri , Trachuraponicus  and Circilonotus  that havAMOVA results and the neighbor-joining analysis did not show significant genetic structure among geographic populations. Ecological characteristics and marine currents may play important roles in shaping the contemporary phylogeographic pattern of marine fishes. For example, the rockfish S.schlegelii is typical of fish that congregate in drifting seaweed during early development (Sebastiscusmarmoratus, a species of rockfish with life history similar to S.schlegelii, is believed to exhibit the same behavior, dispersing with drifting seaweed during November and December and in the following year from February to April (S.marmoratus larvae via drifting seaweed and promote exchange throughout its range.Compared with anadromous and freshwater fishes, marine species are generally expected to show a low degree of genetic differences among geographic regions owing to their high dispersal potential through planktonic drifting of eggs, larvae, or adults and the absence of physical barriers . Our AMOelopment . Sebastito April . The Kurto April . Inflowsto April . Waters S.marmoratus caught by bait in Norwegian waters and thus shown to be actively foraging, a strong indication that S.marmoratus may thrive in unfamiliar conditions.Recent research reveals that the currently most common unintentional pathway for the transport of marine organisms is the ballast water of commercial vessels . As humaS.marmoratus. It has been transported among ports and wharves along the NW Pacific Ocean and into rocky coastal areas with the release of ballast water, where it can easily survive (S.marmoratus have been reported (S.marmoratus. However, this is undetectable by the molecular markers we used in this study. It has been suggested that a genotyping-by-sequencing technique could reveal the occurrence of local adaptation (We may conclude that transportation via ballast water may be a source of genetic homogeneity of  survive . With adreported . It is aaptation .S.marmoratus may have experienced a population contraction with the loss of genes of those dying out and the majority of survivors migrating to more suitable environments. A single branch from the star-like network representing the Hyogo population suggests a likely founder effect (Using genetics to understand biogeography is important to determine patterns influencing distribution of geographically distant populations. Genetic diversity, genetic distribution patterns, and effective population size were also influenced by paleogeological changes and fluctuations as well as life history and marine environment factors . Followir effect .Fst value analysis, which suggests that the deep and semi-open area of inland waters might have an impact on the geographic isolation. Genetic differences among Hyogo, Behai, and Niigata populations and between Hyogo, Behai, and Niigata and the other populations were primarily significant, and possibly relate to convergence evolution (Significant genetic differences were revealed between Hyogo and other populations based on the star-like network tree and volution  and the volution . In addivolution . FurtherS.marmoratus. Information provided by the current study will facilitate its comprehensive management. Future studies should be based on informative nuclear markers to provide additional information on genetic structure and differentiation of populations of S.marmoratus.Climate fluctuations caused by glacial-interglacial alternation, early life-history, and ecological characteristics, combined with transport via ballast water may play important roles in the extensive gene flow among populations and the current genetic distribution pattern of


Adaptation to a hotter climate is vital for future livestock as heat stress can extremely reduce their productivity, health, and fertility , United Arab Emirates (UAE), Kenya, and Bahrain can be the starting point for a genetic improvement in dairy camels if they participate in a common genetic evaluation program. To our knowledge, little communication and collaboration is currently practiced between these dairies, due primarily to competition. However, this lack of collaboration is bound to fade away with the realization that cooperation will improve long term profitability. Under such cooperation, records pertaining to milk production and health traits can be exchanged between the dairies as well as verified pedigree data. This exchange can help create a virtually common nuclear flock which can be utilized for traditional genetic evaluation of sires and dams. At a later stage, genomic selection can be practiced in order to speed up the genetic gain. In addition, genetics of the elite animals can be disseminated to camel owners in respective countries. The realized genetic gain in the camel owners' herds shall encourage them to participate in genetic improvement programs. This will increase the number of participatory herds and the genetic variability accessible to the genetic evaluation program and accelerate genetic improvement. As the numbers of herds increase and more pedigree data become available, the evaluation can be extended to other traits such as beef, racing, and beauty. That in turn will help with the classification of the camel population into beef and dairy individuals and the identification of elite individuals in each category. This classification and will later make it easier for investors and owners to make future breeding decisions and reinforce the industry. An alternative to starting with the camel dairies for genetic improvement is starting with the camel owners themselves by forming cooperative community based breeding programs. These can begin with a nuclear flock formed by the owners that expand to include more owners in future. Such programs are found in developing countries for sheep and goats  was found to profoundly affect the racing speed and stamina . This is due primarily to the difficulty in semen collection and handling . In addition, deep freezing of camel semen has proved to be highly a challenge. Although research groups have tried different buffers and diluents as mediums for freezing camel semen . This is expected given the cost of production and the lack of genetic improvement in camels. It is therefore challenging for small scale producers to survive without government subsidies and support. To increase the market share and potential for such producers, added value products  need to be produced and smart marketing strategies need to be adopted. Such strategies could include awareness campaigns of the health benefits of camel products, attractive product packaging, online marketing and partnership with existing cattle dairies and beef production firms for distribution and marketing. Camel milk can be marketed as a functional food, optimal for infants and elderly Nikkhah, . Focus cIn conclusion, camels have a large potential that is underutilized due to technical, logistic, political, and economic challenges. However, these challenges are not insurmountable, and much can be done to exploit the camels' potential. Genetic improvement is certainly promising in camels but would require the collaboration of all stakeholders and deeper understanding of the potential of this exceptional animal.MA conceived the idea and wrote the manuscript. BF participated in writing and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FST values and revealed a genetic structure mostly driven by male gene flow across villages. This genetic feature illustrates the uxori-matrilocality inherited from the Bantu expansion, though one exception exists in Bandamadji whose historically documented military status implied patrilocality in this locality.Post-marital residence of spouses is one of the architects of population genetic structure. In the present study, we tested how the place of residence of males and females in Ngazidja, Comoros Islands, has unequally channeled, by dispersal among villages, the male and female genetic diversity. Using sequences of the hypervariable segment I of the mitochondrial DNA (mtDNA HVS-I) and six Y-chromosome microsatellites (Y-STRs), we measured the genetic variation and male-to-female effective number of migrants ratios based on  The Comoros Islands are an archipelago of four islands, Ngazidja , Nzduani (Anjouan in Shindzuani), Mwali , and Mahore (Mayotte in Shimaore), located at the northern end of the Mozambique Channel in the Indian Ocean. Historical, social, linguistic, and genetic studies showed that the Comorian population arose from the encounter of East African Bantu metallurgists with Islamic Middle Eastern traders and, to a lesser degree, with Austronesian people –5.hinya. All these conditions create a real socio-economic pressure to stay in the birth village for both men and women [The social structure in Comoros Islands results from the interactions between Islam’s laws and ancestral rites from East Africa, with a mixed distribution of matrilineal and patrilineal traits in various domains of social organization: kinship, inheritance of property, succession to titles, mode of residence, and detention of authority , 6. In tnd women , 6.Previous surveys in matrilocal and patrilocal structured populations have shown a contrasted pattern of within and between-population genetic variation when examined for paternally and maternally inherited genetic markers , 8. HereIn accordance with French regulations of an ethical committee , we collected with consent approval 86 male samples from five villages of Ngazidja: Bandamadji, Hahaya, Iconi, Male, and Mitsoudje. We included only unrelated men for at least two generations back in time and ensured the name of their birth village.Blood collection and lab methods are detailed in ref. . DNAs weFST and RST genetic distances and distribution of the molecular variance (AMOVA) using the ARLEQUIN software [Nmalemmale/Nfemalemfemale from FST values, the ratio between effective numbers of male and female migrants per generation, as described in ref. [m) and/or effective population size (N). Hence, the expected value of genetic differences between populations, FST, could be written were FST = 1/(1 + Nm) for haploid systems [We first estimated the within-village genetic diversity and pairwise-difference (Pw), then tested the inter-village genetic structure with a measure of software . We alsosoftware  and weigsoftware  after ex in ref. . In this systems .We measured the female and male genetic diversity indices in the five Comoros villages understudy Table . The maiFST and RST genetic distances onto a Multi Dimensional Scaling . Finally, one can notice twice more village-specific haplotypes for HVS-I (n = 24) than for Y-STRs (n = 12).We then depicted the sis Fig. , networkp-value = 0.000), but less within-village variation . Due to the distinct genetic pattern of Bandamadji above-mentioned, we ran an additional AMOVA excluding this village. We noticed a strong increase of the contrasting patterns of within-population and between-population genetic variation as seen from HVS-I and Y-STRs, since the female genetic variation among populations reached almost 10-fold the male one .AMOVA points out that HVS-I presents 3.2-times more genetic variation amongst villages than Y-STRs .Lastly, from the es Table . The maiA great variety of human social organizations defined by rules of marriage, residence, descent and mode of subsistence has been described worldwide , which iUniparental genetic markers in Ngazidja indicate a male-oriented repartition of genetic diversity within and amongst villages, which is in accordance with described matrilocality. However, one exception is Bandamadji which behaves as a patrilocal population. Indeed, oral and historical knowledge evokes that Bandamadji was the military basis of one of the great Comorian clan M’Dombozi, where men have sworn fidelity to their sultan Hachim ben Ahmed (died 1889), and hence, had to stay to protect the city ( and KassFinally, the Comorian villages are organized according to the social space in which males and females participate distinctly , and theS1Table


Batagur affinis is one of the world's 25 most endangered freshwater turtle species. The major portion of the global population is currently found in peninsular Malaysia, with the only remnant Indochinese population in southern Cambodia. For more than a decade, wild nests in this remnant Cambodian population have been fenced and hatchlings reared in captivity. Here we amplified 10 microsatellite markers from all 136 captive individuals, obtained 2,658 presumably unlinked and neutral single nucleotide polymorphisms from 72 samples with ddRAD‐seq, and amplified 784 bp of mtDNA from 50 samples. Our results reveal that the last Indochinese population comprised only four kinship groups as of 2012, with all offspring sired from <10 individuals in the wild. We demonstrate an obvious decrease in genetic contributions of breeders in the wild from 2006–2012 and identify high‐value breeders instrumental for ex‐situ management of the contemporary genetic stock of the species.The southern river terrapin,  Decreased genetic diversity can be translated into a loss of long‐term evolutionary potential, and inbreeding depression is one of the most pervasive extinction mechanisms reducing reproductive success and survival in populations   Figure a. Conser) Figure . The sma) Figure . Since 2The turtle conservation program in Cambodia includes in‐situ and ex‐situ conservation measures such as fencing wild nests to protect hatchlings from potential predators, protecting remaining nesting sites along the Sre Ambel River, transferring new hatchlings to secure sites, and rearing of hatchlings in captivity  obtained via double digest restriction associated DNA sequencing (ddRAD‐seq) . Nest searches were conducted in the early mornings to ensure female tracks on sand beaches had not been washed out. Once found, nests were fenced for protection from natural predators and guarded by the field guards. All hatchlings were health checked and transported to the wildlife sanctuary to form the captive population after emerging from within the fences. These individuals comprise the cohorts of 2006–2012, except for 2008 when no turtle nests were found. Every year only one nest was found on either of the corresponding beaches, except for 2006 when three nests were found . DNA concentrations were measured with a dsDNA High Sensitivity Assay Kit by Qubit 2.0 Fluorometer (Invitrogen).2.2Batagur trivittata  following the protocol of O'Bryhim, Chong, Lance, Jones, and Roe  were am and Roe . All sam and Roe . For subThe presence of null alleles was checked with MICRO‐CHECKER  genome as it is the phylogenetically closest available genome for B. affinis, using the SimRAD package within R  were digested using the EcoRI and MspI enzymes, because this enzyme combination yielded a maximum number of in silico RAD loci with the target size selection window of 250–650 bp  were set to 7, 2, and 1, respectively. Highly repetitive RadTags were removed to avoid inclusion of paralogs. The resultant SNP set was used in subsequent analyses as SNP set A.For de novo assembly only the forward read was analyzed and no more than one SNP per locus was admitted to preclude linkage bias. Minimum depth of coverage (m), minimum mismatches in creating individual stacks (M), and mismatches in secondary reads (C. picta bellii) genome using the BWA‐MEM alignment algorithm , expected heterozygosity , and inHs Nei, ) were caHs Nei, . Global Hs Nei, . InbreedHs Nei,  and pairHs Nei,  as well Hs Nei, ) were esHs Nei, , the R pHs Nei,  and RHH Hs Nei, , respectOur assurance colony management strategy depends on minimizing the average PR across the group and IR of individuals included in the group. Therefore, we ran SWINGER , the phylogenetically closest species for which an mtDNA genome is available, by using CLC GENOMICS WORKBENCH 10 (Qiagen). For each individual, aligned sequences >20× coverage were retained. Recovered regions were located between positions 4,399–4,544 in the mtDNA genome, encompassing 145 bp from the ND3 gene.Fifty samples were randomly selected from the genetic clusters obtained from the above‐mentioned population structure analyses. Given that ddRAD‐seq libraries contained numerous mtDNA sequences of the samples that were cut by EcoRI and MspI, de‐multiplexed and cleaned individual sequences of these 50 samples were aligned against the full mtDNA genome of Additionally, a primer set of 5′‐TTTTTCCCCTAGCATATCACCA‐3′ (forward) and 5′‐AGTTGCTCTCGGATTTAGGG‐3 (reverse) was manually designed for the amplification of a 639 bp fragment of the mtDNA control region using the software PRIMER3  were excluded from subsequent analyses as they showed significant levels of null alleles. Six of 10 loci showed deviations from Hardy–Weinberg equilibrium; of these six loci, five showed a significant excess in heterozygotes. Linkage disequilibrium was observed for 23 pairs of loci across the entire dataset, corresponding to the 52% of all pairs. These results are most likely due to the small population size and sampling of sibling groups, not actual physical linkage of sites. None of the loci significantly deviated from neutrality. Therefore, all 10 microsatellite loci were used for the population structure analysis.3.2Samples from 129 individual turtles yielded a total of 598,211,204 bp of raw data. Six of these samples were immediately removed , corresponding to <0.03% of all reads, due to poor data recovery (<250 kb). Eventually, 72 individual samples in SNP set A and 68 samples in SNP set B remained after individuals having >10% missing loci were eliminated.De novo and reference‐based mapping of loci, followed by missing data and MAF filtering, yielded six different SNP datasets Table . We cond3.3The PCoA performed with 10 microsatellite loci provided two loose genetic clusters Figure , whereasCluster 1 and 2 of SNP‐based network analysis matched with cluster 1 and 2 of the SNP‐based PCoA Figure , respectThe first two PCs of the SNP‐ and microsatellite‐based PCoAs were plotted against sampling years. The time series of both types of data show a massive decline in population structure even within the short time frame of 7 years 2006–2012) . The inbreeding coefficient GIT was calculated as −0.025 ± 0.005. Individual inbreeding coefficients ranged from −0.225 to 0.27  than a random distribution of relatedness values  was insignificantly higher than the average relatedness across all parent sets, whereas the average relatedness between parent sets of clusters 3 and 4  was shown to be significantly lower than across all sets at a 95% confidence level , followed by a drop to two in 2007, and to zero (no nests were found) in 2008 Table . Our ped4.3Our sample set mostly includes half‐ and full‐sibs that were sired by <10 breeders in the wild. Consequently, any unrelated individuals to these families will be of highest importance for representing the remnant wild genetic variation within the captive population. Therefore, three of the ungrouped individuals  that were merged with the main clusters of microsatellite‐based analyses but represented in SNP‐based clustering analyses were assigned as valuable breeders for the assurance colony. One of these males belongs to the earliest (2006) cohort from the Sre Ambel population  and it shares the same 784 bp mitochondrial haplotype with other individuals hatched in Sre Ambel. This individual might be the only surviving hatchling from a single clutch belonging to an unknown location in the Sre Ambel River System. The remaining ungrouped male and the female have an unknown origin and had been labelled as Big Boy and Big Mama, respectively. Big Mama was thought to belong to a Cambodian population of unspecified provenance, and Big Boy was thought to hail from the Sre Ambel population . Given that Big Mama and Big Boy share the same 784 bp mitochondrial haplotype that is 0.2% different from the remaining Sre Ambel population, we assume that these two individuals might belong to another maternal lineage in the expanded, pre‐bottlenecked Sre Ambel population. The existence of these unrelated individuals will boost genetic diversity in a self‐sustaining assurance colony.4.4B. affinis population currently remains in Cambodia , the genome‐wide analysis provided a fine resolution in determining population structure, whereas traditional genetic markers failed to do so.The majority of the world's turtles are threatened with extinction and among these B. a. edwardmolli while bridging the gap between in‐situ and ex‐situ conservation. Given that ~70% of the world's most threatened turtles inhabit Asia, with comparable demographic histories to B. affinis, our genomic strategy has the potential to be applied to many other species in the mid‐term future.We applied genomic methods to inform choices for assurance colony management, and to help preserve the genetic diversity of We do not have any conflict of interests.FGÇ, DPB and FER designed the study. BDH and SS assisted in tissue sampling, logistics and fieldwork. AS conducted microsatellite genotyping and analyses under the assistance of FGÇ. FGÇ performed NGS library preparation and conducted bioinformatic analyses. FER supervised molecular work and bioinformatic analyses. FGÇ wrote the first draft with extensive input from FER, and all remaining authors contributed to the review of the manuscript. Click here for additional data file. Click here for additional data file.


Identifying and monitoring locally adaptive genetic variation can have direct utility for conserving species at risk, especially when management may include actions such as translocations for restoration, genetic rescue, or assisted gene flow. However, genomic studies of local adaptation require careful planning to be successful, and in some cases may not be a worthwhile use of resources. Here, we offer an adaptive management framework to help conservation biologists and managers decide when genomics is likely to be effective in detecting local adaptation, and how to plan assessment and monitoring of adaptive variation to address conservation objectives. Studies of adaptive variation using genomic tools will inform conservation actions in many cases, including applications such as assisted gene flow and identifying conservation units. In others, assessing genetic diversity, inbreeding, and demographics using selectively neutral genetic markers may be most useful. And in some cases, local adaptation may be assessed more efficiently using alternative approaches such as common garden experiments. Here, we identify key considerations of genomics studies of locally adaptive variation, provide a road map for successful collaborations with genomics experts including key issues for study design and data analysis, and offer guidelines for interpreting and using results from genomic assessments to inform monitoring programs and conservation actions. Quality of de novo assemblies is assessed using metrics including the length of the contigs and the degree of sequence overlap. De novo assembly is common in studies of nonmodel organisms where reference genomes from the focal species or related species are not available.Ne):Effective population size . Typically, Ne is smaller than Nc due to processes that accelerate drift such as nonrandom mating, unequal reproductive success, and fluctuating population sizes. Ne/Nc is often 1/10 to 1/4, but sometimes much smaller. To simplify slightly, Ne is approximately the number of individuals in a population who contribute to offspring in the next generation.Exome: The subset of the genome that is composed of exons, the parts of genes that are transcribed after RNA splicing occurs .Genetic drift: A change in allele frequencies over time due to stochastic processes (random transmission from generation to generation). Drift occurs in all populations but operates more quickly in small populations . Drift decreases genetic variation and drives alleles toward fixation (frequency of 0 or 1).Genetic markers: Any type of genetic sequence information that can be used to identify differences between individuals, populations, and/or species. Examples include (but are not limited to) microsatellites, fragment length polymorphisms, single nucleotide polymorphisms, and gene sequences.Genomic: A loosely defined term that can refer to the use of large numbers of anonymous genetic markers (thousands to millions), the use of targeted gene sequences, or analyses that account for genomic context such as linkage, recombination, or gene function , even if they are not physically proximal.Local adaptation: Due to the action of natural selection, resident genotypes have higher relative fitness in their local environment than genotypes from other environments.Microsatellites: Anonymous markers whose alleles are defined by polymorphism in the length of the DNA sequence. Microsatellite markers have many different alleles , meaning that genetic variation can be captured by fewer microsatellite markers than would be captured by the equivalent number of SNPs. Therefore, most microsatellite studies have fewer than 30 markers, compared to more than 1,000 markers for studies using SNPs. However, this low number of markers does not provide sufficient genomewide coverage for estimating genomewide parameters.Reciprocal transplant: An experimental approach in which organisms from two different environments are raised in both environments. Traits are compared between environments to determine whether there is a genetic component to the differences between environments (adaptive differentiation).Recombination: The exchange of genetic material either between multiple chromosomes or between different regions of the same chromosome. Recombination typically occurs during meiosis, when homologous chromosomes pair up to be passed on to the gametes .Sensitivity analysis: The process of testing a variety of parameter settings using the same starting data  to compare the results from different parameter combinations. If the results from different parameter settings are qualitatively similar, then the results are likely a real signal. If the data are highly sensitive to parameter settings, it might be worth investigating to see whether there is a major source of bias in the dataset.Single nucleotide polymorphism (SNP): One base pair in a DNA sequence that shows variation among individuals. SNPs are typically biallelic  and occur frequently throughout genomes.Transcriptome: The set of messenger RNA transcripts that are produced in a cell or tissue in response to factors such as the environment or developmental stage. To generate sequencing data for these messenger RNA transcripts, RNA from a particular tissue is converted to DNA and sequenced in short reads on high‐throughput sequencing machines . These short reads are then bioinformatically assembled to create sequences for genes; these consensus gene sequences are the “transcriptome.”Trigger point: A value for an indicator variable that is decided before monitoring begins. When the indicator variable reaches this point, a predefined conservation action will be implemented.Box 21Identifying geographic patterns of local adaptation, the environmental drivers of divergent selection among populations, and genes and their variants involved in local adaptation can inform conservation strategies for species at risk  selectively neutral markers  were commonly used to delineate conservation units. NGS provides increased resolution, while also allowing for characterization of adaptive differentiation among populations. Funk, McKay, Hohenlohe, and Allendorf  genetic diversity by moving individuals between populations  while minimizing financial costs. Under climate change, protecting populations with complementary sets of intraspecific adaptive genetic diversity has become increasingly important, as this adaptive variation is indicative of the evolutionary potential of populations under changing conditions , provide unprecedented opportunities to integrate genomic data into conservation management of nonmodel species . When divergent selection is strong, local adaptation is likely to develop, even in the face of high gene flow , and large 2.2If the project will benefit from understanding local adaptation, several options exist. For species that are amenable to experimental approaches , patterns of local adaptation can be reliably addressed by traditional methods such as common gardens and reciprocal transplants . Additionally, any genomic study is more difficult  and potentially costlier in species with large genomes . Overall, before deciding to embark on a genomic study of adaptive variation, we recommend clearly defining the biological or management questions, identifying how genomic data will help address these questions, evaluating alternative nongenomic approaches, researching any existing genetic resources for the focal or a closely related species (or identifying whether those resources need to be developed), considering biological and genomic characteristics of study species, and evaluating budgetary constraints for both assessment and management.In many cases, however, phenotypic methods will not be feasible for the focal species, and genomics may be the preferred alternative. Many management issues related to local adaptation do not require a complete assessment of adaptive variation, nor the functional validation of candidate adaptive variants. Instead, managers may simply need to characterize geographic or environmental patterns of adaptive variation across populations, information which can be generated for species without prior genomic information  or climatic factors . Environmental characterization may be as simple as collecting weather station data or relevant GIS layers from online databases see Daly . Finally3.1.2exomes  due to sources of error such as PCR bias  for the design of capture probes . Polymorphic loci are then identified and the genotypes of individuals inferred from their reads for these loci  which NGS method will be used; (ii) whether a reference genome is available; (iii) how the genotype‐calling coverage and mismatch thresholds will be set, and whether the sensitivity of the data to those parameters will be evaluated; and (iv) what coverage cutoffs will be used to select loci and assign genotypes to individuals Figure .3.2The first step in analyzing genotypic data collected during the assessment is quality control filtering. Data filtering is a multistep process, with specific criteria dependent on the analyses to be performed  or imputed. The impact of these different strategies on downstream analyses is an area of active investigation  and genotype–environment associations (GEAs). These approaches, recently reviewed in Hoban et al. (FST) across loci. Some commonly used methods include tests based on the island model of migration as proposed by Beaumont and Nichols  Do the data meet the model assumptions? (ii) How is neutral genetic structure incorporated into the model? (iii) Are univariate approaches corrected for multiple testing? And (iv) what are the thresholds for detection? Thresholds for differentiating loci potentially under selection are generally arbitrary  and should be tested and modified based on the study goals  from the risk of type 2 errors , as different sequencing and analytical approaches carry different type 1 and type 2 risks. For example, if the proposed conservation action is genetic rescue, then acting on type 1 error increases the risk of outbreeding depression, whereas acting on type 2 error would minimize the number of available source populations. The conservation team can evaluate the risks of each type of error through sensitivity analysis. While to our knowledge, sensitivity analyses have not yet been used in applications of adaptive genomics in management, the benefit of these analyses is clearly evident in other aspects of conservation planning, including climate change vulnerability assessments  requires a monitoring program. In an adaptive management context, monitoring is a means for both learning more about the system and evaluating the effectiveness of management actions once they are initiated  and temporal frequency of sampling must be designed to detect significant changes in allele frequencies or loss of adaptive variants in key populations , are common aspects of biological changes, but some methods can help predict whether a phase shift is imminent  to determine historical genetic variation conditions and compare those to contemporary and future changes , epistasis (a gene has a different phenotypic consequence when in a new genetic background due to interaction with another gene), or other evolutionary constraints  that is influenced by multiple ecological  and evolutionary processes  that often interact. Therefore, interpreting causes of change (or lack thereof) in indicators over time may be challenging.7In this study, we present a modified adaptive management framework to help managers better understand the process of collecting NGS data and the potential applications for assessment and monitoring of adaptive variation Figure . This frAssessing and monitoring adaptive and neutral genetic variation can be a powerful tool for conservation biologists and wildlife managers, but it has limitations. NGS is not a “silver bullet,” but it may be a useful tool, particularly when the entire adaptive management framework is considered prior to embarking upon a study, and with the understanding that implementation of management will be an iterative process that is likely to require adjustments and improvements over time.There are no data associated with this review.


Genetic testing has been widely introduced for many hereditary disorders. While the attitudes towards these facilities have been evaluated in many countries, there are only a few reports on the knowledge of and the orientation among Iranians.The current study assesses the attitudes and knowledge of pre-marriage individuals toward the availability and use of genetic tests.A comprehensive questionnaire was distributed among 408 marrying individuals. The questions addressed the demographic characteristics along the registration of participant's knowledge, education, and attitude toward genetic testing. The individuals were divided into three groups based on their knowledge: 1) Scored above 80 to 100 were defined as "good" 2) 60 to 80 as "average" 3) less than 60 as "poor" knowledge.Most participants (86%) believed consanguineous marriages increase the risk of genetic diseases; 82.3% knew that thalassemia is a type of genetic disease, only 33.3% could distinguish prenatal diagnosis (PND) from other laboratory tests. The relationship between the participants' knowledge and their level of education was significant , age , and urbanity (p░<░0.01). A prominent relationship was observed between the knowledge  or education  and people’s desire to use the genetic tests before the wedding ceremony. No significant correlations were found between the participant’s attitude and their ages/urbanity. Most of the individuals agreed to arrange a genetic counseling before marriage (0.94%).This study revealed that most individuals were interested in using genetic counseling services and genetic tests before marriage. In the era of next-generation sequencing (NGS) technologywith a vast range of application that provides an analysis of genes toany genetic disease in family, the knowledgeand attitude of people about genetic services areimportant . Also, pThe majority of the Iranian population is Muslim with a high frequency of consanguineous marriages, and it is assumed that this will lead to an increased frequency in homozygosity and genetic disorders . MazandaThe aim of this study was to assess the knowledge and attitudes of pre-marriage individuals in northern Iran toward genetic diseases and genetic testing.2This study was a cross-sectional survey. This investigation used a prepared questionnaire and applied a descriptive study on premarital couples who were referred to the Health Care Center in Ghaemshahr, Mazandaran province, north of Iran. People were first asked if they are interested voluntarily to participate in this research (inclusion criteria), and there was no limitation of age, sex, race, education, or place to live, etc. (exclusion criteria); 88.8% of the volunteer individuals were from Mazandaran and 11.2% of them represented other ethnic groups living in Iran.Cronbach's alpha was measured for the internal consistency of the questionnaire. The value of this index was 0.79 as an acceptable range for consistency for 408 individuals. The construct validity of the questionnaire was determined via factor analysis. The value of KMO index was 0.659, and it represented that sample size was adequate for factor analysis. Also,Bartlett’s Mauchly'ssphericitytest was rejected under alpha 0.05 (p░<░0.05). Questions covered demographic information such as: gender, ethnicity, location, employment, and educational status. Other questions were designed to evaluate the participant’s knowledge regarding genetic diseases like thalassemia , consanguineous marriage risks,and PND.They were invited to answer the questions about their attitude toward genetic counseling, consanguineous marriages, abortion, and religious limitations.The sampling method was accessible as a kind of non-probability sampling approach. The Cochran ratio formula was utilized for estimating the sample size. The sample size was calculated as 384 individuals with p░=░0.5, d░=░0.05, and 95% confidence level;408 questionnaires were filled out by the volunteers. 2.1This study was approved by the ethics committee of Mazandaran University of Medical Sciences (IR.MAZUMS.REC.95.8623) and the procedures followed were in accordance with the ethical standards and in line with the Helsinki Declaration of 1975, as revised in 2008. All the participants have signed consent forms for participating in this project.2.2https://www.R-project.org/)  . The rel33.1There were 408 individuals, including 402 volunteers - 231 females and 171 males;64% of the participants were living in urban areasand 36% inthe suburbs or nearby villages. The majority of the participants werefrom the Mazaniethnic group, and the remaining were from Turkish, Kurdish and Gilaniethnics . The age3.2Individuals were divided into three groups based on their knowledge: 1) those whoscored above 80 to 100 were defined as "good"; 2) above 60 to 80 as "average"; 3) and those less than 60 as "poor"knowledge(Table This study showed that while 23.5% of the participant had good knowledge about genetic diseases and testing (score 80 to 100), 50.2% had an average knowledge (score 60 to 80) and 26.3% had poor knowledge (score less than 60) Table . Most of3.3To evaluate the attitude, participants were divided into three groups based on their scores: “positive” (30–36), “neutral” (25–30), and “negative” attitude (< 25). A strong relationship was achieved between the individuals’ attitude toward genetic testing and their level of knowledge (p░<░0.001) or education (p░<░0.001) (Tables I and II). Interesting results were achieved between the awareness and the attitude of the volunteers. About 40% of the attendants who previously had good knowledge represented positive attitudes, but 43% without previous knowledge had a negative trendtoward genetic testing. On average, about 48% of participants in all groups were neutral, regardless of their knowledge. The differences between negative trends toward genetic testing among high- andlow educated people were also significant with 21% and 34%, respectively. Meanwhile, no significant correlations were found between the participants’ attitudes and their ages or urbanity. There was a considerable relationship between the propensity and gender:29% of the females had a positive while 20% had negative tendencies toward genetic testing compared tothe males with 18% positive and 36% negative (p░<░0.01), respectively.3.4No significant differences were found between men and women's attitude toward consanguineous marriages (p░=░0.069). Also,their knowledge had no effect on their attitudes (p░=░0.090). The majority of the participantsbelieved that consanguineous marriages will hold risks for genetic disease in the next generation. While most of the entirelyagreed that couples should be counseled prior to their marriage, only 0.5% were in complete disagreement with receiving these services. The overall opinion of the participants was positive about the main items in questionnaire such as avoiding consanguineous marriages (86%), using genetic counseling before marriages (94%), and termination of the pregnancy in case of embryonic genetic disorders (73.2%).4This study showed that 23.5% of the participants had good, 50.2% had average,and 26.3% of the population had poor knowledge about genetic diseases and testing. Most of the participants (86%) thought that consanguineous marriages increase the risk of genetic diseases in children. There was a significant relationship between participants' knowledge and their level of education, age, and urbanity. A strong relationship was achieved between individuals’ attitude toward genetic testing and their level of knowledge or education. About 40% of attendants who previously had good knowledge represented positive attitudes, but 43% without previous knowledge had a negative trend toward genetic testing. On average, about 48% of participants in all groups were neutral, regardless of their knowledge toward genetic testing.People with different ethnicities have been living in Iran for a long time and this has made Iran's population a highly mixed one . Like thThere was a significant decrease in consanguinity among high-risk thalassemia families in Iran since the national thalassemia program was established , 22. ThiAmong the individuals with bachelor or higher university degree, 42% showed well attitude and perception about the genetic tests, while people with high school degrees only showed 20% attitude in this regard Table . SimilarResults achieved in this study also showed that 73.2% of women said they would terminate the pregnancy if their fetus borea genetic disorder. Another study in Iran revealed that 91.3% of physicians support abortion in the case of thalassemia . Also, t5The results of this study revealed that most of the participants were interested in using genetic counseling services and genetic tests, so these kinds of services such as PND should be available for everyone. It is evident that professional teams with proficiency in genetics and interpersonal communications are required for increasing the awareness of society in genetic fields, which lead to informed decision-making, especially for parents who intend to have a child.The authors report no conflicts of interest.