One of the few cattle breeds in the world undergoing restoration is the Polish White-Backed breed. The objective of this study was to use autosomal microsatellite markers to investigate genetic diversity in cows of this breed, taking into account their origin, and to evaluate their reproductive parameters. The findings of this work not only confirm the value of protecting local cattle breeds around the world, but may also be of importance in developing selection indices for highly productive breeds, in which reproductive functioning should be one of the most important factors considered.Local breeds are the main reservoir of biodiversity of farm animals. According to FAO, they account for 87% of all described breeds in the world. To ensure that they are adequately protected, they should be continually monitored for genetic variation. Another crucial factor is reproduction, which is the most important guarantee of population growth. In the present study, genetic variation in 372 Polish White-Backed cows was determined using DNA microsatellite sequences, taking into account their parentage. Reproductive parameters were analysed as well, based on data from 3658 lactations of 1128 Polish White-Backed cows. The results indicate that despite the small initial population and the implementation of a moderate selection of animals, the existing population of Polish White-Backed cattle has a high level of genetic variation, reflected in the degree of heterozygosity (0.761). Regarding reproductive traits, despite their late age at first calving, Polish White-Backed cows were shown to be distinguished by very good fertility parameters in comparison to other breeds raised in Poland. These findings not only confirm the value of protecting local cattle breeds around the world but may also be of importance in developing selection indices for highly productive breeds, in which reproductive functioning should be one of the most important factors considered. Restoration and conservation of local livestock breeds possessing unique traits is of importance not only for agriculture and global food security but also for rural development, nature conservation, cultural heritage, and science . The mosThe Food and Agriculture Organization (FAO) reports that among 8190 breeds of farm animals, 87% are local breeds and 1984 breeds are endangered  confirm One of the oldest cattle breeds in Poland is the dual-purpose (milk and beef) White-Backed breed. According to the FAO , this brChanges in genetic variation can be monitored owing to the development of molecular genetic analysis at the level of deoxyribonucleic acid (DNA), using proven and widely used genetic markers. Demonstration of genetic diversity and population structure is also of great importance for the sustainable use of genetic resources. Widely used markers in genetic analysis of populations of farm animals are DNA microsatellite sequences, regarded as an ideal tool due to traits, such as numerous repetitions throughout the euchromatic region of the genome, codominance, and high polymorphism ,17. TheiIn addition to a high level of genetic variation, reproductive parameters are an important factor determining both breeding success and economic success in livestock farming . These dThe objective of the study was to use autosomal microsatellite markers to investigate genetic diversity in Polish White-Backed cows, taking into account their origin, and to evaluate their reproductive parameters.The material for the study consisted of hair bulbs from 372 Polish White-Backed cows protected by a genetic resources conservation programme, selected on the basis of parentage. The hair bulbs were collected for the purposes of breeding work. The cows selected for the study were unrelated, going back two generations. The animals were divided into three groups: G1—cows with no documented origin (178); G2—cows with documentation of the either the mother or the father (86); and G3—cows with documentation of both parents and grandparents of the Polish White-Backed breed (108).DNA was isolated using a commercial kit for isolation of nucleic acids from biological traces (Sherlock AX A&A Biotechnology), according to the procedure described by the manufacturer. DNA was amplified in a multiplex reaction (12 loci) using the Bovine Genotypes Panel 1.2 Kit , containing 12 markers from the ISAG-recommended STR panel .Microsatellite sequences were amplified according to the manufacturer’s protocol, using 12 primer pairs labelled with four fluorescent dyes. PCR products were analysed by capillary electrophoresis in the 3130xl genetic analyser  and separated using the GeneScan-500 LIZ Size Standard (Applied Biosystems). The electrophoretic separation results were analysed using GeneMapper 4.0 software (Applied Biosystems). The analysis included the entire population of Polish White-Backed cows covered by the genetic resources conservation programme during the period from 2003 (when breed restoration was initiated) to 2022. In total, 3658 lactations of 1128 cows were included in the analysis, including 372 individuals analyzed for genetic variation. Information obtained from milk performance evaluation of Polish White-Backed cows conducted by the Polish Federation of Cattle Breeders and Dairy Farmers was grouped according to genotype  and age at first calving .The following reproductive parameters were determined for the groups of animals: age at first calving, days open , calving interval , dry period , calving-to-first-service interval , service period (number of days between the first service and conception), services per conception (number of insemination procedures per conception), and gestation length. 2 test, using GENEPOP 4.0 software [Basic parameters of genetic variation, i.e., observed number of alleles (No) and effective number of alleles (Ne), were estimated for each locus and population using GenAIEx 6.5 software . Observesoftware .p ≤ 0.05; means designated A,B differ significantly at p ≤ 0.01.The information obtained from the reproductive performance evaluation of Polish White-Backed cows was used to create a database. Statistical analysis of the database was performed by one-way analysis of variance (ANOVA) of means for each parameter using StatSoft Inc. STATISTICA 9.0 software. The groups had a normal distribution. Significance of differences between values for groups was determined by Tukey’s test. Means designated a,b differ significantly at p < 0.01), except for loci ETH10, TGLA126, and TGLA227 in G1, loci ETH10, SPS115, TGLA53, and TGLA126 in G2, and loci ETH3, SPS115, TGLA53, TGLA126, and TGLA227 in G3. The average heterozygosity was high in all groups (>0.7). Observed heterozygosity was lowest in the animals with one known parent (G2 (0.702)) and highest in those of known origin for two generations (G3 (0.755)). The locus with the highest observed heterozygosity was BM2113 in group G3 (0.926), while locus TGLA126 had the lowest Ho values in both G2 and G1 . The estimated value of Fis, which describes an excess or deficiency of heterozygotes in the population, ranged from −0.110 (BM2113) in G3 to 0.261 (ETH225) in G2. The average value for this parameter was 0.00 in the group of cows of fully known origin, 0.096 in G1, and 0.131 in G2.The results of the analysis of DNA microsatellite sequences are presented in p < 0.01) shorter in that group than in group R1: calving interval by 16.57 days and days open by 16.15. In addition, in the R3 group, significantly fewer services per conception (1.59) were found in relation to the R2 group (2.01) and the R1 group (1.96), which significantly shortened the period of first service to conception. A different tendency was noted for age at first calving, which was earliest for heifers from group R1 (unknown origin), at 811.43 days, and latest for those with one known parent (R2), at 870.69 days. Cows from group R3 (fully known origin) first calved at the age of 850.35 days. The differences between means were confirmed statistically (p < 0.01). The average milk yield for 305-day lactation in the cows ranged from 4332.26 kg of milk in R1 to 4642.38 kg in R3. The differences in milk yield were not statistically significant.Indicators of fertility were determined in each group of Polish White-Backed cattle: age at first calving, calving interval, days open, calving-to-first-service interval, service period, services per conception, dry period length, and gestation length. The data in In the present study, to describe generational changes in the genetic variation of Polish White-Backed cows, a tested and reliable genetic tool was used—DNA microsatellite sequences. The high average number of alleles per locus in all analysed groups of Polish White-Backed cattle indicates high polymorphism of the microsatellite sequences used and high genetic diversity in the population. A study by Brasil et al.  conducteHowever, a decline of over 28% was noted in the average number of alleles per locus in the group of cows of known origin for two generations relative to the group of animals of unknown origin. The influence of breeding selection on the degree of genetic variation in a breed is well known . Polish Our study showed a high level of expected heterozygosity (>0.7). A similar level was noted in local Senegalese breeds of zebu cattle, ranging from 0.730 to 0.799 , in locaThe Fis inbreeding coefficient is a parameter indicating a reduction in heterozygotes due to non-random mating . In the An important reference point for the results pertaining to genetic variation obtained in the present study is a paper by Sawicka-Zugaj et al.  presentiIn the case of intensive milk production, the optimal age at first calving for maximizing the yield and the productive life of cows in herds is considered to be 24–27 months . In the A study by Bieber et al.  showed tEvaluation of the milk and reproductive performance of cattle in Poland, carried out by the Polish Federation of Cattle Breeders and Dairy Farmers , showed Twenty years after the start of the Polish White-Backed breed restoration programme and the implementation of moderate selection of animals, assessment of genetic variation showed that its level was high in the group of animals of fully known origin. The high degree of heterozygosity and the inbreeding coefficient (Fis) of 0 indicate that in situ conservation of the Polish White-Backed breed was successful. Moreover, well conducted breeding work in the breed restoration process resulted in favourable reproductive parameters, i.e., a much shorter calving interval, fewer services per conception, and, in consequence, shorter periods from first service to conception and days open. The results confirm the need for continuous monitoring of indigenous breeds, for both genetic variation and production parameters, and, at the same time, can provide inspiration and hope for organizations deciding to fight for the survival of local breeds of farm animals—not only of cattle.


Eristalis tenax can be found in temperate and continental climates across the globe, often synanthropically. Eristalis tenax pupae of different generations and different climate zones are thus exposed to vastly different temperatures. In many insects, the ambient temperature during the pupal stage affects development, adult size, and survival; however, the effect of developmental temperature on these traits in hoverflies is comparatively poorly understood. We here reared E. tenax pupae at different temperatures, from 10°C to 25°C, and quantified the effect on adult hoverflies. We found that pupal rearing at 17°C appeared to be optimal, with high eclosion rates, longer wings, and increased adult longevity. Rearing temperatures above or below this optimum led to decreased eclosion rates, wing size, and adult survival. Similar thermal dependence has been observed in other insects. We found that rearing temperature had no significant effect on locomotor activity, coloration or weight, despite evidence of strong sexual dimorphism for each of these traits. Our findings are important as hoverflies are key pollinators, and understanding the effects of developmental temperature could potentially be useful for horticulture.Hoverflies  are cosmopolitan, generalist flower visitors and among the most important pollinators after bees and bumblebees. The dronefly  We reared hoverfly pupae at different ambient temperatures, and measured the effect on eclosion rate, adult size and coloration, locomotor activity and survival. We found that rearing the pupae at 17°C is optimal for high eclosion rate, long life span, and larger flies. The shape of the curve depends on what is being quantified. For example, developmental rates can often be described with skewed TPCs  with a 24.5 or 32.5 cm side for maintenance at room temperature under laboratory lights  on the thoracic dorsum to enable tracking of individuals. We recorded the life span of adult hoverflies from the day of eclosion to their death Figure . Hoverfl2.2For the hoverflies reared at 12°C, 17°C, and 25°C, we assigned a group of individual hoverflies  that were kept in a separate cage and regularly weighed at an interval of 5–15 days until the time of death Figure . The firAfter the hoverflies died, we took high‐resolution photographs of them using an Olympus SZX10 camera equipped with DF PLAPO 1X‐4 JAPAN lens , or Olympus E‐M10 Mark II camera equipped with Olympus ED 12–50 mm f/3,5–6,3 EZ lens . A total of 226 hoverflies were photographed while still being alive using the Olympus E‐M10 Mark II camera, and to prevent their movement during photographing, we covered them with a lid of a tissue culture dish pressed against a soft cellulose square. Camera type and status during photographing  were used as predictors for statistical models to filter out potential systematic variability see Data . Wings wE. tenax color morphs  with 25 mm diameter × 125 mm long Pyrex glass tubes  positioned horizontally. Hoverflies were individually placed in each tube, where the ends were sealed with a cotton ball with water, pollen, and honey . The length of the pupation period was also slightly, though significantly, different between the batches . When the pupae were kept at 10°C, it took almost 2 months (54.5 ± 4.6 days) for them to eclose, whereas it took only 8.3 ± 1.3 days for them to eclose when kept at 25°C , whereas the batches did not differ significantly from each other .Following eclosion, the survival rate of hoverflies reared at four different temperatures was tracked. We found that the rearing temperature significantly affected the adult life span , with the oldest female reaching an impressive age of 231 days  and 25°C . However, there was no difference in wing length between hoverflies reared in 12°C or 25°C , and the overall effect of rearing temperature was small (R2 = 1.53%). Females had longer wings than males  and the wing length differed between batches . The effect of rearing temperature on thorax length was not significant , and thorax length differed between batches .Next, we quantified the size of the hoverflies. We found that wing and thorax length were both affected by sex and batch identity . However, females had a significantly larger wing/thorax ratio than males .When looking at the wing/thorax length ratio, we found that there was no difference between rearing temperatures , whereas males did not . Nevertheless, neither the weight , nor its changes over time , were affected by the rearing temperature. The only exception were males reared in 17°C, which were slightly heavier than other males , and thus, the interaction between sex and rearing temperature was significant .We weighed the flies every 5–15 days until the time of death and found that females were heavier than males . Nevertheless, we found that females .As a final morphometric measure, we looked at abdominal coloration, classified into six categories according to Heal . There ws Figure  were ove3.3p < .001, Table p = .949, Figure p < .001). This was not observed in females . We found no significant effect of rearing temperature  or batch identity . Notably, when we tested if the hoverflies were more active in the temperatures in which they were reared as pupae (interaction between rearing and testing temperature), we found only a weak, borderline‐significant effect .As a measure of fitness, we quantified hoverfly physical activity, using a locomotor activity monitoring system (LAMS). We found that the activity of the hoverflies increased with testing temperature . In contrast, water beetles exposed to low temperatures for 5 days were more active at higher test temperatures than those exposed to high temperatures  take place when the air temperature is 10°C, and the first moving animals are observed when air temperature reaches 11°C , but not on the rearing temperature  suppresses melanin production, which leads to adults with a lighter abdominal coloration Heal, . HoweverEristalis genus, females are often darker than the males ; data curation (lead); formal analysis ; investigation (lead); methodology ; software ; validation ; visualization (lead); writing – original draft (lead); writing – review and editing . Sarah Nicholas: Conceptualization (supporting); investigation ; methodology ; supervision ; writing – review and editing . Karin Nordström: Conceptualization ; data curation ; funding acquisition (lead); methodology ; project administration ; resources (lead); writing – review and editing (lead).This research was funded by the US Air Force Office of Scientific Research , the Australian Research Council , Grant Agency of Charles University (464220/2020), the Faculty of Science Foundation of Charles University, and the Mobility Fund of Charles University.The authors declare no conflict of interest.Data S1:Click here for additional data file.


Fagopyrum tataricum) is an important food and medicine crop plant, which has been cultivated for 4000 years. A nuclear genome has been generated for this species, while an intraspecific pan-plastome has yet to be produced. As such a detailed understanding of the maternal genealogy of Tartary buckwheat has not been thoroughly investigated.Tartary buckwheat  separated by a large single copy region (LSC) and a small single copy region (SSC). Although the structure and gene content of the Fagopyrum plastomes are conserved, numerous nucleotide variations were detected from which population structure could be resolved. The nucleotide variants were most abundant in the non-coding regions of the genome and of those the intergenic regions had the most. Mutational hotspots were primarily found in the LSC regions. The complete 521 Fagopyrum plastomes were divided into five genetic clusters, among which 509 Tartary buckwheat plastomes were divided into three genetic clusters (Ft-I/Ft-II/Ft-III). The genetic diversity in the Tartary buckwheat genetic clusters was the greatest in Ft-III, and the genetic distance between Ft-I and Ft-II was the largest. Based on the results of population structure and genetic diversity analysis, Ft-III was further subdivided into three subgroups Ft-IIIa, Ft-IIIb, and Ft-IIIc. Divergence time estimation indicated that the genera Fagopyrum and Rheum (rhubarb) shared a common ancestor about 48 million years ago (mya) and that intraspecies divergence in Tartary buckwheat began around 0.42 mya.In this study, we The resolution of pan-plastome diversity in Tartary buckwheat provides an important resource for future projects such as marker-assisted breeding and germplasm preservation.The online version contains supplementary material available at 10.1186/s12870-023-04218-7. Fagopyrum) is a genus of plants that are annuals or perennials, with an herbaceous or semi-shrub habit , the divergence time between Fagopyrum and Rheum (44.2 Mya HPD: 25.2–55.2), and the divergence time between Fagopyrum tataricum and Fagopyrum esculentum (7.24 Mya HPD: 5.22–9.27). Following the suggestion of Ho (2007), we assigned a normally distributed prior for the three calibrations, in which standard deviations of 6.7, 6.7, and 1.2 were used, respectively.To perform the phylogenetic analysis, complete sequences of plastid genomes were aligned in MAFFT 7  and manuBelow is the link to the electronic supplementary material.Supplementary Material 1


Pinna nobilis Linnaeus, 1758 is an endemic species of the Mediterranean Sea, protected by international agreements. It is one of the largest bivalves in the world, playing an important role in the benthic communities; yet it has been recently characterized as Critically Endangered by the IUCN, due to mass mortality events. In this context, the assessment of the genetic variation of the remaining P. nobilis populations and the evaluation of connectivity among them are crucial elements for the conservation of the species. For this purpose, samples were collected from six regions of the Eastern Mediterranean Sea; the Islands of Karpathos, Lesvos and Crete; the Chalkidiki and Attica Peninsulas; and the Amvrakikos Gulf. Sampling was performed either by collecting tissue from the individuals or by using a non-invasive method, i.e., by scraping the inside of their shells aiming to collect their mucus and thus avoid stress induction to them. Conventional molecular techniques with the use of the COI and 16S rRNA mitochondrial markers were selected for the depiction of the intra-population genetic variability. The analyses included 104 samples from the present study and publicly available sequences of individuals across the whole Mediterranean Sea. The results of this work (a) suggest the use of eDNA as an efficient sampling method for protected bivalves and (b) shed light to the genetic structure of P. nobilis population in the Eastern Mediterranean; this latter knowledge might prove to be fundamental for the species conservation and hence the ecosystem resilience. The haplotype analyses reinforced the evidence that there is a certain degree of connectivity among the distinct regions of the Mediterranean; yet there is evidence of population distinction within the basin, namely between the Western and the Eastern basins. The combination of both genetic markers in the same analysis along with the inclusion of a large number of individuals produced more robust results, revealing a group of haplotypes being present only in the Eastern Mediterranean and providing insights for the species’ most suitable conservation management.The fan mussel  Pinna nobilis, the largest endemic bivalve of the Mediterranean Sea  and the Eastern Mediterranean (Aegean Sea and Tunisian coasts). Furthermore, it identified the Venetian Lagoon population from the northern Adriatic Sea as a potentially diverging population. Interestingly, two other areas in the central Adriatic Sea, the natural marine parks of Mljet and Telascica, showed greater similarity to the Western Mediterranean samples than to those from Venice  P. nobilis populations exhibited high genetic diversity across various locations, although there was no significant genetic differentiation among these populations, thus indicating a genetically homogeneous population spanning the entire coastline  compare it with similar studies from the whole Mediterranean in an attempt to provide further insights into population structuring of this critically endangered species, which will offer a good estimation on the fitness and diversity of the Greek populations.The aim of the present study was to (a) investigate the genetic diversity of the For the purpose of the study 105 samples were collected within the period of August 2018–April 2021 from six locations of the Eastern Mediterranean Sea and particularly from the Islands of Karpathos, Lesvos and Crete, Vourvourou , Attica Peninsula, and the Amvrakikos Gulf . Depth aD. Karagiannis of the National Reference Laboratory for Mollusc Diseases (Greek Government) provided the samples MYT1-MYT9 from Lesvos, which were collected under a permit from local authorities (MEE/GDDDP89926/1117). Samples TS1–TS6 from Lesvos were collected under a permit by the Department of Agriculture and Fisheries, Decentralized Administration of the Aegean (No. 52321/6-9-2018).The sampling method for Karpathos’ samples was non–lethal, non-invasive and low impact aiming at the minimization of the disturbance towards the bivalves, since the tissue removal may provoke stress and make the animal more susceptible to diseases. Initially, a rod of 0.5 cm diameter was placed at the opening of the valves of each animal by the SCUBA divers in order to keep them slightly open, carefully taking into account the fragility of the shell’s outermost part. Consequently, a sampling brush, resembling a buccal swab was used  to scrapP. nobilis from the parasite H. pinnae. Specifically, 50–100 mg of different tissues  from each individual were removed, preserved in absolute ethanol and stored at 4 °C until further processing. As previously, the shells’ width and height were recorded by the divers, for the majority of the individuals.All the other samples were collected from sacrificed individuals under research permits, since the initial aim of the sampling was the investigation of the infection of i.e., the elution of the DNA pellet, replicate samples were pooled and their concentration was measured in a NanoDrop 1,000 spectrophotometer , 4 µl of 5X KAPA HiFi Fidelity Buffer , 1 µl of each primer (10 um), 0.8 µl of dNTPs (10 mM each), 1 µl of KAPA HiFi HotStart DNA Polymerase (1 U/uL)  in a total volume of 20 ul. Amplifications were performed at a BioRad T100 thermal cycler. The PCR protocol was the same for the two genes; namely a denaturation step at 95 °C for 5 min followed by 35 cycles of 98 °C for 20 s, 53 °C for 30 s, 72 °C for 30 s and a final extension step at 72 °C for 5 min.Amplification of the 16S rRNA yielded in some cases a double PCR product; in this case, purification of both the PCR products was carried out from a 2% agarose gel using the NucleoSpin Gel and PCR Clean-up . For the COI amplicons, a sodium acetate-absolute ethanol cleanup protocol was conducted. All purified PCR products were sequenced in an automated sequencer ABI 3730.P. nobilis, for which sample location information was available, were also downloaded from GenBank and added to the aforementioned alignments   was performed to determine genetic variation between populations using poppr (v.2.9.4) (A map showing the distribution of the most abundant 16S rRNA-COI haplotypes in the different locations was generated using ggplot2 (v. 3.4.2)  and scatv.2.1.7) , pegas (v.2.1.7)  and hierv.2.1.7) . The effv.2.9.4)  and pegav.2.9.4)  packagesv.2.9.4) , phytoolv.2.9.4)  and ggtrv.2.9.4)  packagesv.2.9.4) .http://www.ebi.ac.uk/ena/data/view/OX406989-OX407068 (16S rRNA) and http://www.ebi.ac.uk/ena/data/view/OX407172-OX407248 (COI).The map of the sampling sites was generated with the QGIS software. Raw DNA sequences from the present study are available from the European Nucleotide Archive (ENA)  at http:P. nobilis individuals are classified into age classes based on the total shell’s height ; however, in the scatterplot between the genetic distances and the geographic distances, two clouds of points appeared indicating that there might be distant patches  amplifications of eDNA samples were successful for the 16S rRNA and 33 (out of 60) for the COI gene. All the tissue samples provided successful amplifications for both genes. The first dataset included 100 sequences (N) (concatenation of COI and 16SrRNA genes) of 982 bp from the Greek coastline (Eastern Mediterranean Sea). It revealed 34 haplotypes and 45 polymorphic sites. The haplotypic diversity was high (Hd: 0.91 ± 0.017) while the nucleotide diversity was low (Pi: 0.00304 ± 0.00029) . The hapuite low , which a patches . DAPC, a patches  but over patches . hierBAP patches , which dST values were quite low  and there was one single consistent cloud of point in the scatterplot, without discontinuities indicating patches (The second dataset included 294 concatenated sequences (N) of 714 bp from the Western and Eastern Mediterranean Sea (COI and 16SrRNA). It revealed the highest number of haplotypes (104) and polymorphic sites (72) among all datasets. The haplotypic diversity (Hd: 0.961 ± 0.005) was high and the nucleotide diversity was moderately high (Pi: 0.00511 ± 0.00019) . The hapuite low . When we patches . DAPC, a patches  but over patches . hierBAP patches , which aST values were again quite low  and one single consistent cloud of points in the scatterplot (The third dataset included 450 sequences (N) of the COI gene (243 bp) again from the whole Mediterranean Sea. It revealed 48 haplotypes and 36 polymorphic sites. The haplotypic diversity (Hd: 0.652 ± 0.024) was lower than the one observed in the other two datasets and the nucleotide diversity was moderately high (Pi: 0.00475 ± 0.00028) . AMOVA suite low . Again, tterplot . DAPC, atterplot . Reassigtterplot  and hiertterplot ; the thiP. nobilis are variable based on the availability of zooplankton, which is reflected in the location of the individuals , a fact which was attributed to the very long (up to 6 months) pelagic larval stage of the species  kerathurus:   ; using mP. nobilis specimens from different areas of the Eastern Mediterranean basin. Therefore, it significantly contributes to the knowledge of the genetic variability of the pen shell’s populations; the number of available COI sequences has increased 3-fold, while the number of 16S rRNA sequences has had a 20-fold increase. In light of the MME, coordinated studies on the genetic diversity of P. nobilis throughout the Mediterranean Sea, with the cooperation of researchers, institutes and universities, should be performed towards the aim of the conservation and management of the remaining populations of the species. An orchestrated attempt of a pan-Mediterranean investigation appears to be indispensable. Scientific cooperation and use of common standards should be implemented in order to obtain more FAIR data and therefore lead more efficiently to knowledge  Scatterplot showing the relationship between the shell’s total height and the shell’s width for the P. nobilis individuals sampled for this study.(A) Scatterplot showing the relationship between the shell’s height above sediment with the shell’s height buried inside the sediment for the Click here for additional data file.10.7717/peerj.16491/supp-4Supplemental Information 4The shell’s total height distribution categorized in age classes for the different individuals sampled for this study.Click here for additional data file.10.7717/peerj.16491/supp-5Supplemental Information 5(A) Isolation by distance scatterplot for the 16S rRNA-COI Greece (Eastern Mediterranean) dataset. (B) Membership probability of individuals to populations, as calculated by DAPC, for the 16S rRNA-COI Greece (Eastern Mediterranean) dataset. (C) PCA plot depicting the similarity of different populations for the 16S rRNA-COI Greece (Eastern Mediterranean) dataset. (D) Phylogenetic tree of the 16S rRNA-COI Greece (Eastern Mediterranean) dataset showing the hierBAPS clustering into different groups.Click here for additional data file.10.7717/peerj.16491/supp-6Supplemental Information 6(A) Isolation by distance scatterplot for the 16S rRNA-COI Central, Western and Eastern Mediterranean Sea dataset. (B) Membership probability of individuals to populations, as calculated by DAPC, for the 16S rRNA-COI Central, Western and Eastern Mediterranean Sea dataset. (C) PCA plot depicting the similarity of different populations for the 16S rRNA-COI Central, Western and Eastern Mediterranean Sea dataset. (D) Phylogenetic tree of the 16S rRNA-COI Central, Western and Eastern Mediterranean Sea dataset showing the hierBAPS clustering into different groups.Click here for additional data file.10.7717/peerj.16491/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.16491/supp-8Supplemental Information 8TH: Total height, HS: Height above sediment, HD: Height inside the sediment, W: Greater width, DNA conc: DNA concentration of the samples (ng/µl); where DNA was extracted for multiple tissues, the average DNA concentration is shown.Click here for additional data file.10.7717/peerj.16491/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.16491/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.16491/supp-11Supplemental Information 11p-value (population effect on genetic differentiation): 0.01.Click here for additional data file.10.7717/peerj.16491/supp-12Supplemental Information 12p-value (population effect on genetic differentiation): 0.01.Click here for additional data file.10.7717/peerj.16491/supp-13Supplemental Information 13p-value (population effect on genetic differentiation): 0.01.Click here for additional data file.


Advances in amyotrophic lateral sclerosis (ALS) gene discovery, ongoing gene therapy trials, and patient demand have driven increased use of ALS genetic testing. Despite this progress, the offer of genetic testing to persons with ALS is not yet “standard of care.” Our primary goal is to develop clinical ALS genetic counseling and testing guidelines to improve and standardize genetic counseling and testing practice among neurologists, genetic counselors or any provider caring for persons with ALS.Core clinical questions were identified and a rapid review performed according to Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA‐P) 2015 method. Guideline recommendations were drafted and the strength of evidence for each recommendation was assessed by combining two systems: the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) System and the Evaluation of Genomic Applications in Practice and Prevention (EGAPP). A modified Delphi approach was used to reach consensus among a group of content experts for each guideline statement.C9orf72 assay, along with sequencing of SOD1, FUS, and TARDBP, at a minimum. The key education and genetic risk assessments that should be provided before and after testing are delineated. Specific guidance regarding testing methods and reporting for C9orf72 and other genes is provided for commercial laboratories.A total of 35 guideline statements were developed. In summary, all persons with ALS should be offered single‐step genetic testing, consisting of a These evidence‐based, consensus guidelines will support all stakeholders in the ALS community in navigating benefits and challenges of genetic testing. Rapid progress in the discovery of amyotrophic lateral sclerosis (ALS)‐associated genes, and a growing recognition of the genetic basis of clinically sporadic ALS, has opened the door to an era of gene‐targeted therapies for persons with ALS. Despite the progress in ALS gene discovery, and a wide array of clinical gene testing options, the offer of genetic testing to people with ALS is not yet broadly considered “standard of care” and many people with ALS who desire access to genetic testing are not offered it.C9orf72 expansion.A growing proportion of clinicians offer genetic testing to persons with familial ALS, though only 10–50% of clinicians offer testing in the case of apparently sporadic ALS.SOD1 D90A phenotype.Despite these challenges, recently reaffirmed US care guidelines do not address the offer of genetic testingSOD1 ALS, was recently granted accelerated approval by the FDA.The first gene‐targeted treatment for ALS, an antisense oligonucleotide therapy for We identified core clinical questions to be addressed by the guidelines using the AGREE II instrument, an international tool routinely employed in medical guideline development and evaluation by professional organizations. The AGREE II  instrument is an international system created to advance guideline development in healthcare.What genetic testing should be offered to persons with ALS? What information should be provided to persons with ALS before and after testing? (genetic counseling domain)What test methodologies, reporting, and interpretation standards should be used? (laboratory methods domain)Each of the core clinical questions we identified covers a content domain relevant for the development of ALS genetic testing guidelines: a clinical testing domain, a genetic counseling domain, and a laboratory methods domain. These questions formed the foundation for the rapid review (defined below):The target patient population to which the guidelines are intended to apply are persons diagnosed with ALS. That stated, we recognize that in clinical genetics, the family is often considered the unit of care, and indeed family members of a person with ALS may directly or indirectly receive care or education from the proband's neurologist, genetic counselor, or other clinician.The author group includes a genetic counselor (JR), physician scientist (SK), and clinician researcher (MH), all with expertise and published research in ALS genetics, genetic counseling, testing approaches, outcomes, and laboratory methods, as well as a guidelines methodologist (BE), with expertise in medical guideline development using the modified Delphi approach. Expert Panel participants were purposely recruited to represent each stakeholder group and a range of disciplines, expertise, and geographic representation across the United States. Experts were chosen to represent academic neurologists, community neurologists, genetic counselors, physician scientists, laboratory experts, ALS advocates, and persons with ALS. Persons with ALS were included to represent the patient perspective, which is increasingly recognized as key to effective care.C9ORF72, FUS, OPTN, TBK1, KIF5A, VCP, ANXA11, MATR3, SQSTM1, ANG, hnRNPA2B1, hnRNPA1, CHCHD10, DCTN1, PRPH, TAF15, TIA1 , and “Amyotrophic lateral sclerosis” or “ALS”. The search strategy is presented in the Evidence Summary Document, available in A rapid review of the literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA‐P) 2015 guidelines. Three electronic databases  were searched from inception to March 2022. Search terms included any of genetic predisposition to disease, genetic counseling, genetic testing, genetic variation, genes, mutation, missense, DNA analysis, mutational analysis, mutation rate, penetrance, sequence analysis, pedigree; any of A total of 9541 potentially relevant articles were identified in the initial search. After 1794 duplicates were removed, 7743 citations underwent title and abstract review by JR, MH, and SK. After applying inclusion and exclusion criteria to the title and corresponding abstracts and resolving any nonunanimous decisions, 936 were selected for full‐text review. After a full‐text article review, 263 articles were selected for inclusion, and key data extracted and summarized  was revised according to written feedback provided by the expert group. The revised recommendation, “All persons with ALS should be offered testing with an ALS gene panel that includes C9orf72” reached 100% consensus. The final round (Round 3) consisted of a face‐to‐face meeting to discuss and vote on minor changes to the wording of 7 recommendation statements. See Figure The first two rounds of voting were completed via an email link to a Research Electronic Data Capture (REDCap®) survey hosted at the Ohio State University.nd Round  consisteAt the conclusion of the modified Delphi Process, 43 recommendation statements were finalized and approved, forming our Guidelines. After related recommendation statements were merged for brevity, 35 recommendations remained, encompassing the domains of clinical testing  follows a standardized approach for reviewing genetic and experimental evidence in assigning gene‐disease validity in ALS. Genes classified with “definitive” disease validity are appropriate to include in clinical testing.GRADE rating: D ‐ Expert.Strength: This recommendation is supported by Expert Opinion.Recommendation 7: In the event of an FDA‐approved gene‐targeted therapy, all persons with ALS should be offered testing for that gene.Key points: The opportunity for FDA‐approved gene‐targeted treatment warrants the offer of genetic testing for the corresponding gene. The first gene‐targeted therapy for ALS was recently granted accelerated approval by the FDA.GRADE rating: D ‐ Expert.Strength: This recommendation is supported by Expert Opinion.Recommendation 8: Genetic counseling and education should be provided to all persons with ALS.Key points: Genetic counseling promotes adaptation to the occurrence or risk for disease that may be hereditary, including the nature of inheritance, understanding of genetic testing options, and implications for family members. All persons with ALS should be offered genetic counseling, irrespective of the presence or absence of a family history of ALS.GRADE rating: C ‐ Weak.Strength: This recommendation is supported by at least one study of Level 3 evidence.Recommendation 9. Genetic counseling should precede the offer of testing.Key points: Genetic counseling should be provided before testing, to empower persons with ALS to weigh the potential benefits, risks and limitations of testing, and anticipate the possible impact of testing on themselves and their family members.GRADE rating: C ‐ Weak.Strength: This recommendation is supported by at least one study of Level 3 evidence.Recommendation 10. A pedigree going back three generations at minimum should be documented.Summary of evidence: Although increasing the number of individuals ascertained in family history also increases the likelihood that affected persons will be identified by chance,GRADE rating: B ‐ Moderate.Key points: This recommendation is supported by at least one study of Level 2 evidence.Recommendation 11. The pedigree should ascertain ALS and related motor neuron disorders , frontotemporal dementia, other dementias, movement disorders, and psychiatric disease.Key points: Studies of clinic‐based ALS cohorts and defined patient populations have consistently shown that the incidence of pathogenic ALS variants is higher in probands who have a family history of ALS.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by at least one study of Level 2 evidence.Recommendation 12: Genetic counseling should include personalized risk assessments for the likelihood of a genetic etiology, and the likelihood of positive results on testing of currently known genes.Key points: The likelihood of a genetic etiology in a person or family with ALS varies with clinical features such as family history, age of onset of symptoms, and the presence of additional phenotypes such as FTD.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported with at least one study of Level 2 evidence.Recommendation 13: Genetic counseling should include discussion of genetic heterogeneity.Key points: Pathogenic variants in growing number of genes have been shown to cause or increase the risk for ALS; variant frequencies differ by geoancestry.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by at least one study of Level 2 evidence.Recommendation 14: Genetic counseling should include a discussion of inheritance patterns.Key points: Although ALS variants can be transmitted in an autosomal dominant, autosomal recessive or X‐linked manner,GRADE rating: B ‐ Moderate.Strength of recommendation: This recommendation is supported with at least one study of Level 2 evidence.Recommendation 15: Genetic counseling should include a discussion of penetrance.Key points: Many ALS variants are incompletely penetrant, meaning that not all carriers of the variant will develop disease. Penetrance studies have estimated a high disease risk for some SOD1  and FUS variants, but much lower disease risk for others, including the C9orf72 expansion.GRADE rating: C ‐ Weak.Strength of recommendation: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 16: Genetic counseling should include personalized risk assessment for recurrence in relatives.Key points: For persons with ALS who have no known family history of ALS or FTD, empiric data may be used to estimate the risk that relatives would develop ALS. In European populations, the lifetime risk of ALS in first‐degree relatives of those with apparently sporadic disease appears to be 1–3%.pedigree analysis.GRADE rating: B ‐ Moderate.Strength of recommendation: This recommendation is supported with at least one study of Level 2 evidence.Recommendation 17: Pretest counseling should prepare individuals for possible personal, psychological, and economic impacts of testing on themselves and their family members.Key points: Pretest communication of the potential psychosocial impact of testing helps persons with ALS anticipate possible outcomes and prepare for uncertainty. Individual motivations for testing should be discussed.GRADE rating: C ‐ Weak.Strength: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 18: Persons with ALS should be informed of the range of possible testing outcomes: positive, negative, or uncertain.Key points: Outcomes for clinical ALS genetic testing may include positive, negative, uncertain, and/or indeterminate result interpretations.GRADE rating: C ‐ Weak.Strength of recommendation: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 19: Persons with ALS and their families should be informed that all testing methodologies have limitations.Key points: Technical limitations in ALS genetic testing may produce false‐negative or false‐positive results. Persons considering ALS genetic testing should understand that current technologies may in some cases fail.C9orf72 expansion may fail to identify expanded allele. There is no validated cutoff that differentiates between pathogenic and nonpathogenic alleles, and the clinical significance of intermediate size alleles is unknown. Multigene sequencing panels may fail to detect, erroneously identify, or misinterpret a genetic variant for a variety of reasons. Recommendations for assay use and reporting to address these issues can be found below.GRADE rating: C ‐ Weak.Strength: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 20: All persons with ALS who have genetic testing should receive posttest counseling.Key points: Posttest counseling provides persons with ALS the opportunity to discuss their result and understand the implications in the context of their specific personal and family circumstances.GRADE rating: C ‐ Weak.Strength: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 21: Posttest counseling should inform persons with ALS with a pathogenic or likely pathogenic test outcome that result may or may not allow prediction of disease course.Key points: Genotype–phenotype correlations in ALS are generally limited to trends in grouped data and have low predictive value in individual cases, with some notable exceptions. When a pathogenic or likely pathogenic variant is identified, persons with ALS should understand that the genetic result does not allow prediction of disease course in most cases.GRADE rating: B ‐ Moderate.Strength of recommendation: This recommendation is supported with at least one study of Level 2 evidence.Recommendation 22. Posttest counseling should inform persons with ALS with a pathogenic or likely pathogenic test outcome of the genetic risks and implications for specific family members, including the availability of presymptomatic testing.Key points: When a pathogenic or likely pathogenic variant is identified in a person with ALS, the family history should be reviewed in the context of the likely inheritance pattern of the variant, and implications and genetic risks for close and extended family members should be discussed. Adult relatives are candidates for presymptomatic testing, which is a personal choice and should be performed with appropriate genetic counseling.GRADE rating: C ‐ Weak.Strength of recommendation: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 23. Posttest counseling should inform persons with ALS with a pathogenic or likely pathogenic test outcome of the availability of relevant observational studies, FDA‐approved therapies, and clinical trials.Key points: Persons with ALS identified to have a pathogenic or likely pathogenic variant should be informed of FDA‐approved or investigational therapies which are targeted to their particular gene; opportunities for gene‐targeted interventions are likely to increase in coming years.clinicaltrials.gov and other resources.GRADE rating: C ‐ Weak.Strength: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 24. Posttest counseling should inform persons with ALS with a negative outcome that the result does not exclude a genetic form of ALS.Key points: Current genetic testing fails to identify a genetic etiology in a significant proportion of familial cases, indicating that additional, unknown genetic mechanisms contribute to the etiology of ALS. First‐degree relatives of individuals with familial or sporadic ALS without an identifiable genetic basis remain at increased risk of developing ALS compared with the general population.GRADE rating: D ‐ Expert.Strength: This recommendation is supported with at least one study of Level 5 evidence and expert opinion.Recommendation 25. Posttest counseling should inform persons with ALS with an uncertain result that the variant(s) may or may not be contributing to their ALS.Key points: A significant proportion of patients who undergo ALS genetic testing will receive an uncertain result.GRADE rating: C ‐ Weak.Strength: This recommendation is supported with at least one study of Level 3 evidence.Recommendation 26. Posttest counseling should inform persons with ALS that periodic reevaluation of genetic results may be appropriate, and that the interpretation of their results could change over time.Key points: As new evidence emerges, commercial laboratories and/or clinical teams may change their interpretation of specific genetic variants, and persons with ALS should be informed that reinterpretation of clinical testing may occur.GRADE rating: D ‐ Expert.Strength: This recommendation is supported with at least one study of Level 5 evidence and expert opinion.C9orf72 repeat expansion.Recommendation 27. Testing performed on DNA derived from non‐CNS tissues is sufficient to establish the presence of a Key points: Although the C9orf72 expansion shows extreme somatic instability and produces different sized repeats longitudinally and across tissues in one individual, there are no documented cases where this variability produced normal testing in peripheral tissues despite an expanded repeat lengths in the central nervous system. DNA derived from blood or other non‐CNS tissues can therefore be used to test for the presence or absence of C9orf72 repeat expansions.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by at least one study of Level 2 evidence.C9orf72 testing should use a method with high sensitivity and specificity for expanded alleles.Recommendation 28. Key points: A variety of assays are used in commercial laboratories to detect C9orf72 expansions. In a 2014 blinded study, only 5 out of 14 laboratories reported PCR‐based C9orf72 results in complete concordance with the reference Southern blot result, and both false‐negative and false‐positive results were identified.GRADE rating: A ‐ Strong.Strength: This recommendation is supported by one study of Level 1 evidence.C9orf72 alleles with high sensitivity and specificity.Recommendation 28a. Southern blot is an acceptable method for detecting expanded Key points: Southern blot analysis is considered the gold standard for detecting the expansion and is the only commercial method currently available for sizing large expansions. Southern blot may fail to distinguish intermediate or smaller expansions from normal allelesGRADE rating: A ‐ Strong.Strength: This recommendation is supported by one study of Level 1 evidence.C9orf72 alleles with high sensitivity and specificity.Recommendation 28b. Repeat‐primed PCR, performed bidirectionally in some circumstances, is an acceptable method for detecting expanded Key points: RP‐PCR assays are cost‐effective, rapid, and identify patients with an expanded allele by revealing a “saw‐tooth pattern” when a significant repeat expansion is present. However, sequence variants adjacent to the expansion may result in deviant RP‐PCR curves leading to false‐negatives. In such cases, the use of two RP‐PCR assays, one from either end of the repeat region will resolve both alleles with appropriate sensitivity and specificity.GRADE rating: A ‐ Strong.Strength: This recommendation is supported by one study of Level 1 evidence.C9orf72 alleles with high sensitivity and specificity.Recommendation 28c. Dual‐mode PCR is an acceptable method for detecting expanded Key points: The dual‐mode long read PCR assay described by Bram et al. is capable of amplifying GC‐rich sequence and enables repeat sizing from 2 to ∼950 repeats, detects expansions of >950 repeats in agreement with other assays, and flags sequence variants around the repeat tract.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by one study of Level 2 evidence.C9orf72 alleles with high sensitivity and specificity.Recommendation 28d. Expansion Hunter analysis of PCR‐free whole‐genome sequencing data is an acceptable method for detecting expanded Key points: The Expansion Hunter Software Tool developed and validated by Dolzhenko et al. can identify the presence or absence of the C9orf72 expansion when used on PCR‐free WGS short‐read data, even if the expanded repeat is longer than the read length.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by one study of Level 2 evidence.C9orf72 testing should use a method that accurately sizes normal range alleles.Recommendation 29. Key points: A method that allows determination of the exact repeat numbers of alleles with up to 30 repeats is able to exclude a pathological repeat expansion if two different alleles in the wild‐type range are detected.C9orf72 test.GRADE rating: A ‐ Strong.Strength: This recommendation is supported by one study with Level 1 evidence.C9orf72 repeat, either as a stand‐alone assay or as part of a dual‐mode PCR, is an acceptable method for sizing normal range allelesRecommendation 29a. Fragment size analysis of a PCR that spans the .Key points: Fragment size analysis is highly accurate at sizing normal and intermediate range C9orf72 alleles, whereas standard Southern blot techniques are not. However, it should be noted that optimized Southern blot protocols, such as that described by Buchman et al. could enable accurate sizing of normal range C9orf72 alleles by comparing cloned genomic fragments created by restriction enzyme digests at sites located within and close to the repeat expansion region.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by one study with Level 2 evidence.C9orf72 alleles.Recommendation 29b. Expansion Hunter analysis of PCR‐free whole‐genome sequencing data alone is an acceptable method for sizing non‐expanded Key points: The Expansion Hunter Software Tool developed and validated by Dolzhenko et al. shows high accuracy for normal C9orf72 alleles when used on PCR‐free WGS short‐read data.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by one study with Level 2 evidence.C9orf72 should specify the sizes of non‐expanded allelesRecommendation 30. Testing reports for .Key points: There is currently no validated cutoff that differentiates between pathogenic and nonpathogenic alleles. The size of non‐expanded alleles should be documented on test reports in the event that alleles of a particular size are later determined to be unstable or confer increased disease risk.GRADE rating: A ‐ Strong.Strength: This recommendation is supported by at least 1 study with Level 1 evidence.C9orf72 alleles as “intermediate” or “uncertain” should include a statement outlining up‐to‐date data regarding uncertainty of pathogenicity of these allele sizes.Recommendation 31. Labs that classify Key points: Currently, allele sizes of 20–29 repeats have contradictory evidence of association with ALS; if they do confer disease risk this is likely lower than for longer expansions. Laboratories reporting alleles classified as intermediate or uncertain should summarize current evidence, or lack of evidence, for pathogenicity of such alleles.GRADE rating: A ‐ Strong.Strength: This recommendation is supported by at least 1 study with Level 1 evidence.C9orf72 repeat expansions should include a statement clearly outlining the maximum number of repeats detectable by the assay employed .Recommendation 32. Labs reporting Key points: At this time, all commonly employed methods for detecting the presence of the C9orf72 repeat expansion are unable to accurately determine the number of repeats. The upper boundary of the expansion size should be documented on test reports, given the possibility that expansions of a particular size could be shown in the future to confer specific disease risks or other clinical significance. For example, if we learn that pathogenicity begins at 90 repeats, a report designating a boundary of >55 repeats will allow clinicians to determine that retesting should be performed to determine if the patient has >90 repeats.GRADE rating: D ‐ Expert.Strength: There are no studies addressing this recommendation.C9orf72 ALS genes should utilize simultaneous sequencing methods  rather than sequential gene sequencing.Recommendation 33. The interrogation of non‐Key points: Simultaneous sequencing approaches, including multigene panel, whole‐exome or whole‐genome, reduce the cost and time to genetic diagnosis compared to sequential testing, and also enable identification of cases harboring pathogenic variants in more than one gene.GRADE rating: B ‐ Moderate.Strength: This recommendation is supported by at least one study with Level 2 evidence.Recommendation 34. Based on ClinGen classifications, ALS gene panel reports should clearly differentiate between genes that are causal for ALS and those genes where the evidence is sparse, conflicting or insufficient.Key points: The strength of genetic evidence supporting the ability of specific genes to cause ALS varies widely and changes as additional studies are conducted. This evidence often shifts faster than the disease‐specific panels offered at testing laboratories and results in variants being reported for genes that are no longer considered monogenic causes of ALS. Laboratories should consider dropping these genes from their panels or at least designate the gene‐disease validity of tested genes, as classified by the Amyotrophic Lateral Sclerosis Spectrum Disorders Gene Curation Expert Panel of ClinGen . In particular, genes classified as “Limited” or “Refuted” or not curated should be clearly differentiated on the report.GRADE rating: D ‐ Expert.Strength: There are no studies supporting this recommendation.Recommendation 35. When targeted‐capture, whole‐exome, or whole‐genome methods are used, gene regions that were not adequately assessed should be interrogated further or highlighted in the report.Key points: Due to difficulties generating adequate sequencing coverage or challenges with mapping and alignment, most current sequencing technologies have reduced sensitivity for variants in some genomic regions . It is recommended that laboratories use additional methods to fill these gaps whenever possible. In the event that this is not performed, laboratory reports should clearly identify regions of genes where inadequate sequencing coverage or known issues may have decreased sensitivity for specific types of mutations. These specifications should aim to be interpretable by clinicians without genomic training . This will enable ordering clinicians can accurately assess the likelihood of missed finding in their patients.GRADE rating: D ‐ Expert.Strength: There are no studies supporting this recommendation.These evidence‐based, consensus guidelines for ALS genetic testing, counseling and methodologies are meant to establish a standard of care in clinical practice for individuals with ALS. Currently, persons with ALS in the United States have variable access to tertiary, academic, multidisciplinary ALS clinics staffed by genetic counselors. And while there has been improvement in access to multidisciplinary care clinics worldwide, the majority of people with ALS do not have access to such clinics.Genetic counselors in the United States are board certified by the American Board of Genetic Counseling and offer unique value in the care of individuals with ALS. They provide a perspective and training that neurologists and other medical providers do not typically have, including the treatment of family as the unit of care. These guidelines will support neurologists and other clinicians in providing genetic counseling when a board‐certified genetic counselor is not available and are intended to provide uniformity to the genetic counseling and testing approach of a person with ALS, regardless of clinical setting. Access to board‐certified genetic counselors is scarce, and the ideal scenario of an integrated genetic counselor within the multidisciplinary ALS care team is currently scarcer still. Given the rapidly increasing importance and need for genetic counselors in ALS, and in neurogenetics in general, research into alternative service delivery models is needed. Possible models to expand access to genetic counseling and patient education include telemedicine modalities, patient webinars with follow‐up genetic counseling, and online decision tools.Nonetheless, genetic counselors will become increasingly required as part of multidisciplinary teams in neurology as the technological advances in our understanding of genetic risk and association progresses. In particular, the analysis and complexity of genetic testing results will necessitate expert interpretation and the ability to communicate complexity and uncertainty to patients and clinicians alike. The need for these specialized clinical skills will become ever more acute as gene‐targeted therapies are approved and patient eligibility is determined.Our review of the literature on laboratory practices in commercial ALS genetic testing revealed a lack of consistency in methodologies and clinical reporting of results. It also revealed a paucity of published evidence for the interpretation and reporting of results. Commercial genetic testing laboratories establish their own workflows; however, the guidelines presented here are meant to serve as standards to harmonize the methodologies and reporting for these stakeholders.These guidelines reflect current genomic technology, which will evolve along with our scientific understanding of the genetics of ALS. It is expected that genetic associations with ALS and related disorders will continue expand, and with it, the complexity of results that must be communicated. In the future, associations with the nonprotein coding genome are expected to advance for example, which will require increased genetics sophistication of ALS providers. We view these guidelines as a first step toward a uniform and equitable approach to ALS genetic testing that will require revision periodically as new genetic discoveries and new genetic therapies, both experimental and FDA‐approved, move forward for people living with ALS.JR conceived and designed the study, acquired and analyzed data, and drafted the manuscript and figures. BE acquired and analyzed data and drafted figures. JW acquired and analyzed data. MH acquired and analyzed data and revised the manuscript. SK designed the study, acquired and analyzed data, and revised the manuscript and figures. The members of the Expert Panel (see Acknowledgments) reviewed the evidence summary, voted on each recommendation statement, and participated in a video conference to refine recommendations.JR has served as a consultant for Biogen, Ionis, and Uniqure. MH has served as a consultant for Biogen, Amylyx, Invitae, Guidepoint Global, and MDA. BE, JW, and SK have no conflicts to declare.Table S1Click here for additional data file.


Cervus elaphus). The findings revealed correlations between parameters estimated with both markers that were associated with the level of genetic diversity and genetic differentiation. However, microsatellites showed lower accuracy in representing the distribution of genetic diversity among individuals.Population genetic structure and individual multilocus heterozygosity are vital for wildlife management. Traditionally, microsatellite markers have been used to estimate population genetic parameters, but single-nucleotide polymorphisms (SNPs) have gained popularity due to their greater measurement precision. This study compared genetic estimates at the population and individual levels using microsatellite and SNP markers in red deer (Cervus elaphus) in the Spanish wild population with both 11 microsatellites and 31,712 SNPs. We compared parameters related to population genetic structure and individual multilocus heterozygosity obtained with both types of markers. Our results showed correlations between parameters measured using both microsatellites and SNPs, particularly those related to the level of genetic diversity and genetic differentiation. However, we found notably lower precision of microsatellites in measuring the distribution of genetic diversity among individuals. We conclude that microsatellites can be used to monitor the overall genetic status and detect broad patterns in red deer populations. Nevertheless, the greater precision of SNPs in inferring genetic structure and multilocus heterozygosity leads us to encourage scientists and wildlife managers to prioritize their use whenever possible.The analysis of population genetic structure and individual multilocus heterozygosity are crucial for wildlife management and conservation. Microsatellite markers have traditionally been used to assess these genetic parameters. However, single-nucleotide polymorphisms (SNPs) are becoming increasingly popular. Our goal here was to determine to what extent SNPs can provide better insights than microsatellites into the overall genetic status and population genetic processes in the species. To this end, we genotyped 210 red deer ( Genetic structure plays a crucial role in wildlife management as it provides essential knowledge. The assessment of the spatial distribution of genetic diversity enables the identification of migration barriers , the conMicrosatellite markers (short tandem repeat loci or STRs) have been widely used in conservation genetics to estimate genetic structure ,18 and dIn recent years, the use of single-nucleotide polymorphisms SNPs to study genetic structure has increased ,28,29. DCervus elaphus) is a widely distributed large mammal, ecosystem engineer, and an important game species in Europe [The red deer . For northern Spain, we included a population from the southern Pyrenees . In PYSPYS, see . n = 59), SP2 (n = 7), MO (n = 19), SM1 (n = 75), SM2 (n = 24), and PYS (n = 26). Specimens were hunted between 2020 and 2021 during regular sport hunting and management culling under local regulated hunting plans. This study never provoked hunters to shoot additional deer. We collected a piece of ear cartilage from 210 red deer culled by hunters. Sample sizes for each population were as follows: SP1  according to the manufacturer’s protocol. We genotyped the individuals with 12 microsatellite markers  according to the methodology described in [We isolated genomic DNA from ear tags using the BioSprintribed in . We usedribed in  . Moreover, the relationship between both heterozygosities was determined with the FIS parameter (inbreeding coefficient at population level). For microsatellites, genetic diversity measures were obtained with Genetix 4.05 [ST values and assessed their significance using Genetix (with 1000 permutations) for microsatellites and dartR (with 1000 bootstraps) for SNPs. Pairwise FST values obtained with both types of markers were compared with correlations and a Mantel test  in the vegan package [The genetic diversity of each population was quantified with observed and expected heterozygosities (Htix 4.05 . For SNPtix 4.05  in R [76 package . Princip package . The mos package  for micr package  accordin package . For SNP package . LEA was2), heterozygosity-heterozygosity correlation [2 between multilocus heterozygosity and inbreeding. Standard deviations were obtained after 1000 permutations and 1000 bootstraps for g2, 1000 repetitions for HHC, and 1000 bootstraps for the expected r2. Finally, we quantified the correlation between multilocus heterozygosity obtained with both microsatellite and SNP markers.We inferred the expected relationship between multilocus heterozygosity and inbreeding for both types of genetic markers using the inbreedR package . Inbreedrelation , and theO, while SP2 showed the lowest HE (O and HE) at microsatellite markers. In terms of SNPs, SP2 displayed the lowest heterozygosity (HO and HE), while the highest heterozygosity (HO and HE) was observed in SM2 . In terms of FIS, the values obtained with microsatellites and SNPs were not significantly related . The genetic differentiation between all pairs of populations was significant for both types of markers (p ≤ 0.001). Pairwise F markers  were posIn the PCA obtained with microsatellites, individuals from the same populations tended to have similar PC1 and PC2 scores  were similar to the ancestry coefficients obtained with SNPs : −0.002/0.018; 2 = 0.0049, S.E. = 0.0005, C.I. (2.5%/97.5%): 0.0038/0.0059; 2 between inbreeding level and multilocus heterozygosity (MLH) was only significantly greater than 0 for SNPs  = 0.951/1.000, Identity disequilibrium was not significantly different from 0 when it was quantified with microsatellites . Therefore, our results indicate that the relationship between the parameters describing the genetic structure of populations obtained with microsatellites and SNPs varies depending on the studied species. Moreover, we cannot rule out the potential impact of sample sizes , which might differentially affect the correlation of each genetic parameter [E is sample size dependent, the small sample size might contribute to the low HE in SP2. Therefore, it might affect the low correlations between HO and HE at microsatellites. However, the absence of correlations between genetic measurements obtained with microsatellites and SNPs was not limited to one population  and then salar; ). Howeven salar;  with Arahalleri,  with Armarameter . In thispulation A,C. Testudo hermanni, [Hyla molleri and Pelobates cultripes, [We found differences in the principal component analysis obtained with both types of markers, indicating high levels of genetic differentiation for SNPs compared to the results with microsatellites. Despite these differences, the percentages of explained variance of PC1 and PC2 were low in both types of markers. The low percentages might be due to the lower capacity to detect population-specific genetic variations of microsatellites and the noise provided by irrelevant SNPs. The genetic differentiation observed in the PCA, obtained with SNPs, was further supported by the admixture analysis conducted using SNPs. The cross-entropy analysis in the admixture procedure identified five clusters as the most likely number of genetic groups. The number of clusters and the membership scores can be readily discerned in the PCA obtained using SNPs. In contrast, with the microsatellites, the genetic structure is less discernible in the PCA plot, and the ΔK procedure applied to the Structure results suggested the existence of a smaller number of clusters. Similar discrepancies in the PCA plots between markers were observed in the Mediterranean tortoise . Variatltripes, ). Therefltripes, ,88). The2, HFC and expected r2 with inbreeding when microsatellites were used. However, the C.I. of these values included zero. Contrarily, the positive values of these parameters at SNPs did not include zero, even HHC and expected r2 values were close to one. Despite the significant relationship between multilocus heterozygosity in our sample of red deer at both genetic markers, the use of a high number of SNPs represents a clear advantage in the study of inbreeding in natural populations of red deer. Similarly, Miller et al. [2 and r2 between small numbers of microsatellites and a large number of SNPs. Thus, despite the existence of studies that have detected HFCs with microsatellites in red deer [Concerning the utility of using multilocus heterozygosity at the individual level, we obtained positive values for gr et al.  reportedred deer ,45, the red deer .The results regarding the genetic structure of populations and the multilocus heterozygosity of the individuals, obtained with microsatellite markers, generally correlated with those obtained using SNPs. Despite the heterogeneous sample sizes and the limited number of red deer populations, microsatellites provided insights into the overall genetic status and the existence of population processes in red deer. Nevertheless, the greater precision in inferring genetic structure and the increased power to detect HFCs encourage scientists and wildlife managers to use SNPs wherever possible. In our red deer populations, the utilization of SNPs revealed patterns that were not apparent in the analyses conducted with microsatellites. Moreover, SNP technology holds high potential for detecting inbreeding events in a species like red deer, which has important ecological, health, and economic implications.