Genetic Integrity Research Articles

Missed diagnosis or misdiagnosis: Common pitfalls in genetic testing.

Genetic testing has the power to identify individuals with increased predisposition to disease, allowing individuals the opportunity to make informed management, treatment and reproductive decisions. As genomic medicine continues to be integrated into aspects of everyday patient care and the indications for genetic testing continue to expand, genetic services are increasingly being offered by non-genetic clinicians. The current complexities of genetic testing highlight the need to support and ensure non-genetic professionals are adequately equipped with the knowledge and skills to provide services. We describe a series of misdiagnosed/mismanaged cases, highlighting the common pitfalls in genetic testing to identify the knowledge gaps and where education and support is needed. We highlight that education focusing on differential diagnoses, test selection and result interpretation is needed. Collaboration and communication between genetic and non-genetic clinicians and integration of genetic counsellors into different medical settings are important. This will minimise the risks and maximise the benefits of genetic testing, ensuring adverse outcomes are mitigated.

Rotation manipulation of single-molecule magnetic trapping and gene transcription regulation dynamics

Gene transcription regulation is a key step for gene expression in all organisms and responsible for the transmission of genetic information and genome integrity. As one of the most important mechanisms in gene transcription, an RNA polymerase (RNAP) specifically interacts with and unwinds genome DNA to form a transcription bubble where a nascent RNA transcript is polymerized, taking one of the unwound DNA strands as its template. The RNAP translocates along the DNA to transcribe the whole gene by carrying the transcription bubble. In such a way, an RNAP completes its biological task of gene expression by physically acting as a molecular machinery. Thus, an RNAP molecule can be considered as a research object for physicists who are willing to uncover the mechanisms of life processes in a physical view. To achieve this, single-molecule method has been invented and used widely. As one of these methods, single-molecule magnetic trapping manipulates biological molecules by applying extension force or torque to the magnetic beads tethered through biological molecule to pre-coated glass surfaces by manipulating the position or rotation of a pair of magnets. A linear DNA molecule can be manipulated in such a way to generate plectonemes, i.e. DNA supercoils, under an extension force of 0.3 pN (1 pN = 10<sup>–12</sup> N), possessing the feature that the number of unwound base pairs of a supercoiled DNA can be observed by the changes in the number of supercoils reflected by the DNA extension changes. Thus, the DNA unwound by RNAP, i.e. the transcription bubble, during transcription can be observed in this way. By monitoring the kinetics of the transcription bubble in real time, this method thus allows single-molecule detection with single-base resolution and a high-throughput data collection fashion in the kinetic studies of transcription. Owing to the advantages of the manipulation of DNA supercoils with single-molecule magnetic trapping, one can mimic the mechanistic feature of DNAs in vivo and characterize the kinetics of transcription under such conditions. This method can also be combined with single-molecule fluorescence method which can be applied to studying the mechanism of transcription regulation while monitoring the behaviors of fluorescently labeled biological molecules that interact with functional RNAP molecules, providing examples for studying the mechanisms of transcription regulations in more complex systems.

Retracted: Optimal Control of Whole Network Control System Using Improved Genetic Algorithm and Information Integrity Scale.

[This retracts the article DOI: 10.1155/2022/9897894.].

Stable dingo population structure and purity over 11 years of lethal management

Context Interaction between predators and humans is a key driver of human–wildlife conflicts, and can underpin management of predator populations. Management of the impacts of dingoes on livestock and native species is a prime example of a persistent and contentious predator management issue with potential impacts on the integrity of dingo populations. To manage the potential impacts of dingoes and their control, it is imperative to understand the effects of control approaches on their populations in the short and long term. Hybridisation of dingoes with domestic dogs also threatens the genetic integrity of pure dingoes. It has been hypothesised that lethal control of dingoes can facilitate hybridisation through disrupting pack social structures leading to increased dingo–domestic dog interactions. Aims We aimed to to investigate how dingo population structure and genetic purity have changed, assessing dingo purity, individual relatedness, population clustering and gene flow, particularly across land use types and barrier fences, in the context of ongoing lethal control within the Murchison Regional Vermin Cell area in Western Australia (WA). Methods We tested dingo genetic samples from three distinct sampling periods (2009, 2014 and 2020) for changes in population summary statistics and dingo ancestry. Barriers and corridors to gene flow were also examined. Key results We identified three genetically distinct populations in the study area, consistent with previous genetic studies in WA. We did not find any evidence of change in dingo purity or population characteristics; however, barrier fencing may be influencing recent gene flow. Conclusions The metapopulation of dingoes in the southern rangelands of WA appears to be stable over the 11 years assessed. Implications Because we were unable to demonstrate that lethal control has accelerated hybridisation between dingoes and domestic dogs in the study area over the last 11 years, we have no evidence that lethal control to reduce losses to livestock production and for conservation of native wildlife in the southern rangelands of WA is putting dingo purity at risk. Fencing appears to be an effective management tool because there is some evidence it is congruent with reduced gene flow in areas where the fences are well maintained.

Germination and viability of wild sunflower species seeds stored at room temperature and low humidity for 38 years

The effects of prolonged storage on the germination and viability of seeds of sunflower crop wild relatives stored under less-than-optimal conditions in working genebanks is not known. Seeds of two wild annual species, Helianthus annuus (common wild sunflower) and annual H. petiolaris (prairie sunflower), were stored at room temperature (20 to 22°C) and low humidity of ∼ 22% in the laboratory for 38 years. Germination of stored annual sunflower seeds was 5.5%, while that of prairie sunflower seeds was 0.75%, compared with the germination of freshly harvested seeds of 34.7 and 18.5%, respectively. Tetrazolium, a vital stain, indicated that the seeds were alive. The viability equations were used to predict final viability of the stored seeds. Using the three tests of viability (germination with and without gibberellic acid, and tetrazolium staining), all appeared to underestimate the actual condition of the stored seeds of annual sunflower, but provided better predictions for prairie sunflower. This study indicated that wild sunflower seeds can be stored at less than ideal conditions in working genebanks where viability decreases over time reducing germination, but gibberellic acid treatment can sustainably increase germination to produce a number of plants to maintain the genetic integrity of the original stored seeds often used in long-term prebreeding programmes.

CRISPR/Cas9-mediated generation of hESC lines with homozygote and heterozygote p.R331W mutation in CTBP1 to model HADDTS syndrome

C-terminal Binding Protein 1 (CTBP1) is a ubiquitously expressed transcriptional co-repressor and membrane trafficking regulator. A recurrent de novo c.991C>T mutation in CTBP1 leads to expression of p.R331W CTBP1 and causes hypotonia, ataxia, developmental delay, and tooth enamel defects syndrome (HADDTS), a rare early onset neurodevelopmental disorder. We generated hESCs lines with heterozygote and homozygote c.991C>T in CTBP1 using CRISPR/Cas9 genome editing and validated them for genetic integrity, off-target mutations, and pluripotency. They will be useful for investigation of HADDTS pathophysiology and for screening for potential therapeutics.

Preservation Methods in Isolates of Sporothrix Characterized by Polyphasic Approach.

Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by eight pathogenic species of the Sporothrix genus. Different ex situ preservation methods are used around the world to maintain the survival, morphophysiological and genetic traits of fungal strains isolated from patients with sporotrichosis for long terms. The main aim of the present study was to evaluate the survival, phenotypic and genotypic stability of Sporothrix strains after preservation on PDA slant stored at 4 °C, sterile water and cryopreservation at -80 °C, for a period of time of 6, 12, 18 and 24 months of storage. Eight clinical Sporothrix isolates were identified based on a polyphasic approach consisting of classical macro- and micro-morphological traits, biochemical assays, proteomic profiles by MALDI-TOF MS and molecular biology. According to the final identification, one strain was identified as S. schenckii (CMRVS 40428) and seven strains were re-identified as S. brasiliensis (CMRVS 40421, CMRVS 40423, CMRVS 40424, CMRVS 40425, CMRVS 40426, CMRVS 40427 and CMRVS 40433). In addition, it was observed that the isolates survived after the different time points of storage in distilled water, PDA slant and cryopreservation at -80 °C. For fungi preserved in water, low polymorphisms were detected by the partial sequencing of β-tubulin. Cryopreservation at -80 °C induced morphological changes in one single isolate. The proteomic profiles obtained by MALDI-TOF MS after preservation showed differences among the methods. In conclusion, preservation on agar slant stored at 4 °C was the most effective method to preserve the eight clinical Sporothrix strains. This method produced less change in the phenotypic traits and kept the genetic integrity of all strains. Agar slant stored at 4 °C is a simple and inexpensive method and can be especially used in culture collections of limited funding and resources.

Cryptic persistence and loss of local endemism in Lake Constance charr subject to anthropogenic disturbance.

In the welcome circumstance that species believed extinct are rediscovered, it is often the case that biological knowledge acquired before the presumed extinction is limited. Efforts to address these knowledge gaps, in particular to assess the taxonomic integrity and conservation status of such species, can be hampered by a lack of genetic data and scarcity of samples in museum collections. Here, we present a proof-of-concept case study based on a multidisciplinary data evaluation approach to tackle such problems. The approach was developed after the rediscovery, 40 years after its presumed extinction, of the enigmatic Lake Constance deep-water charr Salvelinus profundus. Targeted surveys led to the capture of further species and additional sympatric normal charr, Salvelinus cf. umbla. Since the lake had been subject to massive stocking in the past, an evaluation of the genetic integrity of both extant forms was called for in order to assess possible introgression. A two-step genomic approach was developed based on restriction site associated DNA (RAD). Diagnostic population genomic (single nucleotide polymorphism [SNP]) data were harvested from contemporary samples and used for RNA bait design to perform target capture in DNA libraries of archival scale material, enabling a comparison between extant and historic samples. Furthermore, life history traits and morphological data for both extant forms were gathered and compared with historical data from the past 60-120 years. While extant deep-water charr matched historical deep-water specimens in body shape, gill raker count, and growth rates, significant differences were discovered between historical and extant normal charr. These resulted were supported by genomic analyses of contemporary samples, revealing the two extant forms to be highly divergent. The results of population assignment tests suggest that the endemic deep-water charr persisted in Lake Constance during the eutrophic phase, but not one of the historical genomic samples could be assigned to the extant normal charr taxon. Stocking with non-endemic charr seems to be the most likely reason for these changes. This proof-of-concept study presents a multidisciplinary data evaluation approach that simultaneously tests population genomic integrity and addresses some of the conservation issues arising from rediscovery of a species characterized by limited data availability.

Haploid gynogens facilitate disomic marker development in paleotetraploid sturgeons.

Acipenseriformes (sturgeons and paddlefishes) are of substantial conservation concern, and development of genomic resources for these species is difficult due to past whole genome duplication. Development of disomic markers for polyploid organisms can be challenging due to difficulty in resolving alleles at a single locus from those among duplicated loci. In this study, we detail the development of disomic markers for the endangered pallid sturgeon (Scaphirhynchus albus) found in North America. One of the strategies for pallid sturgeon conservation is to stock U.S. rivers with offspring of pure pallid sturgeon, but introgression with the sympatric shovelnose sturgeon (S.platorynchus) threatens pallid sturgeon genetic integrity. Currently, 19 microsatellite loci are used to differentiate between both species and their hybrids, but the markers are insufficient to robustly identify backcrosses. We performed double digest restriction site-associated DNA sequencing (ddRADseq) on shovelnose sturgeon haploid gynogens to produce a reduced-representation genomic reference. Contiguous sequences that were heterozygous within a haploid individual were flagged as potentially encompassing multiple loci. Approximately 60 individuals of each species from two management units were sequenced, and reads were mapped to the haploid reference to identify single nucleotide polymorphisms (SNPs) at individual loci. The final data set contained 11,082 microhaplotyped loci which offer at least an order of magnitude greater resolution for species discrimination than the current panel of 19 microsatellites. These markers will be used to examine a larger sample of Scaphirhynchus individuals throughout their ranges to determine the extent and trajectory of hybridization.

Models for Predicting Response to Immunotherapy and Prognosis in Patients with Gastric Cancer: DNA Damage Response Genes.

DNA damage response (DDR) is a complex system that maintains genetic integrity and the stable replication and transmission of genetic material. m6A modifies DDR-related gene expression and affects the balance of DNA damage response in tumor cells. In this study, a risk model based on m6A-modified DDR-related gene was established to evaluate its role in patients with gastric cancer. We downloaded 639 DNA damage response genes from the Gene Set Enrichment Analysis (GSEA) database and constructed risk score models using typed differential genes. We used Kaplan-Meier curves and risk curves to verify the clinical relevance of the model, which was then validated with the univariate and multifactorial Cox analysis, ROC, C-index, and nomogram, and finally this model was used to evaluate the correlation of the risk score model with immune microenvironment, microsatellite instability (MSI), tumor mutational burden (TMB), and immune checkpoints. In this study, 337 samples in The Cancer Genome Atlas (TCGA) database were used as training set to construct a DDR-related gene model, and GSE84437 was used as external data set for verification. We found that the prognosis and immunotherapy effect of gastric cancer patients in the low-risk group were significantly better than those in the high-risk group. We screened eight DDR-related genes (ZBTB7A, POLQ, CHEK1, NPDC1, RAMP1, AXIN2, SFRP2, and APOD) to establish a risk model, which can predict the prognosis of gastric cancer patients and guide the clinical implementation of immunotherapy.

Using the Software DeepWings© to Classify Honey Bees across Europe through Wing Geometric Morphometrics.

DeepWings© is a software that uses machine learning to automatically classify honey bee subspecies by wing geometric morphometrics. Here, we tested the five subspecies classifier (A. m. carnica, Apis mellifera caucasia, A. m. iberiensis, Apis mellifera ligustica, and A. m. mellifera) of DeepWings© on 14,816 wing images with variable quality and acquired by different beekeepers and researchers. These images represented 2601 colonies from the native ranges of the M-lineage A. m. iberiensis and A. m. mellifera, and the C-lineage A. m. carnica. In the A. m. iberiensis range, 92.6% of the colonies matched this subspecies, with a high median probability (0.919). In the Azores, where the Iberian subspecies was historically introduced, a lower proportion (85.7%) and probability (0.842) were observed. In the A. m mellifera range, only 41.1 % of the colonies matched this subspecies, which is compatible with a history of C-derived introgression. Yet, these colonies were classified with the highest probability (0.994) of the three subspecies. In the A. m. carnica range, 88.3% of the colonies matched this subspecies, with a probability of 0.984. The association between wing and molecular markers, assessed for 1214 colonies from the M-lineage range, was highly significant but not strong (r = 0.31, p < 0.0001). The agreement between the markers was influenced by C-derived introgression, with the best results obtained for colonies with high genetic integrity. This study indicates the good performance of DeepWings© on a realistic wing image dataset.

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: The impact of oxidative stress on reproduction: a focus on gametogenesis and fertilization.

Many aspects of the reproductive process are impacted by oxidative stress. This article summarizes the chemical nature of reactive oxygen species and their role in both the physiological regulation of reproductive processes and the pathophysiology of infertility. This article lays out the fundamental principles of oxidative stress. It describes the nature of reactive oxygen species (ROS), the way in which these potentially toxic metabolites interact with cells and how they impact both cellular function and genetic integrity. The mechanisms by which ROS generation is enhanced to the point that the cells' antioxidant defence mechanisms are overwhelmed are also reviewed taking examples from both the male and female reproductive system, with a focus on gametogenesis and fertilization. The important role of external factors in exacerbating oxidative stress and impairing reproductive competence is also examined in terms of their ability to disrupt the physiological redox regulation of reproductive processes. Developing diagnostic and therapeutic strategies to cope with oxidative stress within the reproductive system will depend on the development of a deeper understanding of the nature, source, magnitude, and location of such stress in order to fashion personalized treatments that meet a given patient's clinical needs.

The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells.

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture invitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior invitro or invivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.

Verification of wild apple (Malus sylvestris) nursery stock sold in the United Kingdom

Societal Impact StatementCrop wild relatives are plant taxa that are the progenitors of commercial crops and are an important genetic resource for improving these crops and making them more resilient in the face of environmental change. They play an important role for food security and it is vital to maintain their natural genetic diversity. However, crop‐to‐wild gene flow may reduce the fitness and threaten the genetic integrity of the wild species in the long run. Here we investigate the genetic purity of European wild apple (Malus sylvestris), a rare tree species in Europe that frequently hybridises with domesticated apple (M. domestica), sold by UK nurseries.

Unexpected genetic integrity boosts hope for the conservation of the red-legged partridge (Alectoris rufa, Galliformes) in Italy

The red-legged partridge (Alectoris rufa) is a medium-sized galliform endemic to southwestern Europe. In the easternmost part of the species’ range, the population inhabiting Elba Island (Tuscan Archipelago National Park, Italy) is of undisputed conservation value. While we found nuclear and maternal DNA introgression with the exotic chukar partridge (A. chukar) in previous studies based on microsatellite DNA (n = 25) and two mitochondrial markers (n = 103), respectively, we disclosed a limited or null admixture in a few Elban partridges (n = 4) in a recent genomic investigation relying on 168,675 Single Nucleotide Polymorphisms (SNPs). We herein carried out an extended microsatellite DNA survey including additional 65 samples (total, 90) and six loci (total, 11) to determine both spatial structure and genetic integrity of local A. rufa. A sharp divergence between the subpopulations inhabiting the two sides of the island was disclosed, and the microsatellites indicated that all Elban partridges were not admixed with the chukar, thus fully reflecting the picture inferred using SNPs. We hypothesized that the spreading of chukar genes was constrained by negative selection, with the persistence of only the maternal lineage being indicative of thermal adaptation. The two subpopulations should be treated as distinct Management Units, and an envisaged plan to secure a stock onto nearby Pianosa Island could not only warrant endurance of the Elban population but also establish a source of valuable founders for the ex-situ management of the species in Italy. Our study exemplifies how a suitable samples/loci combination is the key to solve wildlife issues dealing with introgression.

Acute Myeloid Leukemia (AML) Risk Stratification Using the European Leukemianet 2022 Classification in a Real-Life Cohort of Patients from Sardinia, Italy

Introduction Acute Myeloid Leukemia (AML) identifies a clinically heterogeneous group of clonal disorders. Advances in Next Generation Sequencing (NGS) have increased the possibility to define the genetic lesions present in each patient, which has led to improved disease classification, risk stratification and identification of new therapeutic targets. The 2017 European LeukemiaNet (ELN) reccommendations for genetic risk stratification of AML have been widely adopted among physicians and investigators, even though patient responses remain heterogeneous. A new edition of reccomendations for diagnosis and management of AML in adults, ELN 2022, has been recently proposed to better define prognostic significance of genomic abnormalities. Aim of the study was to evaluate the usefulness of the new ELN 2022 genetic risk classification in a series of patients diagnosed with AML at two hematological institutions. Methods A retrospective analysis of 158 newly diagnosed AML patients according to WHO 2016 (100 de novo, 58 secondary) and treated from 2013 to 2021 in two hematological centers of Sardinia, Italy, was performed. Male patients were 96 (61%) and 63 out of 158 (40%) were >70 yrs old. One-hundred-five AML diagnosis samples were analyzed by targeted NGS on Ion Torrent platform using the DNA Oncomine Myeloid Research panel which explores 1256 hotspots in 33 key myeloid genes. Demographic and clinical data, disease characteristics at diagnosis, first-line treatment and clinical outcome data were available for all patients. Cytogenetic and molecular characteristics were used to classify patients into ELN 2017 and ELN 2022 risk groups. Results NGS detected 196 clinically revelant mutations in 87% AML patients with a limit of detection of 2%, while fusion genes were detected in 5% of patients (5/105). The most frequent genetic abnormalities were FLT3ITD/TKD and NPM1 mutations which represented the 29% and 28%, respectively. Co-occurrence of NPM1 and FLT3 driver gene mutations was found with a frequency of 16%. IDH1/2 mutations were observed in 20/105 pts (19%), RUNX1 and K/NRAS in 12/105 (11%), ASXL1 mutated in 11 pts (10%), STAG2 and BCOR mutations were present in 4 and 1 pts, respectively. TP53 mutations were observed with a frequency of 13% (14 patients). ELN 2017 classified 24 cases as favorable (23%), 40 as intermediate (38%), and 41 as adverse (39%) risk group. Sixteen out of 105 (15%) patients migrated to a more adverse risk group when ELN 2022 was applied: from favorable to intermediate (7) or adverse (2), 7 pts from intermediate to adverse. ELN 2022 re-classification assigned at favorable 15 pts (14%), 40 at intermediate ( 38%), and 50 at adverse risk groups (48%). In our cohort with the use of the new ELN 2022 classification no patient was reclassified with a better risk profile. Conclusions The recent ELN 2022 classification of AML with updated genetic integration has been proposed as a useful disease classification which impacts on the choice of treatment and outcome of individual patients. Our retrospective analysis confirms that the ELN 2022 is a robust tool of genetic risk classification and redefines a better genomic profile of AML patients prompting to individualized treatments. Re-classification according to ELN 2022 showed shifting from favorable to a worse category in the 10% of AML patients <60 years of age, who could have been eligible to a more intensive treatment. Furthermore, the implementation of the AML screening gene panel through NGS analysis allows to identify molecular abnormalities, such as IDH1/IDH2 mutations, susceptibles of target therapies.

A genotyping by sequencing approach can disclose Apis mellifera population genomic information contained in honey environmental DNA

Awareness has been raised over the last years on the genetic integrity of autochthonous honey bee subspecies. Genomic tools available in Apis mellifera can make it possible to measure this information by targeting individual honey bee DNA. Honey contains DNA traces from all organisms that contributed or were involved in its production steps, including the honey bees of the colony. In this study, we designed and tested a genotyping by sequencing (GBS) assay to analyse single nucleotide polymorphisms (SNPs) of A. mellifera nuclear genome using environmental DNA extracted from honey. A total of 121 SNPs (97 SNPs informative for honey bee subspecies identification and 24 SNPs associated with relevant traits of the colonies) were used in the assay to genotype honey DNA, which derives from thousands of honey bees. Results were integrated with information derived from previous studies and whole genome resequencing datasets. This GBS method is highly reliable in estimating honey bee SNP allele frequencies of the whole colony from which the honey derived. This assay can be used to identify the honey bee subspecies of the colony that produced the honey and, in turn, to authenticate the entomological origin of the honey.

Epigenetic priming as a mechanism of predetermination of spermatogonial stem cell fate.

In the developing mammalian testis, only a small proportion of fetal and neonatal prospermatogonia give rise to the foundational pool of spermatogonial stem cells (SSCs). Multiple lines of evidence have suggested the determination of which prospermatogonia give rise to foundational SSCs is not random, but is rather predetermined, such that foundational SSCs are ensured to develop advantageous characteristics such as enhanced genetic integrity. Here I suggest that differential epigenetic programing contributes to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs. This would include epigenetic programing that promotes active expression of genes needed to develop advantageous characteristics, as well as differential epigenetic priming, which bookmarks genes that comprise the SSC-specific transcriptome to become activated when foundational SSCs appear in the postnatal testis. I suggest that, together, differential epigenetic programing and epigenetic priming contribute to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs.

Low genetic diversity, local-scale structure, and distinct genetic integrity of Korean chum salmon (Oncorhynchus keta) at the species range margin suggest a priority for conservation efforts.

Chum salmon (Oncorhynchus keta) is an ecologically and economically important species widely distributed across the North Pacific Ocean. However, the population size of this fishery resource has declined globally. Identifying genetic integrity, diversity and structure, and phylogenetic relationships of wild populations of O.keta over an entire species' range is central for developing its effective conservation and management plans. Nevertheless, chum salmon from the Korean Peninsula, which are comprised of its southwestern range margins, have been overlooked. By using mtDNA control region and 10 microsatellite loci, we here assessed the genetic diversity and structure for 16 populations, including 10 wild and six hatchery populations, encompassing the species entire geographic range in South Korea. The analyses showed that genetic diversity is significantly higher for wild than for hatchery populations. Both marker sets revealed significant genetic differentiation between some local populations. Comparisons of six wild and their respective hatchery populations indicated that allele/haplotype frequencies considerably differ, perhaps due to a strong founder effect and/or homogenizing of hatchery populations for stocking practice. Despite its single admixed gene pool for the Korean chum salmon, some local populations housing their own unique lineages should be accorded with a high priority to safeguard their genetic integrities. The results of our comparative analyses of the Korean population with other North Pacific chum salmons (inhabiting regions of Japan, Russia, and North America) revealed a lower diversity but higher contribution to the overall species-level genetic diversity, and also its unique genetic integrity. These findings advocate for the evolutionary significance of the Korean population for species-level conservation.

Propagation of Calendula maritima Guss. (Asteraceae) through Biotechnological Techniques for Possible Usage in Phytotherapy

The genus Calendula (Asteraceae) includes several species that are renowned for their therapeutic properties and/or use as ingredients in the preparation of cosmetics. The rare and critically endangered sea marigold, Calendula maritima Guss., an endemic species from Western Sicily, has also been recognized as a potential “farm plant species” for several important compounds used in cosmetics. However, the few remnant populations of this species are currently threatened with extinction because of several factors, such as hybridization with the congeneric species Calendula suffruticosa subsp. fulgida (Raf.) Guadagno and anthropogenic disturbance of its habitat. Therefore, in order to preserve the genetic integrity from pure genetic lineages, seed-based propagation and seed storage are not recommended for either conservation or massive production purposes. In this paper, we describe a protocol adopted for mass propagation of C. maritima from selected genotypes. Nodal segments collected from selected plants growing in the field were used as starting explants and were cultured for micropropagation on MS medium with and without phloroglucinol. New shoots produced were cultured for rooting under several conditions with the aim of finding the best procedure favoring root induction. The best rooting performance was obtained with zeolite and rooted plants were successfully acclimatized outdoors. The technique described allowed the multiplication of genotypes of interest as well as to overcome the problems of hybridization of this species, hence contributing concretely to the conservation of the sea marigold.