In vivo, induction of peritoneal angiogenesis in sham rats, sham+PD rats, 5/6 nephrectomy (5/6Nx) rats, 5/6Nx+PD rats, and 5/6Nx+PD rats intraperitoneally treated with thalidomide showed that thalidomide was involved in the control of several key endothelial–specific targets, including VEGFR2, VEGFR3, SP4, and STAT3 expression and new vessel formation, confirming the role of thalidomide and STAT3/SP4 signaling in these processes. Taken together, these findings identify a novel mechanism that links thalidomide, STAT3/SP4 signaling, and angiogenesis in the peritoneal membrane.Peritoneal angiogenesis is the key pathophysiological factor that limits peritoneal ultrafiltration during peritoneal dialysis (PD) in uremic patients. Thalidomide has been confirmed to inhibit angiogenesis by inhibiting the secretion of vascular endothelial growth factor (VEGF), but the exact mechanism by which thalidomide inhibits vascular proliferation during PD is still unclear. Here, the objective of the present study was to investigate whether the reduction in VEGF production by human peritoneal mesothelial cells (HPMCs) was controlled by thalidomide. Stimulation of HPMCs with IL-6 in combination with soluble IL-6 receptor (sIL-6R) promoted VEGF expression and secretion, but these effects were attenuated by thalidomide treatment through a transcriptional mechanism that involved signal transducer and activator of transcription 3 (STAT3) and SP4. Conditioned medium from HPMCs cultured with thalidomide inhibited angiogenic endothelial tube formation, which could be further blocked by silencing SP4 and promoted by overexpressing SP4.  Peritoneal dialysis (PD) is an important component of replacement therapy for end-stage renal disease (ESRD), and PD provides the possibility of long-term survival for patients with uremia. However, over time, the withdrawal rate of patients gradually increases, mainly due to ultrafiltration failure UFF; . The incAngiogenesis is a structural change in the peritoneum that has attracted substantial attention in recent years . PatholoVascular endothelial growth factor (VEGF) is currently recognized as the main factor that mediates angiogenesis. VEGF can increase the permeability of blood vessels and promote the proliferation of vascular endothelial cells and angiogenesis . ScholarSignal transducer and activator of transcription 3 (STAT3) is the Janus kinase (JAK) substrate of the JAK-STAT3 pathway. STAT3 participates in many important biological processes in the human body, such as differentiation, proliferation, angiogenesis, and cellular apoptosis, and plays a very important role in the signal transduction of cytokines . StudiesThalidomide is clinically used as a drug to treat multiple myeloma and is known to have anti-angiogenic effects. via transcription factors STAT3/SP4. Inhibiting peritoneal angiogenesis could delay peritoneal dialysis membrane failure and prolong time on peritoneal dialysis. Thus, our study could provide new treatment options for patients with membrane failure and expand the range of therapeutic applications of thalidomide.Based on the results of previous studies and the discussion above, we speculate that thaildomide may inhibit VEGF induced peritoneal angiogenesis 2 (5%) atmosphere. The HPMCs were treated with 100ng/ml IL-6+sIL-6R for 24h and 1, 10, and 100μM thalidomide for 6, 12, or 24h prior to the subsequent assays. The final volume was 200μl, and the thalidomide control group was treated with DMSO alone.Human peritoneal mesothelial cells (HPMCs) were purchased from the American Type Culture Collection (ATCC) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum , 100U/ml penicillin, and 100mg/ml streptomycin (Thermo Fisher Scientific). The cultures were incubated at 37°C in a humidified COvia quantitative real-time PCR (qRT-PCR) 48h after transfection. Three distinct siRNAs against STAT3 or SP4 were designed and synthesized by GenePharma . The siRNA sequence against STAT3 was 5'-GCTCAACGAGTGCTTCATCAAGCTACCCA-3'. The siRNA sequence against SP4 was 5'-AAGTTGTAGTTGTTTGGAAATAT-3'.Human peritoneal mesothelial cells were transiently transfected with STAT3 siRNAs and SP4 siRNA after being cultured in six-well plates overnight. A mixed negative control, namely, an empty vector, was transfected by applying the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and FuGENE® HD Transfection Reagent , following the manufacturer’s instructions. The cells were then harvested to observe the knockout efficiency Total RNA was extracted from cells and tissues with TRIzol reagent as stated by the protocol. All the mRNA was subjected to quantitative polymerase reaction and reverse transcription according to the protocol for the PrimeScript® RT Master Mix Perfect Real Time  and SYBR Green Master Mix , respectively. The qPCR reaction was conducted on an Applied Biosystems 7900HT Real-Time System. The primer sequence information was showed in The collected cells were lysed in RIPA protein extraction reagent  supplied with a cocktail of protease inhibitors . The lysis products were loaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels for electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked in 5% milk prior to incubation with the indicated primary antibodies and secondary antibodies. Autoradiograms were quantified by densitometry, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. Antibodies against VEGFR2, SP4, and STAT3 were purchased from Abcam .The intracellular VEGF release was detected using the enzyme linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.4 cells per well) were seeded onto the Matrigel and cultured in DMEM with or without 10% (vol/vol) conditioned medium from HPMCs treated as described in the figures. The total length of the tube-like structures was measured 24h later under a microscope , and five randomly selected fields from each well were analyzed to determine the total capillary length using ImageJ 1.43 software.56.Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. For the tube formation assay, Matrigel  was added to a 96-well plate (50μl per well) and solidified at 37°C for 30min. HUVECs . All animals were housed in individual ventilated cages, provided sterilized water and food at libitum, and handled under specific pathogen-free conditions in the Institute’s animal care facilities, which meet international standards. Rats underwent 5/6 Nx or sham surgery under ketamine/xylazine anesthesia (100mg/kg). Briefly, the left kidney was exposed, and the upper and lower poles were tied with a polyglycolic acid suture line, followed by right nephrectomy. The peritoneum and skin were then sutured, and the animals were returned to their individual cages. One week later, the sham and 5/6 Nx rats were injected with 20ml of peritoneal dialysate (3.86% glucose) daily for 4weeks. And then the 5/6 Nx+PD rats were injected with thalidomide (200mg/kg/day). The control group was intraperitoneally injected with normal saline. The rats were euthanized at the designated time points , and specimens of peritoneal peritoneum were collected and snap frozen in liquid nitrogen.Paraffin-embedded blocks were cut into 4-μm-thick sections and dewaxed and hydrated. Then, the sections were immersed in distilled water containing 3% hydrogen peroxidase twice to reduce the endogenous oxidase activity. Next, the tissue sections were incubated with CD34 antibodies  for 2h at room temperature, and subsequently, a goat-anti-rabbit antibody was applied to the cells at room temperature for 40min. The degree of staining was developed by diaminobenzidine (DAB) chromogen . Subsequently, the tissues were dehydrated and sealed with gum. Five random fields of view were selected at 100× magnification with a camera using a microscope , and the mean of the microvessel counts was recorded as the microvessel density.t-test or one-way ANOVA. Values of p<0.05 were considered to be statistically significant.All the statistical analyses were executed using SPSS 22.0 software  and GraphPad Prism 5.0 . Differences between groups were analyzed utilizing Student’s **p<0.01). In addition, thalidomide treatment may attenuate the mRNA levels of SP4, STAT3, and VEGFR2 in a significant time- and dose-dependent manner . Furthermore, western blot analysis showed that SP4, STAT3, and VEGFR2 protein levels increased significantly under IL-6 plus sIL-6R treatment and decreased obviously after thalidomide supplementation in the IL-6 plus sIL-6R solution for 48h , but significantly increased in SP4 or STAT3 overexpressing group and decreased in SP4 or STAT3 siRNA group when compared to the scrambled controls , but significantly decreased in conditioned medium from SP4 OE+Thal or STAT3 OE+Thal groups compared to the corresponding OE groups , and further decreased in the medium with SP4 siRNA+Thal or STAT3 siRNA+Thal . These targets included +PD rats –E.*p<0.05; **p<0.01; To investigate the relationship between microvessel density and thalidomide treatment in the peritoneum, immunohistochemistry was performed to assess the positive CD34 signalling in the sham, sham+PD, 5/6Nx, 5/6Nx+PD, and 5/6Nx+PD+Thal rats –E. CD34-in vitro, the use of IL-6 plus sIL-6R significantly stimulated HPMCs to secrete VEGF. This result is consistent with the results of reported in vitro studies  to activate extracellular signal regulated kinases (ERK) and phosphorylate STAT3. The signal transduction pathway causes the expression of VEGF to be downregulated, and this inhibitory effect is obvious with the increase in the concentration of thalidomide, showing a dose-dependent change (via STAT3/SP4 signaling.Thalidomide is a synthetic glutamate derivative. At present, many studies have found that thalidomide plays an important role in immune regulation, anti-inflammatory activity, anti-angiogenesis activity, and collagen synthesis inhibition. Scholar t change . Our resin vivo. Our results show that thalidomide can significantly reduce the expression of VEGF, VEGFR2, VEGFR3, STAT3, and SP4 in the peritoneum of nephrectomy uremic rats after peritoneal dialysis and significantly inhibit peritoneal neovascularization. The decreased microvessel density in the thalidomide-treated 5/6Nx+PD rats suggested that suppressive angiogenesis might account for the long presence of peritoneum observed in the process of peritoneal dialysate following thalidomide administration. The results described above further indicate that thalidomide can be used as a drug to inhibit angiogenesis, inhibit the expression of VEGF in peritoneal tissue, and delay the progression of peritoneal neovascularization.Studies on human endothelial cells have also found that thalidomide can not only reduce the level of VEGF production but also inhibit VEGF-induced cell migration, capillary formation, and angiogenesis . The forVEGFR2, STAT3, and SP4 in HPMCs treated with IL-6 combined with sIL-6R. Moreover, angiogenic endothelial tube formation was inhibited by conditioned medium from HPMCs cultured with thalidomide, which could be further blocked by silencing SP4 or STAT3 and promoted by overexpressing SP4 or STAT3. In vivo, qRT-PCR analysis revealed that thalidomide attenuated the expression of several key endothelial-specific targets; including VEGFR2, VEGFR3, SP4, and STAT3, and immunohistochemistry showed that thalidomide inhibited the formation of new vessels in 5/6Nx+PD rats. Finally, these observations may help clarify the possible therapeutic actions of thalidomide in uremic patients with PD. In addition, SP1 as a transcription factor binding to the VEGF promoter region, also plays key roles on VEGF activity. Some scholars suggested that thalidomide inhibits the binding of the transcription factor SP1 to the promoter of the VEGF gene, which affects the production of VEGF and reduces the formation of blood vessels (Taken together, these results demonstrate that thalidomide treatment reduced the expression of  vessels . What isThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Ethics Committee of Shanghai General Hospital.in vitro experiments and wrote the manuscript. JJ and LG performed the RT-PCR and western blot experiments. HGo, XW, MY, and HGa helped with the writing and revision of the manuscript. WY conceptualized and supervised the entire project. All authors contributed to the article and approved the submitted version.NZ and LW performed all the 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The deep scoping method incorporates the use of a gene bank together with different population layers to reintroduce genetic variation into the breeding population, thus maximizing the long-term genetic gain without reducing the short-term genetic gain or increasing the total financial cost.Genomic prediction is often combined with truncation selection to identify superior parental individuals that can pass on favorable quantitative trait locus (QTL) alleles to their offspring. However, truncation selection reduces genetic variation within the breeding population, causing a premature convergence to a sub-optimal genetic value. In order to also increase genetic gain in the long term, different methods have been proposed that better preserve genetic variation. However, when the genetic variation of the breeding population has already been reduced as a result of prior intensive selection, even those methods will not be able to avert such premature convergence. Pre-breeding provides a solution for this problem by reintroducing genetic variation into the breeding population. Unfortunately, as pre-breeding often relies on a separate breeding population to increase the genetic value of wild specimens before introducing them in the elite population, it comes with an increased financial cost. In this paper, on the basis of a simulation study, we propose a new method that reintroduces genetic variation in the breeding population on a continuous basis without the need for a separate pre-breeding program or a larger population size. This way, we are able to introduce favorable QTL alleles into an elite population and maximize the genetic gain in the short as well as in the long term without increasing the financial cost.The online version contains supplementary material available at 10.1007/s00122-021-03932-w. Truncation selection is often used in genomic selection to rapidly increase the short-term genetic gain of a breeding population. By selecting individuals with the highest genomic estimated breeding values (GEBVs), breeders hope to maximally pass favorable properties to their offspring. The underlying idea is easy to understand and matches the gut feeling of most breeders, making it one of the most popular strategies in plant breeding. Unfortunately, truncation selection is also associated with a loss in genetic variation Jannink . Besidespopulation merit method , which not only takes into account the mean predicted genetic value of the offspring, but also the selection intensity, prediction accuracy and genetic variation of the offspring  scores individuals based on their haplotypes, and can therefore better preserve favorable QTL alleles in the breeding population, increasing the long-term genetic gain introduce alleles and haplotypes into the breeding population, resulting in an increase in the maximum reachable genetic value. A gene bank is an (inter)national collection of different plants ranging from wild specimens to different crop varieties at different stages of selection. To optimally reintroduce genetic variation into a breeding population and thus increase the genetic gain in the long term, the gene bank must show a broad genetic variation Simmonds . The inthaploid estimated breeding value (HEBV) and the UC to select and cross elite individuals with donor individuals. However, the calculation of the UC requires the construction of a covariance matrix, which considerably increases the computational requirements of the simulation while the bridging population can only reintroduce a fraction of the genetic variation into the breeding population. The parental selection can also be guided using genotyping-by-sequencing or related techniques, in which the relatedness of germplasm collections and elite individuals in the breeding population can be quantified and used to preserve the genetic variation in the breeding population , individuals from a gene bank are crossed with individuals of the elite population, reintroducing genetic variation in the breeding population. Next, different layers are added, allowing for a gradual flow of favorable QTL alleles from the first layer to the elite population. Over each layer, the genetic variation is exploited, increasing the genetic gain and maximizing the transition of pre-bred individuals into the elite population.We propose a new method that incorporates the use of a gene bank to reintroduce genetic variation into the breeding population, maximizing the long-term genetic gain without reducing the short-term genetic gain. By using a fraction of the breeding population for pre-breeding, the sizes of both the breeding population and the parental population remain unchanged, avoiding additional costs. This method, coined Hordeum vulgare) from the University of Minnesota (UMN) and the University of North Dakota (NDSU), counting, respectively, 384 and 380 six-row spring inbred lines with 1590 biallelic SNP loci. The same base population was also used by Vanavermaete et al. , containing individuals with high GEBVs, can be distinguished. The H-score E  and should thus be selected as a parent.In a breeding population, an elite subpopulation (denoted The deep scoping method combines truncation selection with the (re)introduction of (new) QTL alleles in the breeding population with the aim of maximizing both the short- and long-term genetic gain. To introduce new QTL alleles, a gene bank is used, containing a population with a high genetic variation, but lower mean genetic value. When individuals of the gene bank are introduced into the breeding population, their lower genetic value prevents them from being selected during truncation selection. This will create a gap between the genetic value of the elite individuals and the rest of the breeding population, isolating them from one another. Although both QTL alleles will still be present in the breeding population, the QTL alleles of the individuals in the elite population will still be fixed causing a premature convergence of the genetic value. Therefore, a three-step selection procedure was designed to not only introduce QTL alleles into the breeding population but also in the elite population. To do so, the breeding population is divided into two subpopulations: the elite population and the pre-breeding population see Fig. . IndividThe breeding population consists of an elite (sub)population containing 500 individuals and a pre-breeding (sub)population containing five different layers with each 100 individuals. In order to create the elite population, 50 individuals with the highest GEBVs are selected. In contrast to truncation selection, the parents are not crossed at random. The individual with the highest GEBV is selected as the P1 parent and is coupled with a P2 parent that minimizes the genetic relationship between both parents. Other crossing block designs have been considered as well, such as crossing the two individuals with the highest GEBVs with each other or crossing the top-50 individuals with the top 51–100 individuals, but both designs resulted in a significantly lower long-term genetic gain.n selected individuals and k different markers coded as −1, 0, or 1 and The pre-breeding population tries to introduce favorable marker alleles into the breeding population and ultimately in the elite population. To select the first parents for Layer 0, the HEBVs for the individuals of the gene bank are calculated. Next, the H-score is calculated for each individual of the gene bank. The five individuals with the highest H-score and thus containing the most favorable haplotype segments are selected as P1 parents. The five P2 parents are selected from the elite population to maximize the genetic value of the offspring. To maximize the genetic variation of the offspring, the scoping method is used instead of truncation selection. The scoping method has been proposed by the present authors . Once the gene bank is available, the newly introduced marker alleles lead to an increase in the maximum reachable genetic value see Fig. . In the In Fig. The HUC method with bridging combines two different parental selection schemes. On the one hand, elite individuals with the highest GEBVs are selected and crossed with each other, maximizing the short-term genetic gain. On the other hand, individuals of the gene bank are crossed with elite individuals to introduce QTL alleles into the breeding population. By crossing the individuals of the gene bank with an elite individual, the GEBVs of the offspring increase such that they can be selected as an elite parent in the next breeding cycle, maximizing the long-term genetic gain. However, when the GEBVs of the breeding population increase, the gap between the genetic value of the elite individuals and the gene bank increases as well, impeding the transition of pre-breeding individuals into the elite population and thus resulting in the convergence to a sub-optimal genetic value. Increasing the number of initial breeding cycles using truncation selection  increases the gap between the genetic value of the breeding population and the gene bank, resulting in the convergence to even lower genetic values see Fig. . The meaThe deep scoping method has been tested in different simulation settings. Each experiment consists of testing 100 different genomes such that the effects of using the deep scoping method or the HUC method with bridging can be studied using different QTL and marker positions. The effect of the heritability and of the number of QTLs on the genetic gain of both methods have also been tested and are shown in Fig. The primary goal of the deep scoping method is to preserve newly introduced marker alleles in the breeding population by allowing for a gradual flow of genetic material from the pre-breeding population into the elite population. To achieve this, the scoping method has been redesigned. In the original scoping method, the first parent was greedily selected to prioritize the genetic progress, while the second parent was selected by maximizing the S-score between both parents, preserving the genetic variation of the breeding population. In the deep scoping method, the individuals with the highest H-scores are selected as P1 parents, prioritizing the preservation of favorable marker alleles in the breeding population. Similar to the scoping method, the second parent is selected by maximizing the S-score between both parents. Since both the H-score and the S-score quantify genetic variation, the second parent is taken from a pre-selected population that contains individuals with the highest GEBVs. This way, genetic progress is maximized as well, facilitating the transition of pre-breeding individuals into the elite population. Moreover, by using the S-score, both alleles of each marker will be preserved in the breeding population to the extent possible, thus minimizing the loss of (favorable) QTL alleles  as truncation selection, the deep scoping method reintroduces genetic variation into the breeding population and maximizes the genetic gain thereof. Only the gene bank could be seen as an additional investment. The gene bank contains 500 individuals that are created by crossing individuals of both the UMN and the NDSU dataset by maximizing the genetic variation between both crosses. This means that the individuals of the gene bank will have different heterozygous markers and low genetic values. This way, the ability to include low-GEBV individuals of both the deep scoping method and the HUC method with bridging was studied. Including more exotic germplasm could require more layers, whereas the inclusion of individuals with a higher genetic value could also be done with a lower number of layers.In genomic selection, GEBVs are often used to select the parental population. To do so, a mixed effect model is used to predict the marker effects, which are used to calculate the GEBVs according to Eq. . The genA parental selection based on GEBVs could also lead to the selection of closely related individuals, fixating several favorable and unfavorable QTL alleles in the breeding population. This could be avoided by penalizing the GEBVs to minimize the rate of inbreeding and reducing the loss in genetic variation  QTL alleles into the elite population. In the HUC method with bridging, the selected individuals of the gene bank only have one breeding cycle to be selected as elite individuals. Therefore, when the difference in genetic value between the gene bank and the breeding population increases, the transition of pre-breeding individuals into the elite population degrades, causing a premature convergence of the genetic value.Although the deep scoping method results in a 4–6% points higher long-term genetic gain compared with the HUC method with bridging, in the short term, a slightly lower genetic value is observed see Fig. . The eliThe deep scoping method uses different layers to guide the genetic progress of the pre-breeding individuals and to facilitate their transition into the elite population. Each layer selects parents from the previous layer and couples them with an individual of the elite population, maximizing the genetic gain of the offspring. Prior to the deep scoping method, truncation selection is used to simulate a realistic breeding population. When the deep scoping method is introduced in the breeding program, an additional breeding cycle per layer will be required before the deep scoping method becomes fully operational. Once that is done, the number of layers represents the number of breeding cycles a pre-breeding individual is allowed to be crossed with an elite individual to increase the genetic value of their offspring and thus to pass on their genotypic information into the elite population. Individuals that are not selected in Layer 4 will be eliminated from the breeding population.Figure In Fig. Regardless of the fact whether the breeding population underwent 5, 10, 15, or 20 breeding cycles of truncation selection, when the deep scoping method is used, for each layer, a similar flow of individuals to the elite population is observed see Fig. . When thEven compared with the scoping method, using the deep scoping method after five breeding cycles of truncation selection will result in a 3% points higher genetic value in the long term (see Table S1)  QTL alleles into the elite population. Replacing the frequently used GEBVs by HEBVs allows for a more accurate selection of individuals containing favorable QTL alleles, maximizing the long-term genetic gain. In summary, the deep scoping method combines an elite population with different layers using the HEBVs, H-score, and S-score, thus maximizing the genetic gain in the short as well as in the long term without increasing the financial cost of the breeding program.Supplementary file1 (PDF 68 kb)Below is the link to the electronic supplementary material.


The traditional folk classification of Kam fragrant glutinous rice by the Dong community is consistent with the biological classification. The traditional naming of Kam fragrant glutinous rice provides an important reference for understanding its genetic diversity. The high level of genetic diversity in Kam fragrant glutinous rice is not only related to the natural environment of the area but also tightly linked with the abundant and diverse Dong ethnic traditional cultures, which has led to protection of Kam fragrant glutinous rice’s genetic diversity.The genetic diversity of rice germplasm is the basis for increases in rice yield and quality. The collection, assessment, and protection of the genetic diversity of rice germplasm is important for achieving sustainable agriculture and assuring food security. Many underdeveloped indigenous areas have abundant and valuable rice germplasm resources. However, in-depth assessments of the genetic diversity of rice germplasm from these areas and studies related to protecting these traditional cultures are not available. In this study, from 2005 to 2016, the authors have conducted in-depth evaluation of the genetic diversity of Kam fragrant glutinous rice germplasm resources in southeast Guizhou by using multidisciplinary comprehensive methods such as ethnobotany, cultural anthropology, and modern molecular markers. In total, 376 Kam fragrant glutinous rice samples from 42 villages in the Dong community in southeast Guizhou were collected. Agronomic traits of panicles were complex and exhibited diversity. Some varieties had good disease resistance and adaptation to cold and wet climates. The Dong people named the Kam fragrant glutinous rice varieties by using seven elements, including diverse traits, growth environment, and origin. Traditional folk classification, in addition to morphology and biological analysis using molecular markers, indicates that Kam fragrant glutinous rice includes 91 varieties. Kam fragrant glutinous rice comprises a very high number of varieties, most of which are  Oryza sativa L.) is cultivated in over 100 countries and provides food for millions of people 34] using2O (14.8 µL), dNTPs (0.4 µL), buffer (2 µL), F (0.3 µL of 20 µM primer solution), R (0.3 µL of 20 µM primer solution), DNA template (2 µL), and Taq (0.2 µL). The following amplification program was used: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 35 s, and extension at 72 °C for 40 s; and a final extension at 72 °C for 3 min.PCR amplification: The SSR fluorescence-labeled primer system  consisted of ddHV/V), and the 9-µL mixture was loaded into the gel. Next, a 10× dilution of 1 µL of PCR product was added. A 3730XL DNA analyzer  was used for capillary electrophoresis. The fragment (plant) analysis in the Genemarker software was used to analyze the primary data. By comparing the location of the size standard in each lane with the location of the peak in each sample, fragment sizes were obtained.Capillary electrophoresis: Formamide and a size standard were mixed at a ratio of 100:1 (Data analysis: According to the format requirement by Convert 1.31 software , data we


Solanum tuberosum L.) is an important staple food and economic crop in many countries. It is of critical importance to understand the genetic diversity and population structure for effective collection, conservation, and utilization of potato germplasm. Thus, the objective of the present study was to investigate the genetic diversity and population structure of potato germplasm conserved in the National Agrobiodiversity Center (NAC) of South Korea to provide basic data for future preservation and breeding of potato genetic resources. A total of 24 simple sequence repeat (SSR) markers were used to assess the genetic diversity and population structure of 482 potato accessions. A total of 257 alleles were detected, with an average of 10.71 alleles per locus. Analysis of molecular variance showed that 97% of allelic diversity was attributed to individual accessions within the population, while only 3% was distributed among populations. Results of genetic structure analysis based on STRUCTURE and discriminant analysis of principal components revealed that 482 potato accessions could be divided into two main subpopulations. Accessions of subpopulation 1 mainly belonged to cultivars and breeding lines. Accessions of subpopulations 2 basically corresponded to wild relatives of potatoes. Results of this study provide useful information for potato improvement and conservation programs, although further studies are needed for a more accurate evaluation of genetic diversity and phenotypic traits of potatoes.Potato ( Solanum tuberosum) is one of the most important tuber crops. It can be used as raw material for starch and alcohol production . Th. Th63]. software . Analysisoftware . The popsoftware ,70 accorsoftware . Bayesiasoftware , testingsoftware . The mossoftware . A membewordcloud package for R software to determine the frequency of potato use. Using results of word cloud analysis, network analysis was performed using tidygraph package for software to identify the relationship among potato accessions.Word cloud analysis was performed using


Genetic testing is associated with many ethical challenges on the individual, organizational and macro level of health care systems. The provision of genetic testing for rare diseases in particular requires a full understanding of the complexity and multiplicity of related ethical aspects. This systematic review presents a detailed overview of ethical aspects relevant to genetic testing for rare diseases as discussed in the literature. The electronic databases Pubmed, Science Direct and Web of Science were searched, resulting in 55 relevant publications. From the latter, a total of 93 different ethical aspects were identified. These ethical aspects were structured into three main categories  and 20 subcategories highlighting the diversity and complexity of ethical aspects relevant to genetic testing for rare diseases. This review can serve as a starting point for the further in-depth investigation of particular ethical issues, the education of healthcare professionals regarding this matter and for informing international policy development on genetic testing for rare diseases. Geneticividuals . This noividuals .A precise molecular diagnosis is essential for the efficient handling of rare diseases in order to provide disease management and treatment options. In addition, it enables informed future family planning decisions and the formation of supportive networks of individuals and families affected by rare diseases . Early aAdvances in genetic testing, especially next generation sequencing technologies (NGS), have positively impacted the likelihood of obtaining a genetic diagnosis in a timely manner . HoweverThe rapid technological advancements in genetics and the lack of education in this field limit the ability of many nonspecialized physicians to partake in the much needed professional discussion of ethical issues in genetic testing . The widThis review is the first, to the best of the authors’ knowledge, to present a profound overview of all ethical aspects of genetic testing for rare diseases as published in the literature. In systematizing ethical problems related to this field this review can assist researches in the field of genetics as well as clinicians and counsellors in enhancing the moral sensibility for issues pertinent to their professional practice. For example, this review systematically gives a list of ethical issues occurring at the micro-level of patient-provider-contact and enables a further in-depth literature analysis of moral problems relevant for the individual reader. It furthermore provides a systematic basis for the ethical education of not only healthcare professionals but also patients, their families and other relevant stakeholders. This review provides a systematic background for further empirical and normative investigations of ethical aspects and is meant as a comprehensive aid to health policy making.This article provides an overview of the full spectrum of ethical aspects in genetic testing for rare diseases based on a systematic review of the literature closely following the methodology used by Strech et al. . The repThe electronic databases Pubmed, Science Direct and Web of Science were searched  . Publica“Ethical aspects” were identified on the basis of the ethical theory of principlism, according to Beauchamp and Childress . This ap“Genetic testing” is defined as an laboratory examination aimed at detecting or ruling out the presence of hereditary illnesses or predisposition to such conditions in a person by directly or indirectly analyzing their genetic heritage  .Up until the completion of this article, no definite set of criteria had been established on how to conduct a quality appraisal for reviews of ethical literature . ConsequNo restriction was applied to the type of publications included in this review. Therefore, not only original articles but also comments, editorials and book chapters were included. In order to display the full spectrum of ethical aspects relevant to the review question, not only argument-based but also empirical literature  was included  Process of testing: Ethical aspects concerning the procedure of genetic testing for rare diseases, the analysis of these tests and the delivery of the results to the patient and/or the family.2) Consequences of the test outcome: Ethical aspects that result from the knowledge of the test result or the decisions made following the disclosure and patient reactions to the test result.3) Contextual challenges: Ethical aspects that are associated with the circumstances and background of the tests, the diseases tested for and the test results.The following three main categories were established: process of testing encompasses 36 ethical aspects in nine subcategories  which they did not consent to but if not disclosed might have an interest to know. This ambiguous situation needs to be extensively addressed and prepared for during counselling:“Ethical challenges are generated when information produced by the results may affect third parties, including family members not directly involved in the process.” ontextual challenges includes 21 ethical aspects in four subcategories . Prior to the final search, an exploratory search of several databases was conducted and the three databases subsequently accessed were identified as delivering the highest number of relevant results. Additionally, the neglect of literature written in languages other than English or German limits this review.No quality appraisal for the included literature was performed due to the lack of quality assessment tools for systematic reviews of ethical literature . TherefoA lack of knowledge and comprehension of the fast-paced developments of genetic testing among professionals poses an obstacle to accessing comprehensive testing . Many phThis review found that not many physicians find themselves in a position where they feel knowledgeable enough to order and conduct genetic testing, especially for rare diseases . An effeHowever, this should, on the other hand, not deviate from the much needed extension of the education of physicians and other healthcare professionals to deliberately cover the advantages and disadvantages of genetic testing in the context of rare diseases, including not only medical subjects but also the ethical and legal issues presented in this review .


Syrmaticus humiae, Hume 1881) at the Doi Tung Wildlife Breeding Center to assess the genetic consequences associated with captive breeding. Analysis of microsatellite loci and mitochondrial D-loop sequences reveal significantly reduced genetic differentiation and a shallow population structure. Despite the low genetic variability, no bottleneck was observed but 12 microsatellite loci were informative in reflecting probable inbreeding. These findings provide a valuable source of knowledge to maximize genetic variability and enhance the success of future conservation plans for captive and wild populations of Hume’s pheasant.Captive breeding programs are crucial to ensure the survival of endangered species and ultimately to reintroduce individuals into the wild. However, captive-bred populations can also deteriorate due to inbreeding depression and reduction of genetic variability. We genotyped a captive population of 82 individuals of the endangered Hume’s pheasant ( The most important concerns in the conservation biology of wildlife are a decrease in genetic variability, an increased likelihood of extinction due to changes in land use, over-exploitation of living resources, and urbanization ,2. The sSyrmaticus humiae, Hume 1881) is distributed in specific habitats throughout the hills of North-eastern India, Northern and Western Myanmar, South-western China and Northern Thailand between 1,200 and 2,285 meters above sea level [Hume’s pheasant  at the Doi Tung Wildlife Breeding Center  in Thailand between October and November 2020. The original founder sources are unknown and wildlife staff did not track label information of mother/offspring. Detailed information on the sampled individuals is presented in 2, 0.2 mM dNTPs, 5.0 μM primers, 0.5 U Taq polymerase , and 25 ng genomic DNA. The PCR protocol was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 45 s, 50–58°C for 45 s , Chinese goral , and water monitor lizards  [A), effective number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and linkage equilibrium were calculated using Arlequin version 3.5.2.2 [t-test, which does not assume equal variance between samples, was used to test for significant differences between Ho and He using the “t.test” function in the package “stats” using R version 3.6.3 [I) and a fixation index (F) were calculated for each locus of the population using GenAlEx version 6.5 [Ne) was estimated as the number of breeding individuals that contributed to the population using the linkage disequilibrium method in NeEstimator version 2.01 [We followed the same approaches as those used in previous studies of Asian woolly-necked stork ti 1768) –16. Alle68 –16. A 3.5.2.2 . Welch’son 3.6.3 ,21. Alleon 3.6.3 , and theon 3.6.3 . Micro-Con 3.6.3 . Polymoron 3.6.3  and calcsion 6.5 . Effectiion 2.01 .r) values were calculated for all pairs , and mean pairwise r values based on allelic frequencies in the population were calculated at captivity using GenAlEx version 6.5 [FIS) with 95% confidence intervals (CIs) were calculated using the LynchRt estimator [r and FIS was conducted under the assumption that the mean did not differ significantly from those of random assortments of unrelated individuals. Parentage analysis and the probability that two individuals shared the same genotype were calculated using COLONY version 2.0.6.6 [To consider the possibility of sibling or parent-offspring pairs in the captive population, we determined whether the captive-population individuals were more closely related than random unrelated individuals. Relatedness  was performed to assess the overall relationship across individuals in the captive population using GenAlEx version 6.5. The model-based clustering method implemented in STRUCTURE version 2.3.3 was used to determine population structure [K) varied from 1 to 25, with 25 replicates for each value of K. The most probable number of clusters was determined by plotting the log likelihood of the information (ln Pr (X|K)) [K values before choosing the optimal K value at which ln Pr (X|K) settled. The ΔK strategy was applied using Structure Harvester [The condition of heterozygosity abundance and changes in allelic frequency appropriations were examined in hereditarily bottlenecked populations using Bottleneck version 1.2.02 . The Wiltio test  was perftio test . The M rtructure . Run-lenr (X|K))  over thearvester .AGGACTACGGCTTGAAAAGC-3′) and PHDH (5′-CATCTTGGCATCTCAGTGCC-3′) [2, 0.2 mM dNTPs, 5.0 μM primers, 0.5 U Taq polymerase  and 25 ng genomic DNA. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 45 s, 56°C for 45 s, and 72°C for 3 min, and final extension at 72°C for 10 min. The PCR products were purified using the FavorPrep GEL/PCR Purification Mini Kit (Favorgen Biotech Corp.). Nucleotide sequences of the DNA fragments were determined by the DNA sequencing service of First Base Laboratories Sdn Bhd . The BLASTn tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search nucleotide sequences in the National Center for Biotechnology Information database to confirm the identity of the DNA fragments amplified in this study. Generated sequences were deposited in the DNA Data Bank of Japan  . PCR ampof Japan .h) and nucleotide (π) diversity [D* [D* and F* tests [Fs [R2, which has greater statistical power for small sample sizes, was calculated using DnaSP version 6 [Multiple sequence alignment of the 100 partial mt D-loop sequences was generated using the default parameters of Molecular Evolutionary Genetics Analysis X . All university  number oiversity . A statiiversity . Demograsity [D* , Fu and F* tests , and Fu’ests [Fs  were calests [Fs . Ramos-Oersion 6 ,46. The Columba livia, Gmelin 1789, GenBank accession number: FJ792695.1). The best-fit model of DNA substitution was determined for each genetic region using Kakusan4 [The mismatch distribution approach, in which an observed frequency distribution of pairwise nucleotide differences is obtained among individuals with expected distributions from an expanding population  or a stationary population (large raggedness index), was performed to test for the genetic signatures of historical population expansion within the captive populations ,48. ThesKakusan4 . The MCMF values of 0.847 ± 0.053. The PIC ranged from 0.048 to 0.749, and I ranged from 0.140 to 1.709  and the He values ranged from 0.048 to 0.780 (mean ± SD: 0.435 ± 0.071) . The AR value of the population was 7.500 ± 0.802. The standard genetic diversity indices are summarized in Tables Eleven captive individuals were genotyped. Ninety-one alleles were observed among all loci, with mean number of alleles per locus of 7.500 ± 0.802 Tables  and S5. to 1.709 . The Ho ) Tables . Welch’sr value of the Hume’s pheasant pairs among the 82 sampled individuals was -0.006 ± 0.089. A total of 2,523 pairs showed -0.25 < r < 0.25 and 798 pairs showed 0.25 < r (r > 0.25). The mean FIS was 0.153 ± 0.179, with individual values of FIS ranging from 0.007 to 1.149   . Simulta–57.100) . All pat100)  on the basis of Evanno’s ΔK, with all Hume’s pheasant individuals grouped into four clusters  (K) revealed one peak (K = 21), which suggests 21 clusters (I–XXI) . The 9 Tables . The M rified by , which i, and δ) . By cont (I–XXI) .The amplicon length and alignment length of the mt D-loop sequences were approximately 801 base pairs with four haplotypes resolved. Overall haplotype and nucleotide diversities of the mt D-loop sequences were 0.365 ± 0.004 and 0.006 ± 0.004, respectively . A complD* value was 1.109, p = 0.852, and the mean Fu and Li’s F* value was 1.090, p = 1.000. The mean Fu and Li’s D* value was 0.844, p = 1.000. The Ramos-Onsins and Rozas’s R2 value was 0.161, p = 1.000 (p < 0.05).Five different tests of neutrality were used to estimate the historical population expansion. The mean Tajima’s  = 1.000 . We thenBased on current rates of deforestation, as many as 30% of Southeast Asia’s bird species are predicted to become globally extinct this century . HuntingSyrmaticus humiae in China  [Tragopan caboti  [Bambusicola thoracica  [Syrmaticus ellioti  [He was significantly higher than Ho in the captive population of Hume’s pheasant. Similar cases were also observed in the captive populations of cracids [Aburria jacutinga, Spix, 1825) [FIS value (mean = 0.153 ± 0.179) and the mean r values in the captive population. The Hume’s pheasant captive population may have undergone a large number of generations in captivity. The similarity in genetic profiles may result from different captive population origins, with distribution of the founding individuals restricted to the northern region of Thailand [M ratio signaled a historical reduction in population size. Bottlenecks with low genetic diversity often occur when a small number of founders are taken from a declining wild population, which results in poor breeding success [Ne to the consensus population (N) enabled us to understand the population fitness, including the risk arising from genetic factors [Ne and Ne/N of the captive population were extremely low and have generally remained low, relative to those of captive populations of other birds [Ne or Ne/N. This finding suggests there is a low potential for recovery of the Hume’s pheasant population in the wild. However, sampling error might have occurred as a consequence of the small sample size [Ne and a management strategy that includes new introductions to the captive population are required to mitigate genetic drift.The current Hume’s pheasant captive population exhibited high AR values compared with those of other bird projects ,56. This n = 73) , Tragopa n = 53) , Bambusin = 180)  and Syrm n = 33) , the lev cracids  and Blacx, 1825) , which i success . The nat success ,63. The  factors . Here, ter birds . This miple size . Null alple size . An incrK = 4), although different Bayesian structural analyses (K = 21) showed 21 subgroups that were likely to be scattered. There is a strong risk that a breeding plan for captive Hume’s pheasant individuals will result in inbreeding depression. Careful examination of intraspecific genetic variability and formulation of breeding plans for species with low reproductive rates [The primary goal of the captive breeding program is to develop a self-sustaining population by minimizing undesirable genetic changes as a result of genetic drift in the captive environment, avoiding the deleterious effects of inbreeding depression, and maintaining a novel perspective on genetic management. Records of pedigree and breeding plans are important for captive management to reduce substantial errors in data assessments of specific mating events, and to minimize parentage exclusions and inbreeding . Analysive rates , or matiNe or Ne/N in the captive population. International collaboration to exchange Hume’s pheasant individuals might be an alternative means of enhancing genetic variability in the captive population. Alternatively, the current rapid development of biological technology, especially long-term storage by cryopreservation, will reduce dependence on populations of living Hume’s pheasants for the preservation of gene pools. Once the habitats of Hume’s pheasant are destroyed, they are extremely difficult to rehabilitate within a short period. Therefore, emergency measures are needed to preserve valuable genetic resources of Hume’s pheasants for a limited effective population size [in vitro fertilization, cloning using somatic cell nuclear transfer, and biobanking or genome resource banking [A primary policy instrument to mitigate the loss of biodiversity and reduce ecological degradation is the creation of statutorily protected or conservation areas. However, the level of protection afforded by such areas is widely acknowledged to be inadequate to achieve biodiversity protection goals in multiple dimensions ,71. Cons banking . ExperieThis study of genetic monitoring of a captive population of Hume’s pheasant provides useful information for conservation management officers. Recommended action plans for mating selection in breeding programs can be adjusted and applied to the captive population. The assessment of genetic variability is an important approach to maximize reproductive success and promote genetic variation in captive-bred individuals. Long-term maintenance of populations requires the implementation of a precise genetic breeding plan, accompanied by research that examines synergistic impacts, together with the twin crises of climate change and loss of biodiversity. Results can be used to modify and improve the implementation of effective measures. Given the small sample size, our results should be viewed with caution before implementation of a strict management or conservation strategy, to avoid genetic drift and ensure that a high proportion of the source variation is stabilized to minimize the loss of genetic diversity. Implementation should be accompanied by research to examine impacts, with results used to modify and improve the implementation of effective measures.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableDetailed information for all individuals is presented in (DOCX)Click here for additional data file.S6 Tablep-values with 110 permutations.Numbers indicate (DOCX)Click here for additional data file.S7 TableData were calculated using Bottleneck version 1.2.02. . Detailed information for all individuals is presented in (DOCX)Click here for additional data file.S8 TableDetailed information for all individuals is presented in (DOCX)Click here for additional data file.S9 TableDetailed information for all individuals is presented in (DOCX)Click here for additional data file.S10 TableDetailed information for all individuals is presented in (DOCX)Click here for additional data file.S11 TableDetailed information for all individuals is presented in (DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.


Further, gene prediction revealed that transformation of malate to pyruvate and lactate anabolism was more active in mash liquid of well‐fermented manual Huangjiu, while acetate accumulation was stronger in mash liquid of poorly fermented manual Huangjiu, which explained acidity excess reason in poorly fermented Huangjiu at gene level.Huangjiu (Chinese rice wine) is brewed in an open environment, where bacteria play an important role during the fermentation process. In this study, bacterial community structure and composition changes in the fermented mash liquid of mechanized Huangjiu, well‐fermented manual Huangjiu , and poorly fermented manual Huangjiu  in different fermentation stages from Guyuelongshan Shaoxing Huangjiu company were analyzed via metagenomic sequencing. And bacterial metabolic difference was analyzed via gene prediction of metabolic pathway enzymes. The results showed that the bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200–400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and  The bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200–400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and lactobacillus was predominant species in well‐fermented Huangjiu while lactobacillus brevis had an absolute dominance in spoilage Huangjiu. Another is made of raw wheat and naturally inoculated microorganisms. Mixtures of these starter cultures are used in the modern brewing process of Huangjiu  and Wu Yi Hong Qu (44.73%), respectively, and detected 213 bacterial genus with low abundance via Illumina‐based metagenomic sequencing.Huangjiu (Chinese rice wine), a traditional Chinese alcohol beverage, is popular among Chinese customers. Huangjiu is honored as national banquet alcohol beverage due to abundant nutrition and health function . Samples were stored at −80℃ before genome DNA extraction. For manual Huangjiu with unstable fermentation, we collected 24 batches of samples at different fermentation stages from 6 batches of samples in 4 workshops during the brewing season. Two representative batches of samples were selected for Macro‐sequencing analysis. For mechanized rice wine, due to its stable quality, we only took a batch of samples from normal fermentation for Macro‐sequencing analysis. Therefore, the samples have good representativeness. Three replicates were performed.2.2Alcohol content and acidity of Huangjiu samples were determined according to the protocol recommended by national standard of Chinese Huangjiu GB/T 13662‐2018  was used to extract genomic DNA from the sample. The extracted DNA was determined by NanoDrop nucleic acid quantitative assay. DNA was re‐extracted from the sample with concentration less than 5 ng/μl. The extracted DNA was stored at −20℃ for metagenomic sequencing. Three replicates were performed.2.4The metagenomic DNA libraries were constructed with 2 μg genome DNA according to the IlluminaTruSeq DNA Sample Prep v2 Guide, with an average of 350 bp insert size  and Gen bank, thus identifying the microbial species and calculating its abundance  was used to accelerate saccharification of glutinous rice in mechanized Huangjiu. The saccharification fermentation speed of mechanized Huangjiu is faster, the alcohol content could usually reach 13%vol on the fourth day   Figure , which iLactobacillus plantarum accounted for a large proportion, while the L. plantarum could secrete bacteriocins with antibacterial activity, which gave it an advantage in fermentation , respectively, which revealed that acetate was the main reason of Huangjiu spoilage. Gene prediction analysis showed that metabolic genes such as malate–pyruvate and biotin biosynthesis were expressed more in fermented mash liquid of well‐fermented manual Huangjiu than those in fermented mash liquid of poorly fermented manual Huangjiu . Metagenomic sequencing technology was applied to the analysis of bacterial community structure in the process of Huangjiu fermentation, which made up for the deficiency of isolation culture and PCR‐DGGE methods in the analysis of bacterial diversity, providing an effective means to further understand the role of bacteria, especially Lactobacillus in Huangjiu fermentation.In this study, metagenomic sequencing was employed to analyze bacterial diversity in fermented mash liquid of mechanized Huangjiu, well‐fermented manual Huangjiu, and poorly fermented manual Huangjiu . The results showed that every sample has 200–400 bacteria species, with LAB dominating the bacterial community. However, excessive proliferation of There are no conflicts of interest in this manuscript.This study does not involve any human or animal testing.Compliance with ethical standards.Software applications are available.Written informed consent was obtained from all study participants.


Genetic testing for hereditary cancer risk is usually arranged by a genetic counselor after talking about possible risks and benefits. To increase access to genetic testing, oncologists have started to order genetic testing. This survey study compared patient outcomes following genetic testing ordered by a genetic counselor or an oncologist. Genetic counselor-mediated genetic testing was associated with higher patient knowledge, as well as higher experience and understanding of genetic testing. Differences were noted in the type of psychological concerns reported, with individuals having genetic counselor-mediated testing being more likely to express concerns about having a hereditary cancer predisposition and those having oncologist-mediated testing more likely to express concerns regarding general emotions. Overall, oncologist-mediated genetic testing appears to provide a streamlined alternative to genetic testing; however, all individuals may benefit from post-test genetic counseling to address any knowledge gaps and provide additional psychological support.p = 0.025) and experience/understanding scores  at the time of genetic testing. Significant differences were noted in the potential psychological concerns experienced, with individuals in the GMT cohort more likely to screen positive in the hereditary predisposition domain of the Psychosocial Aspects of Hereditary Cancer tool , and individuals in the OMT cohort more likely to screen positive in the general emotions domain . The results of this study suggest that OMT can be implemented to streamline genetic testing; however, post-test genetic counseling should remain available to all individuals undergoing genetic testing, to ensure any psychologic concerns are addressed and that individuals have a clear understanding of relevant implications and limitations of their test results.This study compares knowledge, experience and understanding of genetic testing, and psychological outcomes among breast and ovarian cancer patients undergoing multi-gene panel testing via genetic counselor-mediated (GMT) or oncologist-mediated (OMT) testing models. A pragmatic, prospective survey of breast and ovarian cancer patients pursuing genetic testing between January 2017 and August 2019 was conducted at the Princess Margaret Cancer Centre in Toronto, Canada. A total of 120  individuals completed a survey administered one week following consent to genetic testing. Compared to OMT, the GMT cohort had higher median knowledge (8 vs. 9;  In the current era of precision medicine, genetic testing has become integral to the care of cancer patients. Among breast and ovarian cancer patients, genetic testing results may alter surgical management  or systeIn the traditional, genetic counselor-mediated testing model (GMT), individuals are referred to a clinical genetics service, with testing coordinated by a genetic counselor over two visits (pre-test and post-test) . GMT is Colloquially known as ‘mainstreaming’, the OMT model has been implemented in centers around the world, with many groups reporting decreased time to genetic test results, improved cost-effectiveness, and high levels of patient satisfaction ,17,18,19The aim of the current study is to compare the knowledge, experience and understanding of genetic testing, and psychological concerns reported among breast and ovarian cancer patients pursuing genetic testing using GMT or OMT models of care. By evaluating outcomes following consent to genetic testing, we hope to gain a better understanding of where additional support may be required with the expanded use of OMT. A total of 370 breast and ovarian cancer patients were identified and 276 (74.6%) were invited to participate in the study; 83/170 (48.8%) of individuals in the GMT cohort and 42/106 (39.6%) in the OMT cohort consented to participate. Five individuals completed the first study survey after receiving their genetic testing results and were excluded from analysis, providing a total 120 study participants  . Particip < 0.001) (p < 0.001) (p = 0.01) or given in a way they could understand (p = 0.01). They were also less likely to report the information provided helped them understand how genetic testing might impact themselves (p = 0.003) or their family (p = 0.003). Furthermore, individuals in the OMT cohort were less likely to report understanding the possible results of genetic testing (p = 0.001) and only 66.7%  were aware that they could contact a genetic counselor if they had questions before deciding to have genetic testing. When asked if they felt the process of having genetic testing worked well, the majority of participants agreed; however, significantly fewer of those who had OMT agreed (p = 0.02).Overall, participants agreed with a median of 10 items on the patient experience and understanding scale. The GMT cohort had a significantly higher median score  . On univ< 0.001) . When ev< 0.001) , the OMTp = 0.025) (p = 0.009). Regarding specific items .Following consent to genetic testing, individuals in the GMT cohort had a higher knowledge scores compared to those in the OMT cohort, with a median of nine and eight correct responses, respectively (= 0.025) . Univari= 0.025) ; OMT wasic items , both grp = 0.29) (p < 0.001)  compared to OMT (22.5%) ( = 0.29) . While t< 0.001) . p = 0.005), whereas a significantly higher proportion of individuals in the OMT cohort screened positive on the “general emotions” domain (p = 0.007) (p = 0.01) (p = 0.04), while increasing age decreased the likelihood of screening positive on this domain  as well as the “family and social issues” domain .The majority of individuals in both cohorts screened positive in at least one domain of the PAHC tool  . Compare= 0.007) . These f= 0.007) . OMT rem = 0.01) . Additio = 0.01)  showed ip = 0.035). Overall, the majority of individuals (97.7%) were able to correctly recall their test results, and most (94.4%) had discussed their genetic testing results with at least one relative.Of the initial 120 participants, 89 (74.2%) completed a second study survey  one week following disclosure of genetic test results. Given the small number of responses, detailed results are presented in This study provides comparisons of various outcomes reported by individuals with breast or ovarian cancer pursuing genetic testing via GMT or OMT. In our study cohort, GMT appears to result in better knowledge and understanding of genetic testing for hereditary cancer. Differences were also noted in the types of psychological concerns expressed at the time of genetic testing consent, which may be important for oncologists and genetic counselors to consider. Though limited by a low response rate and small sample size, the results of this study reveal potential differences in the level of knowledge and potential psychological impact of genetic testing using GMT or OMT models of care, which can be addressed during post-test counseling. Consistent with the high levels of patient satisfaction and acceptability of OMT reported by others ,14,17,19When evaluating the information provided to patients at the time of genetic testing, our results suggest that the information provided during OMT was less clear, did not provide as much detail about the implications of genetic testing, and did not obviously delineate the possible results of testing. The OMT group also appeared to be less knowledgeable regarding the potential for male transmission of hereditary breast/ovarian cancer risk, a common misconception that our group has previously described . The wriTypically, GMT involves detailed pre-test genetic counseling and formal risk assessment . While tp = 0.022). While genetic testing without pre-test genetic counseling does not seem to increase levels of distress above that related to a cancer diagnosis [p < 0.001) at the time of genetic testing. Thus, while the risk of significant distress following OMT is likely low, additional post-test support may be of particular benefit to individuals with a family history of cancer, irrespective of results. Though the response to our second study survey was limited, interesting trends were noted. In particular, similar MICRA scores were reported among OMT and GMT cohorts, which is consistent with reports from others ,19; yet,iagnosis ,13, publp = 0.035). Congruent with Richardson et al., we did not identify a significant difference in post-test knowledge scores between OMT and GMT cohorts following results disclosure [In addition to psychological support, post-test counseling may also serve to fill in the knowledge and understanding gaps noted at the time of genetic testing consent. Again, limited by small numbers, individuals in the OMT cohort who completed both study surveys had improved knowledge scores following post-test genetic counseling  began ordering multi-gene panel testing via OMT. Initially reserved for high-grade serous ovarian cancer, OMT protocols expanded to include select breast cancer indications in August 2018 .In our GMT model, breast and ovarian cancer patients were referred to the Familial Cancer Clinic at PM for genetic testing. The GMT model was used for breast/ovarian cancer patients who did not meet OMT criteria, or whose oncologist did not facilitate testing directly. All patients were provided personal and family history questionnaires to complete in advance of their pre-test genetic counseling appointment. During this appointment, one of six genetic counselors reviewed each patient’s personal and family history of cancer to determine their eligibility for genetic testing. When appropriate, the benefits and limitations of genetic testing were discussed, and genetic testing was offered. For consenting patients, bloodwork was arranged and a post-test appointment with a genetic counselor was scheduled to review test results and provide appropriate recommendations for cancer screening. https://www.uhn.ca/PatientsFamilies/Health_Information/Health_Topics/Documents/Learn_about_Genetic_Testing.pdf; accessed on 22 February 2021) and initiated a referral to the Familial Cancer Clinic. All patients were pre-booked a single appointment with a genetic counselor to disclose their genetic test results. Recommendations for cancer screening were provided based on the reported family history and available genetic testing results. In the OMT model, oncologists facilitated genetic testing for patients with one of the following diagnoses: (1) high-grade serous ovarian cancer; (2) breast cancer diagnosed at or under 35 years of age; (3) triple negative breast cancer diagnosed at or under 60 years of age; (4) bilateral breast cancer, with the first case diagnosed at or under 50 years of age; or (5) breast and ovarian cancer (any histology). A total of four gynecologic oncologists and 15 breast oncologists ordered genetic testing for at least one breast or ovarian cancer patient during the study timeframe. When offering testing, oncologists provided patients with an information pamphlet  tool is a 26-item questionnaire with 4-point scaling responses divided into six problem domains that may exist at the time of testing. These domains include: hereditary predisposition, family issues, practical & social issues, living with cancer, general emotions, and child-related issue . A scoreBRCA1/2 results and those receiving other genetic testing results  [In addition to the knowledge survey described in 4.2.2, the Multidimensional Impact of Cancer Risk Assessment (MICRA) questionnaire was administered one week after disclosure of genetic testing results. Developed to measure the psychological impact of receiving cancer genetic test results, the MICRA questionnaire is superior to other validated distress scales in distinguishing between individuals receiving positive clusive) ,25. It ip < 0.2. Reliability statistics are reported using Cronbach’s α or Kuder–Richardson (KR20) for continuous or dichotomous items, respectively. Statistical analyses were completed using R  and statistical significance was reported using a two-tailed α = 0.05.Descriptive statistics were used to summarize demographic information and participant survey responses. Bivariate analyses comparing GMT and OMT cohorts were completed using Mann–Whitney U-tests and Pearson’s Chi Square tests for continuous and categorical variables, respectively. Linear and logistic regression models were completed to determine associated factors with continuous and categorical outcomes, as appropriate. Genetic testing group (GMT vs. OMT), cancer type (breast vs. ovary), age at diagnosis, and family history of breast/ovarian cancer (none vs. any) were included in univariable analysis; additional multivariable analyses were completed for any covariates with Overall, the results of this study demonstrate that GMT may provide patients with increased knowledge and understanding of genetic testing at the time of consent. While differences were noted in potential psychological concerns at the time of testing, these can be addressed at the time of results. Overall, OMT appears to be a viable alternative to access genetic testing; however, where possible, post-test counseling should remain available to all patients, to provide additional knowledge and support. This may be particularly relevant for those individuals with a positive family history of cancer, who may have a higher perceived risk of hereditary cancer.


Sambucus wightiana—a clonal endemic plant species of the Himalayan region for understanding its possible survival chances in anticipated climate change. Eight polymorphic microsatellite markers were used to study the allelic/genetic diversity and population structure. In addition, ITS1–ITS4 Sanger sequencing was used for phylogeny and SNP detection. A total number of 73 alleles were scored for 37 genotypes at 17 loci for 8 SSRs markers. The population structural analysis using the SSR marker data led to identifying two sub-populations in our collection of 37 S. wightiana genotypes, with 11 genotypes having mixed ancestry. The ITS sequence data show a specific allele in higher frequency in a particular sub-population, indicating variation in different S. wightiana accessions at the sequence level. The genotypic data of SSR markers and trait data of 11 traits of S. wightiana, when analyzed together, revealed five significant Marker-Trait Associations (MTAs) through Single Marker Analysis (SMA) or regression analysis. Most of the SSR markers were found to be associated with more than one trait, indicating the usefulness of these markers for working out marker-trait associations. Moderate to high genetic diversity observed in the present study may provide insurance against climate change to S. wightiana and help its further spread.Habitat loss due to climate change may cause the extinction of the clonal species with a limited distribution range. Thus, determining the genetic diversity required for adaptability by these species in sensitive ecosystems can help infer the chances of their survival and spread in changing climate. We studied the genetic diversity and population structure of  This range expansion can increase the survival chances of species that might get affected by climate change driven shrinkage of suitable habitats; however, individual species' range responses vary greatly, with some species changing their ranges and/or local abundances rapidly while others do so slowly or not at all4. A growing body of literature seeks to explain this variation in range shifts using species’ ecological and life-history traits, with expectations that these shifts are determined by the capacity of species to disperse, establish new populations, and proliferate in the new environments6. In this race for survival, clonal plants appear to be at a particular disadvantage due to their limited mobility and limited capacity for adaptation7. Although, clonal plants have persisted for thousands, or even millions of years during past environmental changes8, the future climate change is predicted to advance much faster than during post-glacial times9. If a species can neither adapt to the modified environmental conditions nor migrate fast enough, then population extirpation or in the worst-case extinction of entire species is expected11. This extinction can be avoided if the populations undergo evolutionary adaptation14; however, the evolutionary potential of a population in turn depends on the pre-existing genetic variation within the species, and a high level of standing variation may allow a faster response to environmental changes16. Thus, empirical studies that estimate genetic diversity in clonal species with limited distribution ranges are important for understanding their possible survival chances in anticipated climate change.Climate warming is affecting the biodiversity and functioning of plant communities across the globe, and in response to this changing climate, species are now shifting their geographical distributions and expanding their ranges across elevational and latitudinal gradients17, probably resulting in biased estimation of the extent of clonality. Moreover, the low resolution of allozyme markers often leads to an overestimation of the extent of clonality18. On the other hand, due to failure to account for small differences between multi-locus genotypes because of PCR and scoring errors or somatic mutations, the use of high-resolution markers such as Amplified Fragment Length Polymorphisms (AFLPs) may lead to an under-estimation of the extent of clonality19. Some molecular markers, e.g., Simple Sequence Repeats (SSRs) have been the marker of choice due to their high polymorphism detection, high reproducibility, co-dominant nature, cost-effectiveness, and ease of study20. In addition to molecular markers, Internal Transcriber Spacers (ITS) have been frequently used for phylogenetic analysis21 and the study of genetic diversity. ITS ribosomal DNA length variants have been reported for several plant species23. In the present study, eight SSR markers, initially designed for Sambucus nigra, and ITS1–ITS4 region Sanger sequencing, were used to quantify the genetic diversity of Sambucus wightiana  to predict its survival chances and potential spread during anticipated climate change. The species is commonly found in forest gaps making way into the canopy, threatening the suitable habitat of many important medicinal plants, decreasing understory plant diversity, and potentially hindering the natural regeneration of conifer saplings.Although the advent of new molecular tools had a major impact on the study of clonality in plant species, the results from genotyping have quite often been interpreted without consideration of the restrictions of the genetic markers usedS. wightiana accessions/genotypes. Multiple loci were amplified by most of the SSR markers as evident by the fact that these 8 SSR makers amplified 17 loci ranging from 1 locus to 3 loci with an average of 2.12 loci per SSR marker. While counting the number of alleles per marker, it was revealed that a total of 73 alleles were amplified by all the 8 SSR markers using 37 S. wightiana genotypes. The number of alleles varied from 7 for SSR marker “EMSn016” and “EMSn003” to 1 for SSR marker “EMSn023” and “EMSn025” with an average of 9.12 alleles/marker. The average number of alleles detected per loci was 4.29  and the percent polymorphic loci was 70.59%. The analysis of gene diversity data revealed moderate gene diversity (He) noticed for all SSR marker loci, which varied from 0 (for monomorphic loci) and 0.818 with an average of 0.526 (SE = 0.086). Except for the monomorphic loci of Sn023 and Sn025 the average He was high with an average of 0.745. The mean values uHe and I were 0.534 (SE = 0.087) and 1.086 (SE = 0.179) respectively (Table He) and uHe values ranged from 0.206 to 0.445 and 0.275 to 0.475 respectively. Also, Shannon’s information index (I) was calculated for populations with the highest diversity found in Pop1  and lowest in Pop6 .SSR markers used during the present study successfully amplified expected genomic regions in a set of 37 To understand the patterns of variations of genetic diversity by SSR data, two multivariate methods were employed; clustering and principal coordinate analysis.S. wightiana in the Kashmir valley revealed three main clusters  was carried by using the data matrix/scores (1-presence and 0-absence) of all 8 SSR markers that led to the uniform spread of all 37 tes Fig. . PrincipS. wightiana with subtle signs of inbreeding.AMOVA was carried to analyze the distribution of genetic variation among sub-population from 7 different watersheds representing the different populations and among and within individuals/accessions/genotypes. The AMOVA revealed that the majority of the genetic variation is partitioned among individuals  i.e., 90% and only 10% variation is present among populations Table . Based o24. Therefore, efforts have been made during the present study to sequence the ITS region of 37 S. wightiana accessions/genotypes collected from the North-Western Himalayas of Jammu and Kashmir. The sequence data generated during the present study was also compared with ITS region sequencing data of 18 other Sambucus species including S. wightiana. The analysis of sequencing data led to the identification of 10 SNPs (SNP density: 1 SNP/ 57.2 bp) in the 645 bp sequence of 37 S. wightiana genotypes. Among the 10 SNPs, five SNPs were found very promising  region that lies between nuclear small rDNA and nuclear large rDNA is considered most important for phylogenetic inference at the generic and intrageneric levels in plants (Alvarez and Wendel 2003). The ITS sequence data has been used in ~ 66% of studies to analyze the phylogeny and 34% of all published phylogenetic hypotheses have been based exclusively on ITS sequences‘Fasttree’, built using online software tool “Clustal W”, with slow NNI and MLACC = 3 based on phylogenetic reconstructions using the function ‘build’ of ETE3 v.3.1.1 was comparable to MEGA 6 results. The optimal tree by MEGA 6 was constructed with the sum of branch length for ITS1 (0.48689146 for NJ and 0.52255238 for UPGMA) and ITS4 (1.61554106 for NJ and 1.80660053 for UPGMA). Both NJ and UPGMA methods show similar results, yet UPGMA showed the best fit results Suppl  and S3. Sambucus and one S. wightiana sequence downloaded from gene bank (https://www.ncbi.nlm.nih.gov/genbank/) were used as outgroups genotype during the analysis. The cluster analysis led to a clear-cut separation of 17 Sambucus species from S. wightiana  and Mixed Linear Model (MLM) was analyzed in the software program TASSEL led to the identification of a total of 5 SSR markers associated with 11 traits in 29. The prevailing opinion is that clonality reduces genetic diversity30. Many studies document lower levels of genetic variation in clonal species preceding repeated genetic bottlenecks leading to a prolonged lag phase32 and in populations of such species extinction is prevented with more intense vegetative reproduction with extensive human-mediated propagule dispersal36. In contrast to this opinion, comparable, or even higher genetic variation for 21 clonal plant species has been documented than non-clonal ones37. The results of many other studies also confirm this assertion, concerning the high level of genetic variation and genotypic diversity of species with clonal reproduction39. The most plausible explanation for high levels of genetic variation in populations of clonal plants is the presence of gene flow via long-distance dispersal and multiple introductions reducing the ecological and environmental constraints on genetic diversity whereas enhancing the potential of sexual reproduction, even if it is periodic and meagre43. Thus clonality helps in the maintenance and expansion of existing populations while the occurrence of sexual reproduction helps in the recruitment of new individuals/ populations however maintaining the genetic diversity44. Clonal plants are more successful in heterogeneous environments benefiting from clonal integration, which may enhance sexual fitness45. However, clonality does not substitute sexual mode of reproduction and genetic rather than ecological factors prove crucial for long term success48.Type of reproduction has an important effect on the maintenance of particular populations and species persistence in time and space. This trait significantly influences the ecological and genetic structure of populations and in consequence the evolution of species49. However, some studies have shown spreading plant species can maintain a remarkable level of genetic diversity across distances, in such cases the gene flow via long-range seed dispersal or pollen might be more important than commonly suggested51. This may be also associated with the multiple introductions leading to diverse genetic admixtures56. Local adaptation to environmental conditions is also considered one among the other dependent factors providing an advantage to spreading species62. Genetic variation plays important role in the evolution-related traits for local adaptation63. Also, evolutionary modifications to reproductive systems provide the spreading capability for widespread species64.The well-known genetic paradox of how spreading species maintain genetic diversity despite going through founder effects and genetic bottlenecks during the range expansionS. wightiana. Against the anticipated low genetic diversity in clonal plants, S. wightiana showed a remarkable genetic diversity which is consistent with some other studies66. The number of alleles per marker is a good indicator of genetic variability67. The detection of 73 alleles in 37 S. wightiana genotypes by 8 SSR primers with 70.59 percent polymorphic loci indicates moderate to high levels of genetic diversity. The high value of average gene diversity for all polymorphic loci (He = 0.74) also points to a good amount of overall genetic variability. Also, the variable ITS regions are used as a standard to measure genetic variability for a long time. The identification of several SNPs among different S. wightiana genotypes with SNP density of 1 SNP/57.2 bp also indicates high diversity in S. wightiana at the sequence level. The present study revealed a good magnitude of genetic diversity and a moderate differentiation with no geographical pattern among populations68.The non-availability of any data on genetic diversity and the aggressive clonal nature led to the perception of low diversity in S. wightiana individuals from both clusterings, PCoA by SSR scores and phylogeny by ITS markers69. This can be attributed to the mixing of the genotypes across the regions via gene flow between populations70. AMOVA showed that most of the variation is due to individuals/ genotypes only i.e., 90 percent and only 10 percent of the variation between geographically distant populations. These results suggest the recruitment of new genotypes into the populations with equivalent genetic differentiation as of non-clonal species. Yet clonality remains a vital strategy, shown by the deviance from Hardy–Weinberg equilibrium for the studied microsatellite loci71. Clonality associated with perineal growth can moderate the negative effects of stochastic population events like genetic drifts, etc.72. This magnitude of polymorphism at the studied microsatellite loci and the resultant genetic diversity may point to the spreading success of this clonal species. The potential for both sexual and clonal reproduction of a species provides the greater ability for landscape spread47. Significant pairwise Fst values (0.125–0.417) were observed which agree with the Nei’s genetic distance and identity parameters, indicating the substantial differentiation between the populations73.Our study does not find any geographically distinct clustering of S. wightiana genotypes. The presence of two sub-populations indicated differences in allele frequency and population differentiations in different sub-populations, indicating a good diversity available in S. wightiana genotypes in the Western Himalayas. The analysis of genotypic data in combination with trait data of the traits led to the identification of genomic loci/genomic regions that are associated with these traits. Using different models (GLM and MLM) 5 markers/loci were found associated with 11 traits, with some markers controlling more than one trait (pleiotropy). The markers controlling more than one trait are considered the most important markers/genomic loci and may be due to pleiotropy or linkage of multiple genes. Identification of more genomic loci through GLM than MLM is attributed to more stringent criteria and incorporation of kin-ship matrix into the analysis in MLM, in addition to genotypic data, trait data, and population structure matrix. In GLM association analysis, only genotypic data, trait data, and population structural matrix are used to work out MTAs. The genes/genomic regions/loci identified will prove useful in future Sambucus breeding programs and provided insights into the genetics of these traits and the kind of gene action in Sambucus.The structure analysis using SSR marker data during the present study revealed different sub-populations (two sub-populations) in our collection of 37 S. wightiana can acquire a moderate amount of genetic diversity, creating a wider genetic base that can withstand the stress environments like the conditions created by changing climate of the North-Western Himalaya. In this study, no particular genotype was found dominant across the regions. Lack of correlation between genetic diversity and geographic distances in S. wightiana could be associated with successful sexual reproduction between genets followed by long-range seed dispersion by human or animal agents, and subsequent recruitment in isolated S. wightiana populations enabling the gene flow between populations and forming diverse genetic admixtures74. The present study also found the successful transferability of these microsatellite markers, which could be used for further studies at a finer scale.Clonal endemic plant species like Sambucus wightiana Wall. Ex Wight & Arn. is a clonal sub-shrub, mostly found in the forests of Kashmir valley within an altitudinal range of 1700–3300 m.a.s.l. Commonly called Kashmir elder, the species is a rhizomatous geophyte growing up to 2 m in height with white or yellowish-white flowers. The umbels consist of orange or red individual berries that reach full ripeness in late summer. The species is native to the North-Western Himalaya and the primary range spans from Indian administered Kashmir to Chitral in Pakistan75. Recently the species has been reported from other parts of the Indian Himalayas like Uttarakhand, Himachal Pradesh, and Sikkim76, pointing towards its spread due to adaptive evolution in response to climate change61. Sambucus wightiana is closely allied to S. adnata Wall., from which it differs in its glabrous or almost glabrous inflorescence.2), that represents a unique biogeographical province in the Indian Himalayan region77. The altitude of the region ranges from 1600 m.a.s.l. at Srinagar to 5420 m.a.s.l. at Kolahoi peak. Within this altitudinal range, Kashmir elder is found from 1700 to 3300 m.a.s.l. in the forests of Kashmir valley. The climate of the region is predominantly of a continental temperate type with wet and cold winters and relatively dry and hot summers. The temperature ranges from an average daily maximum of 31 °C and minimum of 15 °C during summer to an average daily maximum of 4 °C and minimum of − 4 °C during winter. The region has been reported to be a hotspot for climate change due to its complex topography, enormous glacial and water resources, and quick responding watersheds with intense seasonal and climatic variability over a small spatial scale78.The present study was carried out in the Kashmir Himalaya  were selected, which have also been found informative in Sambucus canadensis79 (Table https://www.ncbi.nlm.nih.gov/genbank/). The primers for these eight SSR markers were synthesized on contract from Sigma Aldrich, Bangalore, India. The PCR was carried out in 15 µL reaction volume with the constituents: 2 µL 20 ng DNA template, 2µL (5.0 pmol) forward and reverse primers, 0.4 µL (2.5 mM) dNTPs, 0.3 µL (1.5 U) Taq Polymerase (Sigma), 1.5 µL buffer containing MgCl2 with a PCR profile of; 5 min at 95 °C, 40 cycles of 95 °C for 1 min, 54 °C for 1 min 30 S of annealing temperature, 72 for 1 min and a final extension of 72 °C for 10 min in a Peqlab PCR machine.Eight polymorphic microsatellite (SSR) markers, previously developed from the 2, and 1.0 U of Taq DNA polymerase (Sigma) by using the following PCR profile; 5 min at 94 °C, 45 cycles of 94 °C for 30 S, 52 °C for 45 S of annealing temperature, 72 °C for 1 min and final extension at 72 °C for 5 min in a Peqlab PCR machine.In addition to SSR markers, the ITS1-5.8S-ITS2 rDNA region was amplified using primers (ITS1: 5-GTCCACTGAACCTTATCATTTAG-3 and ITS-4: 5-TCCTCCGCTTATTGATATGC-3) synthesized on contract from Sigma Aldrich, Bangalore . The PCR was done in a 25 µL reaction volume with the following constituents: 25 ng DNA template, 5.0 pmol forward and reverse primers, 2.5 mM of each dNTPs, 1X buffer, 2.0 mM MgClwww.scigenome.com). All SSR amplifications for 37 S. wightiana genotypes were subjected to PAGE analysis using PeqLab Perfect Blue Dual Gel Vertical Electrophoresis system. The gels were silver stained and digitally photographed  80, for multivariate analysis (clustering and PCoA). Both clustering and PCoA were based on Jaccard’s dissimilarity co-efficient with 1000 bootstraps. Two clustering methods  were followed. The final inferences were made on the Unweighted Neighbor-Joining clustering (UNJ), which provided the best fit to the data, and a dendrogram was constructed81.For the characterization of genetic variation in the 37 https://biology-assets.anu.edu.au/GenAlEx/Welcome.html)82. The parameters included the number of alleles for a given locus (N), Number of effective alleles (Ne), Shannon’s Information Index (I), Observed Heterozygosity (Ho), Expected Heterozygosity (He), Unbiased Expected Heterozygosity (uHe), and Fixation Index (F) along with their mean and Standard Error (SE). GenAlEx was also used to calculate the mean allelic patterns across the populations with the above-mentioned parameters.Analysis of Molecular Variance (AMOVA) based on 999 permutations using FST statistics was performed with the scored SSR fragment lengths (8 markers at 17 loci) to determine the segregation of total genetic variation between, among, and within populations using GenAlEx v.6.5  values of individual primers at all loci were calculated based on the formula PIC = 2 × F (1 − F)85.Since our study was confined to a small number of samples, pairwise Nei’s Genetic Distance, Nei’s Genetic identity, and corresponding unbiased Nei’s Genetic Distance and Nei’s Genetic identity values were calculated across populationshttps://web.stanford.edu/group/pritchardlab/structure_software/release_versions/v2.3.4/html/structure.html)86. Population structure was analyzed by setting the number of sub-populations  from 1 to 10 and each run was repeated five times. The program was set to 300,000 burn-in iterations, followed by 600,000 Markov Chain Monte Carlo (MCMC) replications along with the admixture model. The STRUCTURE HARVESTER web version v0.6.94 (http://taylor0.biology.ucla.edu/structureHarvester/)87 was used to derive the appropriate number of sub-populations using a modified delta K (∆K) method88.An analysis of population structure was done using the software STRUCTURE v.2.3.4 (https://tassel.bitbucket.io/)89. Kinship matrix was also used in addition to genotypic data, trait data, and population structure information. The kinship matrix was calculated from genotypic data using the software program TASSEL.A study of Marker-Trait Associations (MTAs) was done using genotypic data, trait data, and population structure information using General Linear Models (GLM), and Mixed-Linear Model (MLM) in the software program TASSEL  and phylogenetic reconstructions were performed using the function ‘build’ in ETE3 v.3.1.190, as implemented on the GenomeNet (http://www.genome.jp/tools/ete/). The tree was constructed using the function ‘fasttree’ with slow NNI and MLACC = 3 (to make the maximum-likelihood NNIs more exhaustive)91. The values at nodes are SH-like local support. To compare these tree results for validity and reliability, MEGA 6 92 was also used for tree construction. The evolutionary history was inferred using the UPGMA method93 and Neighbor-Joining method94. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method95 and are in the units of the number of base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches96. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 528 and 537 positions for ITS1 and ITS4 respectively in the final dataset. Also, the aligned sequences of all 37 accessions were analyzed in BioEdit v.7.2.6 (http://en.bio-soft.net/format/BioEdit.html)98 for SNP identification.The Sanger sequences of ITS1 and ITS4 were manually checked for sequencing errors. The sequences were aligned using the online ClustalW tool (Supplementary Figures.