A genetic diagnosis was confirmed in 48% of those who underwent testing. Those with no genetic diagnosis underwent significantly more genetic tests than those with a confirmed genetic diagnosis , more sequence-level tests and a longer, more expensive testing period than those with a genetic diagnosis . A genetic diagnosis was not established for 52% of children. Integrating genome-wide sequencing into clinical care may improve diagnostic efficiency and yield in this population.Little is known about the pattern of genetic testing and frequency of genetic diagnoses among children enrolled in structured complex care programs (CCPs). Such information may inform the suitability of emerging genome diagnostics for this population. The objectives were to describe the proportion of children with undiagnosed genetic conditions despite genetic testing and measure the testing period, types and costs of genetic tests used. A retrospective analysis of 420 children enrolled in Toronto’s Hospital for Sick Children’s CCP from January 2010 until June 2014 was conducted. Among those who underwent genetic testing ( Children with medical complexity (CMC) have chronic conditions resulting in high family-identified service needs, functional impairments, multiple subspecialist involvement, and elevated health service use . These cAs the number of clinically available genome-based tests grows, optimizing their application for CMC warrants consideration. Improved detection rates ,12,13 anThe objective of this study was to describe the use of genetic testing in a cohort of children in a structured CCP. Specifically, in order to gauge the potential suitability of exome/genome sequencing-based technologies for routine use in CCPs, we aimed to determine the proportion of children remaining without a confirmed diagnosis despite undergoing genetic testing, as well as to characterize the duration of the genetic testing journey, and the types and costs of genetic tests used.The CCP at the Hospital for Sick Children (SickKids) was established in 2006 and is the largest structured CCP in Canada; over 550 children at SickKids and five community-hospital locations have been enrolled since inception. Clinical care is provided by an interprofessional complex care team comprised of a nurse practitioner, a complex care physician, and other allied healthcare professionals ,19. ChilThis study protocol was approved by the Research Ethics Board at the Hospital for Sick Children, and a waiver of consent was obtained for this retrospective review.n = 319) and those who did not (n = 101). Using computer-generated random numbers, 20% of the children who underwent genetic testing were selected to form the study cohort of 63 children and were further divided into two groups: those for whom a genetic diagnosis was confirmed and those with no genetic diagnosis. A confirmed genetic diagnosis was defined as a diagnosis validated by a positive genetic test result that explained all major clinical findings. For most cases, genetic diagnoses were confirmed in the genetic consultation letters of the patient charts and all cases were then subsequently reviewed by a clinical geneticist to verify variant causation. All other children who underwent genetic testing but did not meet these criteria were classified as no genetic diagnosis. Included among those classified as no genetic diagnosis were children for whom variants of unclear significance were found on genetic testing but who did not have a positive genetic test result explaining major clinical findings. To gain a better understanding of the overall genetic diagnoses of the children in the entire CCP and to determine the total number of children in the CCP that remained without a diagnosis despite undergoing genetic testing, the diagnoses of the entire population of children that underwent genetic testing (the parent sample) were also further described. Similar to the study cohort, the children in the parent sample were divided into confirmed genetic diagnosis and no genetic diagnosis. The diagnoses of those that received a confirmed genetic diagnosis were then divided into groupings based on the type of genetic diagnosis.Children were identified for inclusion from SickKids’ Complex Care Database, based upon CCP enrolment from the date of database inception (1 January 2006) until 11 June 2014. Children discharged from the CCP or deceased prior to 2010 were excluded in an effort to ensure that all participants had had the opportunity to pursue chromosome microarray. In 2010, chromosome microarray analysis became the recommended first-line test for the diagnostic assessment of children with multiple anomalies, non-syndromic developmental and intellectual disabilities and autism spectrum disorders  compared to those for whom a genetic diagnosis was confirmed [median (IQR): 0.35 years (0.12–3.04)],  < 0.001 . Both groups had a median start date  of September 2009. p < 0.001 (Costs for genetic testing per child were higher among children with no genetic diagnosis [median (IQR): C$8496 ] compared to those children for whom a genetic diagnosis was confirmed [median (IQR): C$2614 (C$1605–C$4080)],  < 0.001 . SequencThis study describes the high utilization of genetic testing among children enrolled in a large structured CCP. The majority of children used high volumes of genetic testing (median of four genetic tests) over a median time period of greater than 2 years and most (52%) remained undiagnosed. Compared to those for whom a diagnosis was achieved, undiagnosed children pursued a greater number of genetic tests overall and a greater number of high-resolution tests such as sequence-level testing.Our findings suggest that genetic testing is utilized at high rates in complex pediatric populations. Other studies have characterized the use of all diagnostic tests  in neurology clinics for children with both broad  and specThe suitability of exome/genome sequencing incorporates a combination of test performance, cost, and acceptability to the population. A recent costing study of next-generation sequencing for autism spectrum disorder estimated costs to be C$1655  for whole exome sequencing and C$2851  or C$5519  [A number of study limitations are notable. This was a single center study. Although this program shares similarities in patient characteristics with other complex programs , findings may differ in other CCPs based on their specific population. Furthermore, costing estimates and coverage of genetic testing reflected those of the province of Ontario, where whole exome sequencing and whole genome sequencing are not yet licensed as clinical tests, and may differ in other jurisdictions. The primary analysis was conducted on a random sample; although the characteristics of the sample were similar to those of the total clinic, nevertheless, this may have led to some degree of imprecision of estimates. Duration of follow-up and types of genetic tests available to the children enrolled in the CCP were not consistent over the study period. However, children with no genetic diagnosis and children with a confirmed genetic diagnosis had similar median start dates for testing (September 2009) so this lead time bias did not affect the length of follow-up of the two groups. Six cases in the undiagnosed group died or were lost to follow-up and may have been a source of attrition bias. If anything, this shortened follow-up interval in the undiagnosed group would lead to an underestimation in duration of testing for this group. Additionally, charges including mark-ups were used to approximate dollars spent on all genetic testing  and may over-estimate actual costs. We were unable to consistently ascertain whether charges reflected actual expenditures from the payer’s perspective. Although this study demonstrated that substantial charges for genetic testing are incurred for children enrolled in CCPs, the total costs may reflect a conservative estimate as several associated costs were omitted: metabolic testing, physician consultation and allied health service use , parental testing, targeted testing and counseling for other family members, and costs borne by parents . Furthermore, for patients with no diagnosis, the total number and costs of genetic tests excluded future tests that could be conducted after the study end date as they continued on their diagnostic quest.Despite these limitations, this study highlights the resource intensity and time associated with conventional genetic testing in a CCP. Currently, chromosome microarray remains the recommended first tier diagnostic tool for children with multiple congenital anomalies and developmental delay . As such


A set of 700 well-conserved Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) clones from six provinces in southern China in the ex situ gene bank of Longshan State Forest, was analyzed using 21 simple sequence repeat markers, with the aim of assessing the genetic diversity of these germplasm resources. Genetic analysis revealed extensive genetic variation among the accessions, with an average of 8.31 alleles per locus and a mean Shannon index of 1.331. Excluding loci with null alleles, we obtained a low level of genetic differentiation among provinces, consistent with the interpopulation genetic variation (1%). Three clusters were identified by STRUCTURE, which did not match the individuals’ geographical provenances. Ten traits related to growth and wood properties were quantified in these individuals, and there was substantial variation in all traits across individuals, these provide a potential source of variation for genetic improvement of the Chinese fir. Screening large collections for multiple-trait selective breeding programs is laborious and expensive; a core collection of 300 accessions, representative of the germplasm, was established, based on genotypic and phenotypic data. The identified small, but diverse, collections will be useful for further genome-wide association studies.Large  Many plant species are composed of a large number of individuals distributed across vast areas and thus may be rich in diversity. Understanding their genetic variability is important for efficient selection and maintenance of germplasm collections4; this is especially true for tree species, which are perennial, woody, contain a large number of individuals, and are usually cross-pollinating5. A considered approach to the preservation of plant genetic resources is vital. Ex situ germplasm collections are essential for the conservation of plant genetic resources, and they generally involve significant diversity. Appropriate evaluation of ex situ collections of trees, including an estimate of the genetic structure and diversity of populations, provides not only an understanding of their genetic relationships7 and an ability to establish core collections9 but also important information for association mapping11.Genetic variation is essential for the adaptability of a population and is the basis for the evolutionary potential of a species13. Among various molecular markers, simple sequence repeat (SSR) markers are the most popular, as they are co-dominant, hypervariable, neutral, and highly informative14. Association analysis, which is based on relating genes or loci to traits, is often used to identify relationships between morphological characteristics and molecular markers or candidate genes to improve the efficient management and utilization of plant genetic resources. However, association mapping of large germplasm collections is laborious. To improve conservation and the effective use of genetic resources, core collections15 are often used as materials for association analyses17.To assess genetic variability, morphological characteristics are often used, although they can be affected by environmental conditions. As an alternative, molecular markers are more stable and reliable for use in the characterization of germplasm resources and have been used to characterize the genetic variability of various species at the DNA levelCunninghamia lanceolata (Lamb.) Hook], belonging to the Taxodiaceae family, which is diploid (2n = 2x = 22)18, is the principal indigenous tree species in subtropical southern China. It is an economically valuable conifer with high yield, good wood quality, high pest resistance, and many uses, including as furniture or paper material. The species is monoecious, with a predominance of outcrossing, although self-pollination is also likely19. Studies on the genetic modification of Chinese fir have been performed since 1957, including provenance tests, plus-tree selection, clonal tests, and so on; thus, many seed plantations, progeny forests, germplasm banks, and so forth have been built, which have provided many good germplasm resources20. In recent years, the area of artificial afforestation of Chinese fir has expanded consistently, and fourth-generation seed orchards have now been established. These provide potential sources of beneficial alleles for Chinese fir breeding and improvement. Resolution of the genetic structure and relatedness of the accessions would allow proper quantitative analysis of the progeny and would also form the basis of molecular marker-assisted selection breeding. Hence, a more comprehensive analysis of genetic diversity and population structure in Chinese fir is essential. Previous studies have focused on characterizing the genetic variation26 of Chinese fir; however, the number of samples evaluated was relatively small, consisting of fewer than 150 accessions. In 2004, a set of 700 Chinese fir trees from the provinces of Guangxi (GX), Jiangxi (JX), Hunan (HN), Guizhou (GZ), Fujian (FJ) and Guangdong (GD) were conserved in the ex situ gene bank of Longshan State Forest Farm, Guangdong Province, China . Understanding the genetic background of these germplasm resources and their appropriate evaluation is important for their effective use. In addition, a core collection is needed to establish a program of molecular marker-assisted selection breeding to improve the efficient use of Chinese fir in the future.The Chinese fir  (1997) also indicated that HN is most distant from other provinces, whereas GD-GX and GD-JX were most closely related. Further analysis among the three clusters yielded a mean value for FST of 0.010, while the mean Nei’s genetic distance was 0.034, indicating weak differentiation among them.Population genetic parameters, including el Table . Among tT) Table . Most ofte Table . RoussetAMOVA was performed for all Chinese fir accessions of the six different provinces to analyze the distribution of genetic diversity among and within the provinces. The results revealed low variation among the provinces Table . Nearly 3. Tracheid length had the lowest coefficient of variation (11.9%). The maximum tree height (15.25 m) was more than 10-fold that of the minimum tree height (1.50 m), and the average height was 7.71 m. Further analyses of the phenotypic variation in the three clusters are shown in Table ST.Ten growth- and wood-property traits were measured in all 700 clones. The growth traits included tree height (H), diameter at breast height (DBH), bark thickness (T), and stem volume (V). The wood-property traits included proportion of heartwood (P), wood basic density (WBD), hygroscopicity (Hy), tracheid length (L), tracheid diameter (D), and the ratio of L to D (L/D). Abundant phenotypic variation was detected among the 700 clones Table . Data fr height 1.25 m wasTo determine the appropriate sampling strategy and optimal core size, different sampling percentages from the whole collection were designed, and this was combined with an M strategy and random sampling method. The average build results for five repeats Fig.  revealedCV among the measured traits was only 17.71%. Additionally, t-tests performed for all phenotypic data showed no significant differences between the core collection and the whole collection, except for H 26, and determined using the same microsatellite loci despite the sample size of this study being almost five times that of the previous study. Wang et al.31 also obtained similar heterozygosity values for different numbers of wild and semi-wild apricots, using the same microsatellites. Number of loci, rather than the number of populations, affects the estimate of genetic diversity, consistent with the report by Ferrer et al.32. The mean values for Ho and He obtained using SSR markers in this germplasm were higher than those reported for other Chinese firs26 and other conifers35, but lower than those in Abies chensiensis and Austrocedrus chilensis37; this suggests that the level of genetic diversity in this germplasm is moderate. Outcrossing and wind-pollination may explain the considerable level of polymorphisms in this species41. Of the 21 SSR loci used in this study, 14 showed lower values of Ho than He, indicating a deficiency in heterozygotes at these loci. The same heterozygote deficiency was observed at the population level excluding null alleles. The results are consistent with previous findings in the Chinese fir26 using the same 21 SSR markers. In general, natural selection occurring throughout the life cycle of a tree appears to favor heterozygosity42, and hybrids show excess heterozygotes43. In this tree species, however, heterozygote deficiency may be explained by self-pollination41 or by subpopulation structure30. The marker data also showed that the genetic distance between some clones was very small, such as between 25 and 26, 516 and 532, 242 and 244, 185 and 32, 122 and 123, 153 and 154, 533 and 535, 625 and 631, 55 and 61, 171 and 172, 10 and 260, 297 and 305, 96 and 253, 289 and 386, 393 and 400, 515 and 544, 24 and 285, 251 and 266, 529 and 541, and so on. Heterozygosity plays an important role in the response to environmental changes44, and therefore maintenance of heterozygosity and retention of heterosis are vital, although further analyses such as Mendelian inheritance testing1 are needed to verify heterozygote deficiency. Previous analyses of the phenotypic traits of this species identified 98 relatively fast-growing genotypes with relatively high wood basic density45, and all of them had distant genetic distances. These genotypes can be chosen to be the parent in cross breeding. In the future, it will be necessary to further expand the Chinese fir breeding base and increase gene communication between the trees. The presence of private alleles in this germplasm may indicate useful rare variants and also provides the opportunity to select useful recombinants in the future.Characterization of the germplasm is essential for germplasm conservation and collection activities. A large sample of Chinese fir individuals from six different provinces was collected, grafted, and grown at a single location. We surveyed this germplasm for growth and wood-property traits, which are all important for the economic value of woody stems26. The high number of alleles identified in this study may be due to the large sample size1. The relatively low FST value (0.015) of loci observed indicated a low level of genetic differentiation. Correspondingly, only 1% genetic variation was seen among provinces, as confirmed by AMOVA. These results confirmed those from a previous study showing that outcrossing in woody plants resulted in increased genetic diversity and reduced genetic differentiation among populations31. Furthermore, the levels of genetic diversity in each province except HN were similar, although there was a large disparity in sample size. These results indicate that genetic diversity may not be influenced by the number of samples when a large sample size of Chinese fir is available. The FST values between pairwise provinces were significant but low, which indicated that most have mixed ancestry and therefore clustered together. Rousset’s genetic distance values showed that HN was genetically the most distantly related among the provinces, which may have been due to its small sample size, while the most closely related were GD-GX and GD-JX, which may have been due to their geographical proximity or to human activity, such as artificial cultivation.In this study, a mean of 8.31 alleles per locus from 700 Chinese fir accessions was obtained, a value higher than those in previous reportsFST values obtained. That may explain the small phenotypic variation obtained among provinces in a previous study45. The low divergence in this resource could be due to several factors. The materials used in this study were all plus trees, including excellent genetic materials, good local-type materials, excellent provenance materials, and hybrid materials, and may have been widely used. This may have resulted in gene flow under past anthropogenic activities, lowering the differentiation among populations. Additionally, the relatively small geographic scale of our survey may have also been a factor. Previous studies have shown that the influence of climate on population structure is stronger than that of the geographical distance influence in P. tomentosa, and P. tomentosa clones were generally assigned to three different climate regions, whereas most of the geographical proximities were clustered into different groups1. Wang et al.31 also reported that geographic distance was not the principal factor influencing genetic differentiation in the Siberian apricot. The trees investigated in this study were all from similar climate zones, and a sufficient number of individuals may not have been sampled from all local populations, obscuring the genetic structure of Chinese fir, which may have weakened the genetic structure to a certain extent. In addition, outcrossing rate, population size and life-history traits can also have a strong influence on the genetic structure of plant populations40. A history of genetic drift may also contribute to the phenomenon of non-conspicuous genetic differentiation26. Chinese fir is an economically valuable and widely used conifer that is widely distributed in southern China, covering more than 9. 11 × 106 hm2. To obtain greater economic benefits, directional selection is used in breeding, and random sampling from small groups may lead to genetic drift that may contribute to low genetic differentiation. Such past anthropogenic activities may have significantly influenced the present population structure and patterns of genetic diversity in Chinese fir. Additionally, Chinese fir is a wind-pollinated tree species with a low level of inbreeding, which can lead to genetic drift. The samples of this germplasm were derived from different provinces; therefore, a comprehensive study of genetic structure should improve our ability to assess the distances over which differentiation can occur in Chinese fir40. Understanding the genetic structure of Chinese fir could facilitate the selection of trees for breeding to maximize genetic diversity as well as to enhance the potential gain from selection, which may have an impact on the ecological adaptation and evolution of this species in the future40.STRUCTURE identified three non-distinct clusters among the accessions of Chinese fir. The membership coefficient of the whole accession revealed that only 15.43% of individuals had ancestry values >0.80, and nearly half of the individuals had ancestry values <0.60. ANOVA also revealed no significant differences in most traits among the three clusters. Cluster assignment did not match geographical provenances, indicating that the Chinese fir accessions had a mixed ancestry, consistent with the low Association analysis in multiple-trait selective breeding programs is a breeding strategy that can accelerate the breeding process; however, association mapping of large germplasm collections is laborious. At the same time, to avoid using too small of a sample to assess the correlation between phenotypes and genotypes we selected 300 accessions from different genetic backgrounds, representing the maximum variability of 700 Chinese fir accessions, for future association mapping studies. The t-tests performed for most phenotypic data and all genetic parameters showed no significant differences between the core and the whole collections, indicating that the core collection is a good representation of the original germplasm. The core collection developed in this study will be useful for genome-wide association studies in the future to accelerate breeding programs for the Chinese fir.46. These materials come from excellent provenance and a family gene collection area established in 1983, a Chinese fir-type gene resources collection area constructed in 1989, and an excellent hybrid materials genetic resources collection area of a second-generation seed orchard constructed in 1992, including more than 50 provenances with a broad genetic basis. The number of Chinese fir individuals in each provenance ranged from 1 to 67. The selection criteria of original plus trees considered growth indices and morphological parameters, including volume of wood, height–diameter ratio, crown diameter ratio, percent of bark, disease resistance, stem straightness, the natural level of training, crown vice, collateral thickness degrees, growth vigor, and so on. The dominant comparative method and comprehensive evaluation method were used to choose plus trees, and the distance between any two individuals was more than 50 m. From 2004, scions of these trees were grafted onto 2-year-old Chinese fir rootstocks in the ex situ gene bank of Longshan State Forest Farm, Guangdong Province, China , which is located in a subtropical region with a moderate climate throughout the year and ample rainfall47. Each clone had at least four ramets of similar size and vigor. Grafted ramets were planted randomly at the site at a spacing of 3 × 3 m. This study was performed in strict accordance with the recommendations in the Guide for Observation and Field Studies48. Total genomic DNA was extracted from the mature leaves of each clone using a QIAGEN Plant DNeasy Kit  in 2014. The quality and concentration of the extracted DNA were measured using a NanoDrop 2000 Spectrophotometer.Chinese fir is widely distributed in southern China, including Guangdong, Fujian, Zhejiang, and 14 other provinces, as well as in Taiwan Figure . In 200449 were used to genotype the 700 clones. Amplification was performed in a 25-μL reaction volume containing 1.0 μL genomic DNA (~ 100 ng), 1.0 μL forward primer (10 μM), 1.0 μL reverse primer (10 μM), 12.5 μL 2X QIAGEN Taq Plus PCR MasterMix, and 9.5 μL double distilled water. The reaction was performed, as described previously26, in a T100™ thermal cycler, with an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 56/58/62 °C  for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. The forward primer of each pair was labeled with a fluorescent dye  during synthesis. PCR products were separated by capillary electrophoresis using the ABI3730xl DNA Analyzer . Genotypes were determined using Gene-Marker 2.2.0 software .Twenty-one previously identified microsatellite markerset al.50. V was calculated according to the formula V = 0.000 058 777 042 × D1.9699831 × H0.89646157. Additionally, a wood core was drilled at breast height from each tree using a tree growth cone and then placed in a plastic tube, which was not completely sealed, to prevent wet rot. P and WBD were measured using the method described by Duan et al.50. Hy was evaluated using the formula Hy = (W1 −W2)/W2, where W1 and W2 represent the water-saturated weight and oven-dry weight, respectively47. L and D were measured using the methods described by Huang et al.51.Ten growth- and wood-property traits were measured in all 700 clones in 2014 in Longshan State Forest Farm, with at least three randomly selected ramets per clone. The growth traits included tree height (H), diameter at breast height (DBH), bark thickness (T), and stem volume (V). The wood-property traits included proportion of heartwood (P), wood basic density (WBD), hygroscopicity (Hy), tracheid length (L), tracheid diameter (D), and the ratio of L to D (L/D). Growth traits, including H, DBH, and T, were measured during field surveys using the method described by Duan CV). Detailed sampling, measurement methods, phenotypic variations, and phenotypic correlations for these 10 traits were described in a previous study45.Microsoft Excel 2010 and SAS ver. 8.1  were used to examine trait differences in the phenotypic traits; these analyses excluded abnormal data obtained from weak grafts, including the mean value, standard error, amplitude, and coefficient of variation , effective number of alleles (Ne), Shannon index (I), observed heterozygosity (Ho), expected heterozygosity (He), gene flow (Nm), and F-statistics calculations , were calculated using GenAlEx 6 software53. Polymorphism information content (PIC) was calculated using PowerMarker v. 3.2554. Hardy–Weinberg equilibrium (HWE) for all loci was assessed using Arlequin version 3.555 with 100,000,000 steps in the Markov chain and 100,000 dememorization steps56. Null alleles were detected using Microchecker 2.2.357. A neutrality test for all loci was performed in Popgene version 1.3258. An analysis of molecular variance (AMOVA) was performed to partition the genetic variance among and within the provinces using GenAlEx 6.553 together with Microsoft Excel 2010.Microsatellite data were converted into various formats using Convert 1.3.153. The FST values were calculated to evaluate the genetic differentiation between any two provinces using FSTAT version 2.9.359. Nei’s genetic distance was estimated by GenAlEx 6.553. The pairwise genetic distance [FST/(1 − FST)]60 (1997) among any two provinces was also estimated.Genetic variation among the samples obtained from six different provinces was evaluated by calculating genetic parameters using GenAlEx 6.561. The software implements the Markov chain Monte Carlo (MCMC) algorithm and a Bayesian framework under admixture model, correlated allele frequencies. Subgroups were identified based on distinctive allele frequencies, and individuals were placed into K clusters by estimated membership probability (Q). In this study, the optimum value of K was determined using the model developed by Evanno et al.62, with an ad hoc statistic (ΔK), based on the second-order rate of change in the log probability of data between successive K values. The algorithm was run 10 times with a burn-in of 100,000 iterations, followed by 1,000,000 iterations for each value of K and subpopulations (K) ranging from 1 to 20. The height of this modal value was used as an indicator of the strength of the signal that was detected using Structure Harvester63. To further compare the evidence for a range of K values, we set Kmin to 1 and Kmax to 20, and the main repeats were 10, using Marverick software with the default parameters64. The CLUMPAK web server65 was used to visualize the bar plot of the probability of membership from the results of Q-matrix and to evaluate modality.The population structure of the clones was analyzed using the Bayesian model-based clustering algorithm in STRUCTURE ver. 2.3.154 based on M and random sampling strategies. The M strategy is also known as the maximum number of alleles strategy67. The random sampling strategy was the same for all materials, and a certain number of samples was randomly assigned from germplasm materials68. The two sampling strategies were compared among 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, and 400 individuals to assess the efficiency of allele sampling using molecular data. For phenotypic data consisting of continuous variables, 100% of the diversity can be sampled based on the precision of classification using PowerCore software69. From the two comprehensive sets of results, a core set available for future association mapping was constructed. Simple t-tests were performed for all phenotypic data and for the genetic parameters of the entire collection and the core collection using SAS ver. 8.1 .A core collection was constructed. The optimal sampling method and amount were determined using PowerMarker v. 3.25Supplementary informationTable S3Table S4


In Europe, there is a wide variety of genetic tests that various private companies offer to patients or to consumers. More and more people have become curious about their genetic predisposition and susceptibility. Most public health-care systems, however, are not adequately prepared for responding to these new demands and to the results of these genetic tests as, quite often, there is no available therapy for the identified genetic condition. This discrepancy between the newly emerging expectations and the insufficient responses contributes to a further rift between the public and private sectors of health care. Individual genetic test results may also trigger the need for personalized medicine and may open up a competition between the two fields in offering further genetic tests and medical exams. Pro-active patients may need a different kind of information on genetic tests and their implications. In this context, how should the public health system deal with the challenges of private testing? Will private genetic testing transform health care from a solidarity-based system to an individualistic one? In this paper, I would like to explore the emerging legal and ethical issues related to genetic testing and the relevant legal framework that has developed so far. In the conclusion, I will examine the possibilities of further legal development. What is the role of the public and private sector in providing better care and information for patients based on genetic tests? Should direct-to-consumer (DTC) testing be controlled or should regulators give a wide scope of individual autonomy? What is the threshold of reliability? If further collection of the population based genetic data is necessary, how to balance between public and private interests? Could private companies represent public interests? Just looking at the long list of these questions, one can see that the core of the ethical and legal dilemmas today is to what extent can be considered genetic data as private and to what extent it can be used for public purposes? Furthermore, do personalized medicine and pro-active patients require some changes in the content of informed consent? It seems that people are more and more curious about their genetic data, and in this process, testimonies of the celebrities played an important role.The New York Times, in which she started out with an account of the decade-long fight her mother had waged against ovarian cancer, at the end of which Jolie’s children had lost their 56-year-old grandmother. The article then took a startling turn, when the movie star—considered by many as one of the most beautiful women in the world, who at the peak of her career seemed to be in perfectly good health—informed the public that during the past few months her breast tissues had been surgically removed . Although she had not actually been diagnosed with cancer, she decided to have a series of radical, prophylactic operations, which she intended to follow up with the surgical removal of her ovaries, pledging to publish the regimen of the medical procedures on a webpage.Angelina Jolie made an important statement and even published an article in the May 14, 2013 issue of To protect her family and children, she has opted for a preventive surgery to eliminate the chances of developing a cancer that has not even been diagnosed—and in doing so she has provoked a heated debate. Jolie’s coming-out, along with the public debate it has sparked, has ramifications in a range of issues as diverse as the boundaries between healthiness and illness, the precise definition of prevention, the financing of, and access to, genetic tests and the possible evaluation of the results, the expectations about the female body, the media representation of the celebrity world, the yawning gap between the health-care options available to the rich and the poor, as well as the prejudices and stigmas associated with cancer.Although genetics promises to be the science of the future, this is the first time that a genetic test has driven a patient  to have a series of preventive medical procedures as radical as this. This is true even when we take into account that prophylactic mastectomies are much more frequent in the US: the American women’s decision is made easier by the fact that in such cases the cost of breast-reconstruction comes under health insurance coverage according to a federal law passed in 1998.BRCA tests that can examine all the mutations. Located on chromosome no. 17, the gene BRCA1 has approximately 1,180 different variants, with each one of them being capable of disrupting the proper functioning of the gene. Thanks to the huge genetic databases created by the private company Myriad Genetics of Utah, the risk factors can be identified with a relatively high certainty. In other words, it has become possible to estimate the probability of an individual’s developing ovarian cancer or breast cancer before the age of 70. According to the test results, Jolie belonged to the group with the highest risk factors.  The mutation discovered in her gene had already been found in her mother who died prematurely of ovarian cancer. For this reason, a number of people suggested psychological trauma or a pathological fear of cancer as possible motives for her decision. Actually, the truth is, she made a very rational decision: Jolie did not want to run the risk being diagnosed with a serious illness already at an advanced stage.In recent years, considerable advances have been made in the field of genetics: now it has become possible to conduct detailed The public seems to expect that celebrities conceal or deny their personal weaknesses and imperfections up to the hilt. It was this tacit agreement that Jolie has abrogated by going public with the news of her willfully chosen mastectomy—a medical procedure that so far has always been a hushed-up and divisive method of protection. But there is more. Jolie’s case questions the existence of a firm division between healthiness and sickness, considering that one would be hard-put to find the right expression to describe her status in this story: is she ill? Is she a patient? Is she a conscious consumer going out shopping on the health market? Is she a spokesperson for biotechnological advances? The expressions “patient” and “ill” would suggest that Jolie has been the forbearing sufferer who meekly submitted to a medical procedure prescribed by her doctor, instead of taking control of the situation, of planning and executing her own course of action.But in fact that was not what happened here: Angelina Jolie overturned the traditional relationship between doctors and patients; one that is characterized by the latter’s vulnerability, helplessness, and dependence. In this case, it has been the patient who dictated the terms: she has availed herself of the medical services, had her tests, asked for consultation, weighed her options, and made her own decision. All this is in stark contrast with the helplessness of a cancer patient who, after much anxiety and long procrastination, finally makes up her mind to ask for an appointment with a specialist, patiently waits for weeks until her turn, goes to have a mammography, waits again to be scheduled for biopsy and then surgery; begs and bargains, and then, after a series of various operations and chemotherapy, she discovers that the idea of having reconstructive surgery will always remain an unrealizable dream for her. Before this, we saw mastectomy, or an invasive surgery, as a sacrifice ordered by the doctor and rendered by the patient, while considering the invasive medical action as the stigma of a serious illness, cancer, the antechamber of death, rather than the means to secure our life and health.Jolie’s attitude has ushered in the prototype of a new category of patients—proactive consumers who plan preventive measures, who wish to extend and enjoy life, who consider genetics not as an act of fate but as an opportunity—in other words, conscientious biotechnological consumers. Cancer patients carry multiple disadvantages, as the stigma of a serious illness is usually accompanied by the stigma of poverty also.  However, at the moment, this remains closely bound up with the exclusivity and high price-tag of the tests.Breast cancer is the second leading cause of death among American women; it is estimated that in the US the genetic testing designed to reveal the presence of BRCA1 gene is performed on about 250,000 women annually. The basic version of the BRCA test costs between 3,000 and 4,000 dollars—and the simpler tests do not even cover all the potentially fatal mutations. Studies of the mutant genes responsible for the development of breast cancer get more and more accurate and reliable as the number of the tested mutants increases. To evaluate the tests, one needs huge databases, which can only be set up by private biotechnological companies and clinics where extensive data collection has been carried out for decades. The precise interpretation of the genetic tests results also requires a great amount of work and money. The crucial questions that now must be asked are as follows: how much should be paid for these still highly expensive tests and who should be paying for them? How should the price of the tests be determined, and by whom, so as to ensure that the actors of this market stay in business on the one hand, and do not exploit their monopoly on information on the other? And in a broader context: to what extent is it permissible that the patients’ prospects to live a long and healthy life be commensurate with their financial circumstances?BRCA1 and BRCA2 are human genes that produce tumor suppressor proteins. These proteins have a vital role in repairing the damaged DNA. When one these genes does not function correctly, DNA damage may not be repaired properly, and as a consequence, cells may develop additional genetic mutations that can lead to cancer.According to the US National Cancer Institute, specific inherited mutations in BRCA1 and BRCA2 increase the risk of female breast and ovarian cancers, “and they have been associated with increased risks of several additional types of cancer. Together, BRCA1 and BRCA2 mutations account for about 20–25% of hereditary breast cancers and about 5–10% of all breast cancers. In addition, mutations in BRCA1 and BRCA2 account for around 15% of ovarian cancers overall.”Furthermore, “a BRCA1 or BRCA2 mutation can be inherited from a person’s mother or father. Each child of a parent who carries a mutation in one of these genes has a 50% chance of inheriting the mutation. The effects of mutations in BRCA1 and BRCA2 are seen even when a person’s second copy of the gene is normal.”The US National Cancer Institute provided comparative statistical figures on the significant differences between the occurrence of cancer in the general population and cancer in the population that has some genetic predisposition. “About 12% of women in the general population will develop breast cancer sometime during their lives. By contrast, according to the most recent estimates, 55–65% of women who inherit a harmful BRCA1 mutation and around 45% of women who inherit a harmful BRCA2 mutation will develop breast cancer by age 70 years.”The legal approach to all these problems has previously been anchored to the instrument of patent protection: the standard practice of private companies specializing in genetic technology so far has been to obtain patent protection for the DNA sequence they have successfully isolated—this has been the corner stone of their business model. It is a small wonder, then, that nowadays the debates about studies and patents associated with predisposing genes for breast cancer and ovary cancer heat up. In early 2010, a total of 19 plaintiffs, including physicians, researchers, patients, the American College of Medical Genetics, the American Society of Clinical Pathologists, and several researchers at the University of Pennsylvania, formed an unprecedented joinder of parties to file a case against the US Patent Office and Myriad Genetics. In its claim against the company holding the patent, the American Civil Liberties Union (ACLU) argued that genes are the products of nature, rather than of human beings, and thus fall outside the jurisdiction of patent law. In addition, the strict legal protection, ACLU claimed, severely limits the researchers’ work and is, therefore, in violation of the patients’ rights. Representatives of Myriad, the company holding the patent rights, asked the Court to reject the petition, claiming that their company was the first one to isolate these genes within the DNA and, therefore, has acquired the right to place them under patent protection. According to the plaintiffs, through its practice, Myriad keeps the prices high and prevents the patients from checking the test results.According to the Transparency Market Research,It can hardly be argued though, that genetics, and our knowledge of our own genes, in itself has an emancipatory effect. It is quite obvious that in these difficult questions only the well-informed and active patient can make a carefully weighed decision. As it has been demonstrated by a number of examples already, the model of a patient condemned to a role of passive acceptance is no longer a given in the field of genetics.  The case of Angelina Jolie certainly fits into this trend. The effect that the coming out of such a world-renowned celebrity has exerted on the knowledge of medicine, genetic tests, and the expectations of health care of millions of women all over the world could easily have been more profound than a myriad of official health campaigns put together with scientific thoroughness. The reverberations of her announcement are still unclear at this point; but we may certainly hope that it will inspire more and more healthy women and men to act as conscious biotechnological consumers; it may convince them to take their genetic lottery tickets and cash them. This conscious behavior may shake up and reorganize the balance of power in the field of health care, resulting in a number of positive developments. The less fortunate outcome of Jolie’s act may be the increased commercialization of biotechnology, which, through the extraordinary publicity, could lead to the higher pricing of genetic tests, further widening the gap between patients of different financial status.Genetic data can be used for a number of purposes, including the establishment of paternity or personal identity, genealogical research, and the confirmation of exceptional talent. And as the full potentials of genetic testing became ever more apparent, people’s imagination began to go wild. In Italy, for example, it prompted huge media interest and full television coverage when members of a family by the name of Merisio were subjected to oral mucosal swabs to prove their claimed genetic descent from the painter Caravaggio. But genomics’ glamorous association with fame had already begun in 2010. This was when the public found out that the first woman with a known identity to have her full genome analyzed was the famous American actress, Glenn Close. Compared to the $500 million that such a comprehensive procedure had cost at the beginning of the Human Genome Project, the price has now climbed down to $48,000, thanks to new advances in technology.For a large number of people, genetic data represent the most guarded private secrets, in view of the fact that they offer a glimpse of not only possible current illnesses but also of risk factors and personal susceptibility. By contrast, many people do not feel the need to conceal any of their genetic data. The Personal Genome Project (PGP), which involved the participation of a large number of volunteers, was conducted in a completely open manner. PGP has encouraged us to rid ourselves of suspicions and prejudices fixed in our minds and to contribute to a monumental project of genetic research by giving our name and donating our medical data. The findings were later made available to the participants themselves. The home page of the project features 10 researchers, all in apparent good health, who reveal their genetic data to the public. They have no reason to conceal that they suffer from myopia or asthma, or that they sometimes have trouble sleeping because of too much traveling.As a result of what is known as trans-generational effect of genetic information, the findings of genetic testing may also be of concern to people other than the one tested. As Jolie’s suspicions were originally raised by her mother’s illness, it is probable that Jolie’s children will also have to go through genetic testing at some point. Their chances of making the right choices could even be better than their mom’s, considering that the conclusions in their case will have been drawn from data provided by two consecutive generations. We can only hope that by that time they will have better options than invasive surgery.Article 10 (2) of the Oviedo Convention states clearly that “[e]veryone is entitled to know any information collected about his or her health. However, the wishes of individuals not to be so informed shall be observed.”In connection with genetic research, there has emerged a growing demand for the establishment of genetic databanks operated in national or other institutional frameworks. These genetic databanks call for special regulations even if they are involved only in data storage and data management, or operate as tissue banks. Genetic tests provide specific challenges to the protection of private life .Twenty years ago, it was a widely shared opinion that anonym data could guarantee the highest level of data protection for genetic information. Later on, however, experts identified a wide array of ethical and legal problems. One of the issues is that fully anonymized genetic data cannot be compared with the health data of the specific patient and, consequently, the data are not very useful for scientific research. Anonymization is also problematic for the patients/research participants, since it would be impossible for them to receive feedback on their data provided, due to the anonymization itself.One difficulty that laws on biotechnological advances always face is how to balance between legal and scientific definitions of certain terms. While science may develop comfortably without laying down strict legal definitions of the terms “biobank” or “genetic data,” lawmaking requires these definitions to be exact and understood unequivocally, even across the related legal instruments. This, however, brings about the problem of inflexibility: as biotechnological inventions develop, such as in the fields of cloning or stem cell research, new discoveries and technologies might not fit the old definitions.monogenic disorders (caused by the defect or deficiency of a single gene). The accuracy of and potential for using these data are highly variable. Information on any hereditary disease has a serious impact on the lives and decisions of family members who may not have wanted to subject themselves to test examinations.Genetic information has brought about specific legal problems in data processing, as well as in data protection . The scoGenetic data have their implications on lifestyles, routines, selection of spouses, decisions to have children, career preferences, and even the learning ambitions of people. Whoever decides to become acquainted with these parameters has the opportunity to become informed within the limits that the health-care system can offer. In legal terms, the question is rather how the right of disposal over genetic data can be preserved .The main argument behind genetic exceptionalism is that personal genetic data should be processed under special legal guarantees, as genetic data can be considered not only as a simple health-care dataA key condition of the legislative act on data protection is that any data processing should be bound to a specific purpose. Thus, personal data may be processed only for stated purposes, toward the proper exercise of rights and fulfillment of obligations. Data processing should comply with the given purpose in all its phases. Furthermore, only such personal data can be managed that are essential to accomplish the given objective of data management, suitable for the purpose stated, but only to the extent and for the duration actually required for the accomplishment of the underlying objective. So in strict interpretation medical data—not to mention genetic information—generated for medical purposes and—for instance—forming parts of a given documentation, cannot be disclosed in their original forms to insurance companies, but may be revealed only to the extent they are absolutely necessary for the insurance activities.Many distinguished social scientists, even bioethiciststabula rasa and eager to seek specific law that governs their situation so that they do not have to worry about often vague legal interpretations of general legal norms on data protection.Legal scholars are also hesitant to jump to enact special legal provisions to genetic data . UsuallyIt follows from the spirit of data protection norms that even rules on biomedical research have to be developed in order to respond to different kinds of uses of genetic data. For instance, consent for collection and for specific use of genetic data is a precondition for the research. But then legal policy should differentiate whether the research material still carries personal information or not. When human tissue samples are anonymized in the sense that it is no longer possible to trace back the identity of the person from whom these tissues have been harvested then legal concerns are minimized to governing the tissue management.S. and Marper,“[…] the Court finds that the blanket and indiscriminate nature of the powers of retention of the fingerprints, cellular samples and DNA profiles of persons suspected but not convicted of offenses, as applied in the case of the present applicants, fails to strike a fair balance between the competing public and private interests and that the respondent State has overstepped any acceptable margin of appreciation in this regard. Accordingly, the retention at issue constitutes a disproportionate interference with the applicants’ right to respect for private life and cannot be regarded as necessary in a democratic society.”In the case of Although this case was dealing with the genetic data in the forensic context, nevertheless, its legal reasoning is an important step in the legal thinking on genetic data as this Court decision realized that not only genetic data but also genetic samples should be protected under privacy and data protection norms.Personalized medicine and DTC genetic testing would demand a more active role from the patient in health care. When patients are informed about genetic tests and treatment from the web, subsequent information, provided by the health-care professionals should be different than before. On the one hand, doctors may save some time by leaving the explanation of basic health information to the patient’s internet search, in the same time, information found on the web may have been better explained. Doctor and patient may become really good partners in health care, but when information found on the web is misleading or the doctor disagrees with the patients’ choices then an additional attempt has to be made by providing arguments for the test or treatment that he finds medically appropriate. With genetic consultation and by vigorous monitoring of genetic tests, there would be lots of advantages to this new model. Patients would be more conscious of their chosen life style and would be better educated in general about various genetic and non-genetic components of their illness. Nudges might be still necessary in many fields. Doctors should express their doubts when patients insist to a test that is medically not necessary or not appropriate. Medical indifference can be harmful just as much as total ignorance of the patients’ choices or preoccupation with certain familiar or genetic risks. Personalized health care may require new ethical and legal norms to ensure a better communication between the doctor and the patient and a better quality of health care. Furthermore, public and private genetic services should be harmonized in terms of quality and scope.In recent decades, legal rules of the informed consent have tended to follow the individual information model to a growing extent: it means that information is provided only to the patient provided that s/he is capable to act, while family members may be informed only if the patient expressly consents so. It seems, however, that genetic information will potentially re-establish the role of information provision to the family . In the Munro v. Regents of University of California case,Safer v. Pack case,Pate v. ThrelkelIn the In case of genetics, the individual consent model is also often challenged . It follUnited Nations issued declarations. UNESCO has a priority in standards settings in the field of bioethics within the UN. UNESCO adopted three major declarations, two of them had specific content in the field of genetics: the Universal Declaration on the Human Genome and Human RightsIn the field of biomedicine, several special organizations within the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and BiomedicineAdditional Protocol on the Prohibition of Cloning Human BeingsAdditional Protocol concerning Transplantation of Organs and Tissues of Human OriginAdditional Protocol concerning Biomedical ResearchAdditional Protocol concerning Genetic Testing for Health PurposesThe Council of Europe played a very important role in adopting key international norms in the field of biomedicine. The most important one is the While general data protection is provided in the field of protecting genetic data, the most specific legal instrument in Europe was adopted in 2006: the Recommendation Rec(2006) 4 of the Committee of Ministers to Member States on Research on Biological Materials of Human Origins. The extension of the language of human rights to the field of human tissues is clear in the stated objective of the Recommendation. As such, Article 11 of the Recommendation harmonizes the rules on the research on human beings with the rules on human tissues. “An intervention should only be carried out to obtain biological materials for storage for research purposes if it complies with the Additional Protocol concerning biomedical research ….”The Additional Protocol to the Convention on Human Rights and Biomedicine concerning Genetic Testing for Health PurposesSimilarly to the Oviedo Convention, its Protocol also emphasizes the primacy of the human being. “The interests and welfare of the human being concerned by genetic tests covered by this Protocol shall prevail over the sole interest of society or science.”One of the most interesting provisions of the Protocol can be found in Article 7, which prescribes “individualized supervision” by stating that “a genetic test for health purposes may only be performed under individualized medical supervision.”As to the European Union law, the Charter of Fundamental Rights of the European Union of 2000 should be mentioned. The importance of the Charter cannot be underestimated, it is the Bill of Rights of the European Union, and it lists fundamental rights shared by European Union member States. In the field of bioethics, especially Article 1 on human dignity, and more specifically, Article 3 on the right to the integrity of the person are of great relevance. As to the sources of secondary legislation, the following documents are relevant: regulation (EC) No 45/2001 of the European Parliament and of the Council of 18 December 2000 on the protection of individuals with regard to the processing of personal data by the Community institutions and bodies and on the free movement of such data, the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions, and finally, Directive 95/46/EC of the European Parliament and the European Commission on the protection of individuals with regard to the processing of personal data and on the free movement of such data.Finally, one should also mention the opinions and statements of the European Group on Ethics in Science and New Technologies (EGE). It made highly authoritative texts in questions related to the ethical aspects of umbilical cord blood banking (Opinion No. 19.)In the field of the application of genetic knowledge, legal issues are mostly focused on the problem of how new genetic information affects our basic human relations, family ties, decisions over the reproduction, insurance, employment, and intellectual property. To be more precise, how should genetic information be qualified, what kinds of rights can be established on this knowledge, who should hold this knowledge, and who is to control this intrinsically individual, wide-ranging information that can easily be obtained by others? The fine-tuning and detailed scrutiny of our biological and genetic view on humans, as well as the mapping of the entire human genome, originate from a generally understandable scientific drive, and at first sight it produces mostly neutral information. Serious ethical and legal dilemmas arise especially when this knowledge is applied in a broader social context.Now, almost 15 years after, the landmark research on the Human Genome—perhaps in a less passionate manner than at the beginning of the twenty-first century—we can better see the use of various tests but we are facing a new dilemma, how to preserve the European solidarity based health care, but also how can we respect individual choices in genetic testing. As we have seen the legal framework is still sporadic, and although basic human rights and data protection aspects of the genetic testing are regulated the connection between public and private sectors and using genetic tests from one sector to another needs more reflection. In the future, hopefully not only celebrities can benefit from the advances of genetic testing and with a more nuanced interpretation of genetic test genetic information will no longer be interpreted as destiny.The complicated relation between private genetic services and public health genomics has many aspects. The more genetic test results will be available, the more we know about the treatment of the genetically linked diseases. As it follows, there are collective dimensions of the private tests as well, provided that there are scientific publications about the results and methods of testing and databanks follow the same rules. As it follows regulation in this field would guarantee not only patients; rights but also the validity and comparison of the data. In the field of genetics, there are many serious but rare conditions that would require an international co-operation both in terms of testing, as well as finding personalized care.All aspects of manuscript completed by corresponding author JS.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MD and the handling editor declared their shared affiliation.


Ensuring the availability of the broadest possible germplasm base for agriculture in the face of increasingly uncertain and variable patterns of biotic and abiotic change is fundamental for the world's future food supply. While ex situ conservation plays a major role in the conservation and availability of crop germplasm, it may be insufficient to ensure this. In situ conservation aims to maintain target species and the collective genotypes they represent under evolution. A major rationale for this view is based on the likelihood that continued exposure to changing selective forces will generate and favor new genetic variation and an increased likelihood that rare alleles that may be of value to future agriculture are maintained. However, the evidence that underpins this key rationale remains fragmented and has not been examined systematically, thereby decreasing the perceived value and support for in situ conservation for agriculture and food systems and limiting the conservation options available. This study reviews evidence regarding the likelihood and rate of evolutionary change in both biotic and abiotic traits for crops and their wild relatives, placing these processes in a realistic context in which smallholder farming operates and crop wild relatives continue to exist. It identifies areas of research that would contribute to a deeper understanding of these processes as the basis for making them more useful for future crop adaptation. The collection, maintenance, and classification of genetic resources of plants used in agriculture and forestry are vital processes underpinning the steady improvement of crop yields and humankind's ability to feed, clothe, and house an ever‐increasing global population. In response to these needs, protection of germplasm resources has received more or less international attention for the better part of a century . Most events (>95%) showed no change in specificity; of the remainder, only four events were explained by the appearance of unaccountable novel specificities. The occurrence of these novel specificities aligns well with extensive occurrence of resistance gene analogs in a diversity of plants  genes, the majority of which are characterized by a consistent nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) motif  or slow the rate of spread within the plant . Because many of these traits are continuous in their response, small changes in resistance are often difficult to detect Burdon, . Further2.4Linum marginale L.; Thrall et al., There are very few studies that provide direct evidence of temporal change in the resistance structure of wild populations even though this may occur with surprising rapidity  each of small phenotypic effect and in which genotype‐by‐environment effects are often very strong R.Br.) in the Sahel to recurrent drought over the last quarter of the 20th century, no major changes were detected in the main cultivated varieties. However, common garden comparisons of landraces collected at the same locations 27 years apart found significant shifts in adaptive traits—reductions in plant size, spike length, shorter life cycles, and an increase in the frequency of a flowering gene known to affect development  landraces of mainstream agricultural crops, and underutilized and neglected crops, as well as (ii) wild crop relatives and forest tree resources. While these two categories have a number of issues in common, in reality there are also significant differences. In situ conservation of wild relatives and forest tree resources focuses on responding to the drivers and pressures that threaten the natural populations so as to maintain the genetic diversity and geographic range of species, thereby maximizing their potential to respond to natural or human‐made environmental change. In contrast, in situ conservation of landraces of mainstream agricultural crops and of underutilized and neglected crops represents a more complex selection environment where the impact of response to naturally occurring selective forces is overlain with conscious selection by farmers, with deliberate movement and incorporation of germplasm from close and more distant sources (including both more advanced cultivars and wild relatives), and with a range of cultural practices.Solanum tuberosum L.) in Peru  in China  Moench) in Cameroon  in Guyana  by scientists at the French National Institute for Agricultural Research (INRA) under an approach they call dynamic management (DM) of crop diversity. For a review summarizing their results see Enjalbert et al. , and institutions , as well as more concerted and systematic efforts that build on the best available biological and social sciences. This may require the creation of mechanisms to monitor the status and trends of crop diversity, adaptation, and evolutionary processes, based on methodologies and mechanisms to target where and with whom to carry out the monitoring and how to identify useful variation  are wild species living and evolving in natural, semi‐wild, and/or human‐made habitats where their genetic diversity is affected by a wide range of factors including habitat fragmentation and degradation  in southwest Mexico  D. A. Webb; Delplancke et al., Hordeum vulgare subsp. vulgare), significantly higher levels of diversity are encountered in wild, compared to cultivated forms  and its wild relatives has been so intense that there is no evidence of a genetic bottleneck in the cultivated form  Cheesman), gene flow from the wild population to the crop occurs through the regular incorporation of seedlings within cultivated plots  J. Miege) is regularly re‐domesticated as farmers collect, test, and select plants from neighboring natural populations  Buxb. is also undergoing frequent wild‐to‐crop introgression through the regular incorporation of cuttings collected in the wild  may become increasingly important, within‐species evolution of more complex traits  is still well beyond the horizon. For less well‐collected species such as many crop wild relatives and the large numbers of neglected and underutilized species that have little or no representation in ex situ collections, these concerns are magnified many times.In situ conservation on‐farm remains a vital part of ensuring germplasm availability for use by future generations. Evolution in these highly important situations is determined by a complex of interactions between crop, environment, and humans at a range of spatial scales. Social factors involving the full gamut of interactions from relationships between adjacent and more far‐flung communities, to taste preferences and traditional beliefs, ensure that farmers and landraces constitute a complex coevolving sociobiological system. There is strong circumstantial evidence that even without the added human dimension, evolution can lead to the de novo appearance of novel alleles or the selection of favorable gene complexes that adapt plants to changes in their biotic and abiotic environments.The added human component that is an integral part of in situ conservation on‐farm can drive evolution at an even faster pace through measures that lead to repeated introduction of additional genetic variation, while simultaneously enforcing tough selection pressures through active management of less desirable characteristics. Understanding the extent of this process and its impact on the genetic identity of landraces used in subsistence agriculture is a vital component in ensuring the maintenance of diversity into the future. Click here for additional data file. Click here for additional data file.


Pygospio elegans shows variation in developmental mode not only between populations, but also seasonally within populations. We investigated the consequences of this developmental polymorphism on the spatial and seasonal genetic structure of P. elegans at four sites in the Danish Isefjord‐Roskilde‐Fjord estuary at six time points, from March 2014 until February 2015. We found genetic differentiation between our sampling sites as well as seasonal differentiation at two of the sites. The seasonal genetic shift correlated with the appearance of new size cohorts in the populations. Additionally, we found that the genetic composition of reproductive individuals did not always reflect the genetic composition of the entire sample, indicating that variance in reproductive success among individuals is a likely explanation for the patterns of chaotic genetic patchiness observed during this and previous studies. The heterogeneous, unpredictable character of the estuary might maintain poecilogony in P. elegans as a bet‐hedging strategy in the Isefjord‐Roskilde‐Fjord complex in comparison with other sites where P. elegans are expected to be fixed to a certain mode of development.Poecilogonous species show variation in developmental mode, with larvae that differ both morphologically and ecologically. The spionid polychaete  Our aim was to determine whether genetic differences among individuals and cohorts are associated with the population dynamics we described in Thonig et al. , and primers were designed to amplify these loci using WebSat software .DNA was extracted from whole individuals using the Qiagen DNeasy Blood & Tissue Kit following the manufacturer's protocol for animal tissue. We developed two multiplex reactions to amplify ten microsatellite loci in 2.3FST,”, “Force mean FST,”, and “100,000” simulations. This test indicated that Pe385 might be subject to positive selection and Pe294 might be subject to balancing selection.To ensure the quality of the data, every allele that occurred only once in the data set was double‐checked and confirmed in the raw data. Individuals missing information for more than two loci were discarded. Three loci had more than 5% missing data  and were suspected to have null alleles. We used Micro‐Checker , gene diversity, and FIS averaged overall loci were calculated using Arlequin v.3.5.2  and the mean number of alleles for each locus showed a similar trend (data not shown). Thirdly, the model‐based clustering method implemented in Structure v.2.3.4  was performed in Arlequin v.3.5.2 , organic content, C/N, mean temperature (correlating significantly with standard deviation of temperature r = .905), mean salinity, and standard deviation of salinity. The Spearman rank correlation between the two matrices was calculated using RELATE, and the environmental parameters best explaining the observed genetic differentiation were determined via DistLM based on the Bayesian information criterion (BIC) using 9999 permutations.We compared the observed genetic structure at four time points  with the environmental parameters described previously  compared to the divergence of the third cluster from the other two . Analyzing every location separately resulted in a single cluster for both Herslev and Lynæs, whereas two clusters were the best solution for the samples at Lammefjord and Vellerup (graphs not shown). In both cases, the first cluster included the samples from March, May, November, and February, while the samples from August and October belonged to the second cluster.The cluster analysis in Structure revealed three genetic clusters when analyzing the whole metapopulation Figure . The firP. elegans, because we observed high and significant FIS values and P. elegans also is able to reproduce asexually. This program recommended two clusters as a best explanation for the data according to deviance information criterion (DIC). The program Flock determines genetic clusters by partitioning the sample and reallocating genotypes. Several runs starting with a different initial partitioning are performed for a different number of clusters. The number of genetic clusters k is reached when an identical final partitioning is obtained for more than six runs. For k = 2–9 cluster, no more than three identical partitions were obtained for our sample, indicating that either no population structure is present or our data do not contain enough information, that is, too few microsatellites, to infer the number of genetic clusters. Of the three methods used to estimate genetic clusters, the three‐cluster solution from Structure reflects the GST′ values best.Genetic clusters were also estimated with the program InStruct, which accounts for inbreeding and might be more suitable for 3.3G’ST suggests a genetic difference between cohort 2 and 3 (G’ST  = 0.0138). Most individuals in the cohorts at Herslev were assigned to genetic cluster 1 (68%), but some individuals in the size cohorts were assigned to genetic cluster 2 (23%) and genetic cluster 3 (9%). Likewise, at Lynæs, all four size cohorts are primarily composed of individuals assigned to a single genetic cluster: cluster 2 (67%). Nonetheless, individuals assigned to the other two genetic clusters also exist and vary in frequency. Note that 31% of the individuals of the third size cohort belong to genetic cluster 3, whereas individuals assigned to genetic cluster 3 make up only 7%–14% of the other size cohorts. A significant G’ST value indicates that cohort 3 differs from all other cohorts (G’ST ranging from 0.0124 to 0.0243). In contrast to relatively stable genetic composition of size cohorts in Herslev and Lynæs, Lammefjord shows a different pattern: the first and third size cohorts are dominated by individuals assigned to genetic cluster 2 (~70%), while the second size cohort is dominated by individuals assigned to genetic cluster 3 (80%). The genetic difference between size cohorts is evidenced by significant G’ST values between cohorts (G’ST ranging from 0.0694 to 0.0826). These patterns reflect the seasonal variation noted in the initial structure analysis  between genetic and environmental differences between samples. The environmental parameters best correlating with the genetic differentiation and explaining 74.02% of the genetic variation are median grain size, mean temperature, and mean salinity. These results are displayed in a distance‐based redundancy analysis (dbRDA) in Figure We observed a significant moderate correlation  among n et al. . Chaoticn et al. . One likn et al. . AlthougP. elegans alone  might explain the patterns of chaotic genetic patchiness we observed in the Isefjord‐Roskilde‐Fjord estuary complex. Heterogeneous environments can promote the evolution of phenotypic polymorphisms, depending on the accuracy of both genetic and environmental cues that influence development of the phenotype . In contrast, at Lynæs and Lammefjord, the sediment was fine and well‐sorted, and specimens were smaller, occurred in lower densities, and had higher percentage of asexual reproduction. These populations were dominated by the second genetic cluster (57% at Lynæs and 47% at Lammefjord). Salinity was only lower at Herslev (~15 PSU) compared to the other sites (~20 PSU). Herslev was also the only site where no switch in developmental mode and genetic composition of reproductive individuals was observed. Other environmental variables not measured here might be more effective in explaining the patterns of genetic differentiation we observed here.Diversifying selection is another mechanism that can cause CGP in unstable and patchy environments  can lead to emigration, while low‐quality habitats (sinks) with a demographic deficit might not persist without immigration, can go extinct, and be recolonized (Dias, P. elegans, the different population dynamics we observed suggest that Herslev and Vellerup would be characterized as source subpopulations, while Lynæs and Lammefjord would be sinks, according to criteria described by Jolly et al. (P. elegans as a bet‐hedging strategy in the Isefjord‐Roskilde‐Fjord estuary complex in comparison with other sites where P. elegans are expected to be fixed to a certain mode of development (Bolam, Zakas and Hall  proposeded Dias, . If suchy et al. . Howevery et al.  and Jolly et al.  proposedt Bolam, .5P. elegans in the Danish Isefjord‐Roskilde‐Fjord estuary complex, but seasonal genetic structure varied among the four study sites. When present, the seasonal genetic switch correlated with the arrival of new size cohorts. Phenotypic variation in larval developmental mode of P. elegans contributes to patterns of chaotic genetic patchiness observed in the estuary metapopulation. We found that the genotypes of individuals bearing gametes did not resemble the genotypes of the whole sample, indicating a possibility for variance in reproductive success. However, the genetics of larval cohorts and the effects of pre‐ and postlarval settlement on the population genetics are yet to be determined. Diversifying selection could lead to poecilogony in P. elegans as a bet‐hedging strategy to allow persistence in the unpredictable estuarine environment resulting in chaotic genetic patchiness among populations.We found spatial and seasonal population genetic structure of GU321899, GU321900, and GU321906. Genotype and phenotype data for Pygospio elegans analyzed in this study are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.9c5s0 .Microsatellite loci described in this study are available in GenBank under accession numbers MG021816 ‐ MG021822; None declared.AT carried out the field work, phenotyping, genotyping, and data analysis and drafted the manuscript. KEK optimized the molecular markers and participated in data analysis and preparation of the manuscript. KEK, GTB, and BWH designed and coordinated the study. All authors made contributions to the final manuscript and approval for publication. Click here for additional data file.


Beta vulgaris L. ssp. vulgaris) is one of the most important European crops for both food and sugar production. Crop improvement has been developed to enhance productivity, sugar content or other breeder's desirable traits. The introgression of traits from Crop Wild Relatives (CWR) has been done essentially for lessening biotic stresses constraints, namely using Beta and Patellifolia species which exhibit disease resistance characteristics. Several studies have addressed crop-to-wild gene flow, yet, for breeding programs genetic variability associated with agronomically important traits remains unexplored regarding abiotic factors. To accomplish such association from phenotype-to-genotype, screening for wild relatives occurring in habitats where selective pressures are in play  are the most appropriate streamline to identify causal genetic information. By selecting sugar beet CWR species based on genomic tools, rather than random variations, is a promising but still seldom explored route toward the development of improved crops. In this perspective, a viable streamline for sugar beet improvement is proposed through the use of different genomic tools by recurring to sugar beet CWRs and focusing on agronomic traits associated with abiotic stress tolerance. Overall, identification of genomic and epigenomic landscapes associated to adaptive ecotypes, along with the cytogenetic and habitat characterization of sugar beet CWR, will enable to identify potential hotspots for agrobiodiversity of sugar beet crop improvement toward abiotic stress tolerance.Sugar beet ( Beta vulgaris L. ssp. vulgaris, cultivar group Sugar Beet) is one of the most important crops, being within the 2013 top 10 world commodities, with Europe contributing with 68% production  was extensively used as a source of resistance gene   Frese, .The widespread use of genetically uniform crop varieties has caused agricultural crops to lose some of the genetic diversity present in their wild progenitors. CWR offer important sources of useful agronomic traits, including: intermediate C3-C4 photosynthetic activity; tolerance for cold, salt and drought conditions, and nutraceutical characteristics, i.e., plant-based compounds with health-protective roles.  and other endemic species within Beta (B. macrocarpa and B. patula), as well as Patellifolia . Considering the available yet unexplored wild germplasm from Beta and Patellifolia species occurring in this region, their potential for supplementing sugar beet breeding pool is easily recognized, mainly due to their occurrence in habitats of extreme conditions. Generally, crop yield reduction is a consequence of increasingly abiotic stresses  that holds some of the most important CWR of sugar beet, namely the sea beet  and Patellifolia (GP3) is postulated, and within GP1 and GP2 an ecological divergence between West and East Mediterranean Beta species was identified , allows a high-throughput sequencing approach of multiplexed samples that associates genome-wide molecular marker discovery and genotyping  C.F.Gaertn] occurring in distinct habitats , such as the use of different types of DNA probes, from repeated DNA sequences and BAC clones to microdissected chromosomes, pachytene spreads, extended DNA fibers, among others— confirmed these results, with P. patellaris being mainly tetraploid, while P. procumbens and P. webbiana being diploid. However, the cytogenetic diversity in certain regions/taxa was higher than expected: the ploidy of P. patellaris was variable with diploids being found in southeastern Spain and mainland Portugal. Also, in Tenerife, P. patellaris and P. procumbens co-occurred and seemed to cross and form a hybrid swarm, as supported by the occurrence of diploid, triploid and tetraploid plants and by the high morphological diversity. These results indicate that cryptic diversity and interspecific hybridization generates novel genetic variation within the genus, which benefits species survival as it may broaden the adaptive potential and also generate genetic variants of interest to plant breeding. The possible presence of cryptic diversity may also explain why the delineation of the three species is a challenge to genetic resources collectors and genebank curators.In the Buttler, , clearlyo et al.  revealedg Szota,  also quend Cueto  studied Beta-Patellifolia species complex (Castro et al., Considering the importance of CWR for supplementing crops gene pool, species conservation actions in geographical regions encompassing mixed-ploidy populations, as recently reported in the In conclusion, we presented how the application of genomic tools could help uncovering new traits in CWR and how such diversity can be disclosed using high-throughput methodologies to identify new genomic information for breeding application. Such innovative tools will provide crucial genetic/epigenetic/cytogenetic elements to breeding programs. From identification to breeding application is a challenging step and will likely benefit from the emergence of genomics-breeding approach that is still in its infancy.FM and MR conceived the manuscript. All the authors improved upon successive versions.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. The reviewer CB and handling Editor declared their shared affiliation.