Overall, the co-formulation showed greater efficacy compared to either pydiflumetofen alone or current fungicide chemistry. This greater activity was demonstrated over a wide range of spray timings (flag leaf fully emerged to late flowering). The availability of an SDHI with good activity against FHB and the resulting DON contamination of harvested grain will give growers an additional tool within an IPM program that will provide a greater flexibility of spray application windows and reduce fungicide resistance selection pressure.Fusarium head blight (FHB) is an important disease of small grain cereals worldwide, resulting in reduced yield and quality as well as the contamination of harvested grains with mycotoxins. The key mycotoxin of concern is deoxynivalenol (DON), which has legislative and advisory limits in numerous countries. Cereal growers have a number of control options for FHB including rotation, cultivation, and varietal resistance; however, growers are still reliant on fungicides applied at flowering as part of an IPM program. Fungicides currently available to control FHB are largely restricted to triazole chemistry. This study conducted three field experiments to compare a new co-formulation of pydiflumetofen (a succinate dehydrogenase inhibitor (SDHI) with the tradename ADEPIDYN Fusarium species and Microdochium nivale and M. majus [Fusarium species are known to produce mycotoxins. Of the mycotoxins produced, the trichothecene, deoxynivalenol (DON) is the most commonly detected. Numerous entities have set advisory or regulatory limits for DON in cereals and cereal products intended for use as food or feed. The European Union sets a limit of 1.25 mg/kg in wheat intended for human consumption, lower limits for cereal products for direct consumption, and guideline limits for cereal intended for feed and compound animal feed in 2006 [Small grain cereals, and wheat in particular, are a key component of food and feed worldwide. Wheat is the second largest grain crop after maize, with a worldwide production of over 750 million tons . The yieM. majus ,5, with M. majus . All FHB in 2006 . These l in 2006 .Fusarium graminearum is the most important FHB pathogen worldwide and is frequently associated with severe epidemics and high mycotoxin concentrations [trations . During trations .FHB and resulting mycotoxins are best controlled by integrated pest management (IPM) incorporating several components including host resistance and cultural control (rotation and tillage) as well as fungicides. There are few fungicides with activity towards FHB and these are primarily demethylation inhibitors (DMI) of the triazole family, including propiconazole, tebuconazole, metconazole, and prothioconazole . StudiesFHB infection occurs at flowering and numerous studies have shown that fungicide application is most effective when applied from early to mid-flowering (Zadoks GS61-65 ) with stQuinone outside inhibitor (QOI) fungicides have been shown to be ineffective and in some cases can increase mycotoxin levels ,18. EarlPydiflumetofen is due to be launched to the European market in 2022 as a co-formulation with a different triazole, prothioconazole (APN + PTZ). As prothioconazole has greater efficacy against FHB than propiconazole, it was deemed useful to compare pydiflumetofen alone and in co-formulation with prothioconazole to prothioconazole alone  and prothioconazole in co-formulation with two SDHI: bixafen and fluopyram . As early results showed a high efficacy of pydiflumetofen products in FHB and DON control, it was deemed useful to identify if this extended the window of application around the typical application at flowering (T3) and if an earlier foliar application at full flag leaf emergence (T2) contributed to a greater reduction in FHB infection and DON.p = 0.259 and 0.344, respectively). For both measured parameters, fungicide timing and product were very highly significant (p < 0.001). The untreated control had 29% incidence of FHB resulting in 4.75 mg/kg DON in harvested grains. All timings resulted in a significant (p < 0.05) reduction in %FHB, with the most effective reduction at GS61 (93%) and the least at GS69 (35%)  a. Simila69 (35%) b. All fu69 (35%) c. This p69 (35%) d. Howevep < 0.01) at the early application (GS55) but only the APN + PTZ co-formulation resulted in a significant reduction (p < 0.001) in FHB incidence compared to the untreated control when applied late (GS69). For DON, APN and the APN + PTZ co-formulation gave a significant (p = 0.035 and p < 0.001 respectively) reduction at the earlier timing, but for the later application only the APN + PTZ co-formulation gave a significant (p < 0.001) reduction compared to the untreated control.The ANOVA contrast function  was used to compare individual treatments at early and late timings to identify differences in fungicide performance away from the optimum timing. For FHB incidence, all fungicides significantly reduced disease compared to the untreated (p = 0.955) or DON (p = 0.993). There was a very highly significant (p < 0.001) reduction in %FHB incidence and DON concentration in harvested wheat grains from the application of fungicides at both T2 and T3. For T2, the co-formulation of APN + PTZ resulted in the greatest reduction in %FHB (68%) and DON (55%) and this reduction was significantly greater than AscraXpro for disease incidence and for both AscraXpro and Proline for DON  and T3 (GS61) for either %FHB  greater reduction in %FHB compared to Proline . In Experiment 2, there was a prolonged dry spell in late spring and very few mature perithecia were visible by early flowering (mean untreated DON 1.4 mg/kg). In Experiment 3, there was a warm, wet spring with high numbers of mature perithecia by wheat head emergence, followed by several rain events during flowering (mean untreated DON 23 mg/kg). One drawback of using spawn inoculum rather than applying spores of Fusarium active triazole (200 g/ha prothioconazole compared to 125 g/ha propiconazole). Results from this study have shown that the new SDHI fungicide, pydiflumetofen in co-formulation with prothioconazole, was significantly better at reducing FHB disease and resulting DON contamination of grain than either pydiflumetofen A21857B) or prothioconazole (Proline) alone. There are few published studies on the efficacy of pydiflumetofen against FHB and these are either on it a solo product or as a co-formulation with propiconazole (MIRAVIS ACE) 857B or p. These sF. graminearum occurs after a rainfall event when temperatures range from 20 to 30 °C with at least 16 h of wetness [The first experiment showed that early flowering (GS61) is the most effective timing for control of FHB and resulting DON, with an earlier application at mid-head emergence (GS55) more effective than a later application at late flowering (GS69); this in in agreement with several previous studies ,21,23,24 wetness . Therefo wetness . Consequ wetness ,26. If tF. graminearum. The lack of any additional control from AscraXpro compared to Proline agrees with previous studies showing low activity of SDHI in general against F. graminearum [F. graminearum will reduce selection pressure on the pathogen to develop resistance to either active ingredient.A previous study showed that Proline, when applied at the earlier foliar timing of T2  at a 0.75 maximum single field rate, caused a significant reduction in both FHB (58%) and DON (49%) . The curminearum ,27. In tminearum . Any benIn conclusion, the availability of a SDHI with good activity against FHB and resulting DON contamination of harvested grain will give growers an additional tool within an IPM program to provide more effective control and greater flexibility of spray application windows and to reduce fungicide resistance selection pressure.Fusarium inoculum, application of specific fungicides at earlier growth stages (Plots (12 × 2 m) of winter wheat were sown (Crusoe cultivar in Experiment 1 (1918), Extase cultivar in Experiment 2 (2020), and KWS Zyatt cultivar in Experiment 3 (2021)) and maintained per standard agronomic practices at Harper Adams University, apart from the application of h stages , fungiciFusarium spawn inoculum was prepared from five isolates of F. graminearum grown on potato dextrose agar  for 5 days. Five mycelial plugs (5 mm diameter) were used to inoculate sterilized oat broth . Cultures were incubated for 7 days in an orbital incubator at 120 rpm. One kilogram of oat grains was added to an autoclave bag  with 200 mL deionized water, mixed, folded into a swan neck, and sealed with autoclave tape before autoclaving at 124 °C for one hour. Bags were left 24 h, then re-autoclaved. Each oat broth culture was added to 1.25 L sterile potato dextrose broth  and mixed; 100 mL was aliquoted into each bag of autoclaved oats, mixed, folded into a swan neck but not taped, and incubated for three weeks. Every few days, the bags were mixed to avoid the inoculum clumping. All incubations were conducted at room temperature (ca. 18 °C). An equal number of bags of each F. graminearum isolate were combined in large bags and well mixed to supply sufficient inoculum for one block of each field experiment. Inoculum was applied to all plots at a rate of 25 g/m2 in spring when the wheat was at early stem extension (Zadok’s growth stage 31 [stage 31 ). BlankeThe first experiment was a two-way factorial (timing × fungicide) with an untreated control in a complete randomized block design with four blocks. The timings were mid-head emergence (GS55), early flowering (GS61), and late flowering (GS69). The fungicides were all applied at the maximum single field rate except for APN, which was applied at 2.65 L/ha, which is the maximum field rate for the APN + PTZ co-formulation. Fungicide products included: Proline 275 , APN , and APN + PTZ co-formulation .The second experiment was a split plot design with the main plot (five sub-plots of 2 × 12 m) treated at full flag leaf emerged  and the central three sub-plots treated at early flowering . To avoid spread of inoculum between the main plots, only the central 6 m length of the three central sub-plots were assessed and harvested. This resulted in a minimum 4 m gap between sub-plots treated with different fungicides at the earlier application (T2). Fungicides applied at T2 were at 0.75 of the maximum single field rates and included: untreated, Proline 275 , AscraXpro , and APN + PTZ co-formulation . Fungicides applied at T3 were at 0.5 of the maximum single field rates and included: untreated, Proline 275 , and APN . All fungicides at T2 were supplemented with Mirror . The third experiment was a direct repeat of the second experiment.n = 10) for the whole experiment. Once ripe, the crop was harvested with a plot combine ; a 1 kg sample was milled  and mixed in a tumbler mixer for 5 min before analysis for DON using an AgraQuant® DON ELISA assay  according to the manufacturer’s instructions.After treatment at early flowering (GS61), plots were mist irrigated for 5 days from 5 AM to 10 PM each day for 1 min every 15 min. After misting, the untreated plots were monitored for FHB symptoms  and plots were assessed for FHB once incidence had peaked, but before the wheat heads started to senesce naturally . FHB incidence was assessed by counting the number of infected heads in ten grids (33 × 33 cm) per plot. This was converted to %FHB incidence based on the average number of wheat heads per grid . To obtain normally distributed residuals, the incidence data was logit transformed and the DON data was log10 transformed before using ANOVA. A Fisher LSD test was performed post-hoc to identify differences between individual treatments within a factor and the contrast function was used to compare individual treatments to the untreated control. Data presented are the back-transformed means and percentage control compared to the untreated plots.


Together with Cayapas, the Tsachilas constitute the oldest population in the country of Ecuador and, according to some historians, they are the last descendants of the ancient Yumbos. Several anthropological issues underlie the interest towards this peculiar population: the uncertainty of their origin, their belonging to the Barbacoan linguistic family, which is still at the center of an intense linguistic debate, and the relations of their Yumbo ancestors with the Inca invaders who occupied their ancient territory. Our contribution to the knowledge of their complex past was the reconstruction of their genetic maternal and paternal inheritance through the sequencing of 70 entire mitochondrial genomes and the characterization of the non-recombinant region of the Y chromosome in 26 males. For both markers, we built comprehensive datasets of various populations from the surrounding geographical area, northwestern South America, NW, with a known linguistic affiliation, and we could then compare our sample against the overall variability to infer relationships with other Barbacoan people and with other NW natives. We found contrasting patterns of genetic diversity for the two markers, but generally, our results indicated a possible common origin between the Tsachilas, the Chachi, and other Ecuadorian and Colombian Barbacoans and are suggestive of an interesting ancient linkage to the Inca invaders in Yumbo country. Our knowledge of the pre-colonial history of South America native peoples, which was formerly handed down by oral tradition, began to be recorded on written documents by the first chroniclers at the time of the conquest. Hence, it is characterized by several fragmentary and contradictory tales.In this context, genetic investigation, which has become increasingly refined in recent years, have shown important insights not only on the first colonization and migrations events into the southern continent ,2,3,4 buHowever, the origin of peoples who lived at the edge of the empires that dominated the Andes in the past centuries remained entrusted to the oral transmission for a longer time, hence the knowledge of their past is even more difficult to inquire ,9. The gColorados, that has several interesting reasons to be anthropologically investigated.Our study focuses on the genetic history of a small Ecuadorian population, the Tsachilas, also known as the Caras, a population from the isthmus probably belonging to Chibcha linguistic family, conquered the Kingdom of Quito and over the next 200 years, occupied the entire region northwest of the current province of Pichincha, where the rivers Silanchi, Toachi, Blanco, and Caoní converge. During the Late Period, the Caras mingled with the natives of Quito and, over time, the Caras culture gave birth to the Yumbos, the ancestors of the current Tsachilas and Chachi people [First, the Tsachìlas constitute, together with the Chachis (or Cayapa), the oldest population in the territory of Ecuador. They currently live in the Pichincha province in the Canton of Santo Domingo de los Colorados at the western foothills of the Andes. According to some historians, during the year 980 CE, the i people ,11. Tsaci people .Second, the Tsachilas speak a Barbacoan language related to the languages of the Caranquis, the Pastos, and other groups in northern Ecuador and southern Colombia, which some linguists have included in a larger Chibcha-Paez family ,14 althoTahuantinsuyo [Third, some historians have also found traits of cultural (but not linguistic) affinity between Yumbos and the Incas who occupied the area at the time of the northward expansion of ntinsuyo  althoughsierra and went to live in the tropical forest of Pichincha: the Tsáchilas (Colorados) of Sto. Domingo are, to some extent, the last descendants of the Yumbos and are probably of other ethnic groups from the western forests. Nowadays, their population comprises 2950 people, according to the last census [Based on the analysis of linguistic studies and the existing archaeological and ethnohistorical information, Lippi  proposedt census , and thet census . Similart census .At the time of the sampling mission, the Tsachilas were still quite isolated from the surrounding areas, and they have had only limited contact with the national culture and with non native people, probably retaining much of the original genetic variability.In order to obtain a genetic perspective to integrate these archeological, ethnohistorical, and linguistic scenarios, this study focuses on uniparental markers: mitochondrial genomes (mtDNA) and the non-recombining portion of the Y chromosome.The mitochondrial data for South American populations are mostly represented by control region sequences ,26, whicWe expect that these uniparental markers will help to clarify the relationships that the Tsachilas have with the Indigenous Chachi, who are currently living on the coast in the Esmeralda province ; whetherBlood samples from 70 unrelated individuals were collected during the year 2000 during an anthropological mission conducted by Gianfranco De Stefano in the canton of Santo Domingo de los Tsachilas, Ecuador , from tw® Y23 System . The amplified products were analyzed with an ABI PRISMR 3130 Genetic Analyzer (Applied Biosystems) following the recommended sequencing kit protocols and were then analysed with GeneMapperR ID Software v3.2 which, identifies an allele for each of the 23 loci. All the successfully genotyped individuals (26) were assigned to a haplogroup through a Bayesian inference on their Y-STR profiles [TM Multiplex kit (Applied Biosystems) following the protocol described by Sevini et al. [The full mtDNA genomes were amplified with 11 overlapping PCR fragments using a set of primers, and the Cycle Sequencing was performed using a set of 32 nested primers, as described in Torroni et al. . The seqprofiles , and onli et al.  to assigi et al. .st distance matrices used for MDS analysis (φst for mtDNA haplotypes and Rst for Y-STRs), the shared haplotypes for both the mtDNA and Y chromosome data, and the AMOVA measures were performed with Arlequin v3.5 [http://cran.r-project.org (accessed on 28 January 2021)) to calculate the mean TMRCA values.The calculation of the molecular diversity indices shown in uin v3.5 . To visuuin v3.5  on a datuin v3.5 ,53,54,55uin v3.5 ,57,58; muin v3.5 ,30,40,55uin v3.5 , and meduin v3.5 . The varuin v3.5 . Finallyuin v3.5  with an uin v3.5 , and a muin v3.5 , and posWe generated 70 novel sequences of complete mitochondrial genomes. All of the mtDNA profiles belonged to the main Native American haplogroups A2, B2, C1, and D1.Mitochondrial haplogroups B2 and D1 were the most frequent lineages in Tsachilas samples, as shown in st), assigning each haplotype to one of the 15 Ecuadorian, Peruvian, Bolivian, or Colombian populations on the basis of ethnicity or language in order to elaborate a nMDS plot . In order to find such signals of lineage transmission , our 24 novel Y-STRs haplotypes were entered into a database that included 775 Y-STRs profiles belonging to more than 20 populations from all over the northwestern area of the sub-continent, and we selected only 14 loci to include an adequate number of profiles. Following the same logical proceedings used in a previous study , we calcWe also estimated the TMRCA using an exponential growth model in BATWING for the DC6 cluster to infer the possible timing for the origin of the cluster, and we obtained an interesting result dating the cluster back to about 1450 BCE, which was during the final period of the Inca empire.The Tsachilas seem to have preserved much of the ancient diversity that spread along the continent from north to south, which is mostly evident from the mitogenomes analysis. Indeed, they have retained some of the old mitochondrial sub-haplogroups, A2ac, A2k, B2b, D1f , that liAn old classification of South American languages ,70 claimWe did not find any haplotype shared with other populations, indicating a high level of endogamy , even ifThe haplotype-level analyses were also useful to clarify the relationship between the Tsachilas and the populations of the surrounding area, both Barbacoan and not, at a finer scale and we also useful to deepen the question concerning their hypothetical origin. The MDS analysis confirmed a genetic proximity with the Chachi (or Cayapa) on the maternal side . HoweverThe MJ network of the maternal lineages showed a closer relationship with the Pasto people , a population from Colombia whose language became extinct; they were probably linguistically affiliated to the Barbacoans together with Cara people, with both of them being of Andean origin, and this could support the oral tradition of Tsachilas regarding their own origin, although documentation is scarce . HoweverAs other studies have previously showed ,75, the Generally, we found that the genetic structure was more influenced by the linguistic affiliation in mtDNA than in Y chromosome data, as it emerged from MDS  and AMOVThe increase of inter-ethnic marriages is documented in the recent history of Tsachilas, who were originally endogamic communities . HoweverWe wondered about the possible explanations for this difference. Assuming that the different resolution of the genetic markers analyzed  could have a role in the observed differences, we found that some authors reported that the basic Tsachilas social unit is the nuclear or extended patrilinear family and that between neighboring ethnic groups, the kidnapping or exchange of women was quite frequent , this coThe male data, on the other hand, indicate a greater similarity with non-Ecuadorian populations (mainly Peruvians), and we aimed to deepen the question with further analyses on the Y chromosome.Since Y chromosome STRs seem to be informative for more recent contact effects between different populations, we used this marker to infer past Y chromosome lineage expansion events by patrilineal descendants. We found only one descent cluster thatincluded Tsachilas haplotypes among those selected according to the procedure described in the Data Analysis section. Interestingly, this spans from the western foothills of the Ecuadorian Andes to the Peruvian Andes and the highland regions of the Peruvian Amazon , indicatThese data are certainly intriguing since several archaeological studies indicate a possible relationship between the Inca and Yumbo during the northern expansion of the Tahuantinsuyo in the final stages of the Inca empire and a hypothetical certain degree of admixture between natives and invaders in Yumbo Country ,76,77. ASummarizing, the molecular analysis leads us to confirm the Tsachilas as members of the large Barbacoan family, which extends from Colombia to Ecuador, according to linguistic family trees . ParticuWe believe that studies on full mitochondrial genomeconducted at higher molecular resolution s and with a higher number of Y-STRs and Y-SNPs may help in the future to disentangle the origins of the Barbacoan and Chibcha-Paez populations. In fact, high resolution molecular data integrated with linguistic and historical information will help to unravel questions regarding (i) the genetic affinities between the Barbacoan and the macro Chibcha-Paez language families; (ii) the timing of genetic diversification of the Proto-Barbacoan groups; and (iii) the degree of genetic affinity with Inca people and whether this could have eventually altered the genetic legacy of present-day Ecuadorian natives.Nowadays, Tsachilas communities are little larger than at the time of the sampling mission, but they are also more integrated into the urban population and admixed with people of non-native ancestry, so the present study represents one of the last pictures of the native genetic landscape of pre-Columbian Ecuador.


With the advancements in genetics and genomics in the twenty-first century, genetic services have become an integral part of medical practices in high-income and upper-middle-income countries. However, people living in low and lower-middle-income countries (LICs and LIMCs), including Bangladesh, are rather underprivileged in receiving genetic services. Consequently, genetic disorders are emerging as a significant public health concern in these countries. Lack of expertise, high expense, the dearth of epidemiological data, insufficiently updated medical education system, poor infrastructure, and the absence of comprehensive health policies are the main factors causing people living in these countries not having access to genetic services. In this article, the authors took benefit from their professional experience of practicing medical genetics in the area and reviewed existing literature to provide their opinions. Particularly, it reviews the current knowledge of genetic disorders' burden and their causative factors in Bangladesh. It focuses on why providing genetic services is challenging in the context of the country's cultural and religious sentiment. Finally, it proposes a physician-academician collaborative framework within the existing facility that aims to tackle the challenges. Such a framework could also be useful for other LICs and LMICs to address the challenges associated with providing genetic services. To date, more than 6500 inherited disorders have been identified globally, and common genetic diseases are increasingly emerging as a major health challenge , 2. EarlCurrently, available management strategies for most genetic and multifactorial diseases are rather palliative, as curative treatment strategies are not commonly available . AlongsiIn this review, we provide an overview of the challenges associated with the provision of genetic services in the LICs and LIMCs, specifically in Bangladesh. We have discussed the current health-care system in Bangladesh and reviewed the incidence of genetic and multifactorial diseases in the population, cultural factors impacting genetic disease prevalence, and the role of religious practices in setting boundaries on acceptable management guidelines for pregnancies identified to be affected with a genetic disease. We endeavored the key challenges to harnessing genetic information in order to reduce the burden of genetic diseases in Bangladesh. This article proposes a framework for delivering genetic services in the country, which includes dissemination of genetic literacy to the population at large and fostering international collaboration between local physicians, academics, and international experts. The proposed framework could be a model for implementing genetic services in other LICs and LMICs having similar health and cultural status.Health-related socio-demographic features of Bangladesh are listed in Table There is no recognized Government medical genetics center available in Bangladesh, except for one small national Department for Forensic Medicine in Dhaka . Further, the current medical education system  fails to form geneticists in Bangladesh; essence to incorporate knowledge of genetics in both undergraduate and postgraduate levels of medical curriculum . As the As in many LIC and LIMCs, Bangladesh has no comprehensive epidemiological data on the prevalence of most genetic diseases. Though inhabitants suffer from many hereditary disorders and congenital malformations , it is dDespite the fact that Bangladesh lies in the so-called thalassemia belt  and hereditary hemoglobinopathies are the most common genetic diseases in Bangladesh, the information on different aspects, including epidemiology, clinical course, mortality, complications, and treatment outcomes are scarce due to the lack of definitive national data , 25, 26.Over the last decades, mortality caused by the ‘fatal four,’ i.e., cardiovascular disease (CVD), cancer, chronic respiratory diseases, and diabetes mellitus (DM), is increasing at an alarming rate. In all four, genetic risk factors are being identified , 28.CVD has been reported as the leading cause of mortality in Bangladesh, which probably has the highest CVD incidence in the South of Asia , 29, 30.Cancer is a major health burden worldwide and the sixth common cause of mortality in Bangladesh . By 2030DM is considered a major public health issue in Bangladesh due to its high prevalence, which may exceed 16.8 million by 2030 . A studyIn 2013, 12% of mortality in neonates and children of under-five years of age was estimated to be due to congenital anomalies . As in oThe entire South Asian region, including Bangladesh, is considered a hotspot of hemoglobinopathies . As mentDown syndrome is the most frequently reported chromosomal disorder in Bangladesh , 52. OthConsanguineous marriage is considered one of the predisposing factors behind the burden of genetic diseases in Bangladesh . It signIn general, consanguinity is influenced by geographic, demographic, religious, cultural, and socio-economic factors. This custom is common in the Muslim community throughout the world. Islam allows but always discourages first cousin marriages . In BangIn addition to genetic factors, environmental factors play a crucial role in the occurrence of genetic diseases. Environmental factors can lead to disease-causing or putative de novo mutations –82. In BGenetic counseling is considered an essential part of the management of patients and families in which a genetic disorder has been diagnosed or who are at risk for such a disease. It provides patients the necessary information about the disease and their genetic profile. Also, it calculates the risk or probability of a genetic disorder appearing in their family, and provides support, diagnosis, and management facilities and—if available—treatment. In LMICs, such as Bangladesh, most inhabitants are unaware of the inheritance patterns and risk factors of genetic diseases, which poses additional challenges for implementing genetic services for them. Thus, along with developing infrastructure for genetic service delivery, comprehensive public awareness programs should be introduced throughout the country to spread knowledge on the deleterious effects and possible preventive measures of genetic diseases, and the role of consanguinity. Moreover, suitable measures need to be taken to ensure proper treatment and follow-up for patients with genetic diseases.Premarital counseling is regarded as the first step to minimizing genetic disease transmission from parents to offspring . It is iPrenatal screening could be followed by premarital screening as the second step for early diagnosis of any congenital abnormalities . The chrNewborn screening (NBS), finally, is a simple test at birth that can identify genetic conditions that may affect a child's long-term health or survival but are treatable at birth . In manyTo identify the biochemical, chromosomal, and/or molecular etiology of a genetic disease, only a few private hospitals, clinics, and diagnostic centers are recently trying to provide comprehensive lab testing in Bangladesh. Except for some minimal facilities that can perform such diagnostic tests, their diagnostic service is mainly confined to collecting and preparing patient samples and exporting them to foreign diagnostic centers, mostly in India, where the tests are performed. There are currently neither molecular labs nor community projects run by the government of Bangladesh nor medical genetics departments. The limited molecular genetic testing that happens in the country is predominantly restricted to sophisticated laboratories like in the public universities, the International Centre for Diarrheal Disease Research (ICDDRB), the Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) . These iThe curriculum of medical colleges in Bangladesh is not sufficiently focused on the study of genetics and heritable disorders as well as modern diagnostic tools and strategies. The undergraduate medical curriculum of Bangladesh, introduced in 2012, includes only 4-h of basic genetics in Anatomy class . The posRapid evolution in technological possibilities to analyze the human genome and increasing knowledge of its function provides a wealth of new options for dealing with genetic diseases, but also several issues that must be addressed by the genetic services provider, patients, as well as the society. Artificial insemination, genetic screening on embryos, in vitro fertilization, sex selection during prenatal testing, surrogate motherhood, fetal transplantation, and gene therapy raises ethical issues; genetic counselors can play a vital role in the way they are understood and acted upon.A few social and ethical issues accompany the practice of genetic counseling, related to the confidentiality and privacy protection of the patients. The need for confidentiality of sensitive information about a person’s family history, carrier status, and particularly, the risk for a genetic disease, which can lead to social stigma, calls for disciplinary control over medical practitioners, as is done by the Medical and Dental Council of Bangladesh (BMDC). Although this organization is in place, attempting to control malpractice, misbehavior, and violation of the ethical code by medical practitioners, people opt to report directly to court instead. BMDC is a semi-autonomous body under the ministry of health and family welfare; one of its departments is the ethical committee whose function is to review ethical aspects of research protocols before approval. But the BMDC mostly deals with research of medical colleges situated in Dhaka. Further, there are issues regarding its own poor functioning and lack of monitoring , 104.Among factors that affect a couple’s willingness to receive genetic counseling, religions issues that guide marriage habits and reproductive behavior are important. Some aspects of genetic counseling, such as the discussion of a diagnosis, prognosis, and preconceptual options, are quite often sensitive in a like Bangladesh where educational status is suboptimal, and the majority of inhabitants is Muslim, guided by Islam , 106. ThThe development of the preimplantation and prenatal diagnosis of genetic disorders has offered couples an alternative to prenatal diagnosis to avoid selective abortion. According to Islamic jurisprudence, if diagnostic tests prove that a fetus is affected by an untreatable life-threatening disease or carries some severe disability, therapeutic abortion is permissible and lawful, given that the pregnancy is terminated before the time of breathing the soul, i.e., within 120 days of gestation . Muslim The recent progress of genome editing technologies, e.g., CRISPR/Cas9 and antisense technology, has set off new horizons for translational research and treatment of genetic and multifactorial diseases, including cancer, AIDS, thalassemia, and muscular dystrophy , 117. GeGood parental information is essential for the success and ethical acceptability of any newborn screening program. The choice that a couple makes about their progeny heavily depends on the stage at which information about the risk of genetic disorder becomes available to them, and thus on the timing of the prenatal diagnosis, which has come to be known to be barely sufficient, in our experience.In many low and lower-middle-income countries, the options discussed during genetic counseling to prevent inherited genetic disorders, such as prenatal diagnosis, carrier screening, and selective abortion, often have legal prohibitions . For insAs mentioned earlier, the uptake of genetic services is used to be constrained because of the expenses of genetic testing. Coverage of the cost of genetic testing by health insurance is beneficial in ensuring genetic services not being hampered due to cost issues . HoweverOver the years, Bangladesh has built a good foundation for social security; however, unplanned growth of the social safety net portfolio has caused fragmented implementation, with 123 programs under 25 ministries . BesidesA few private companies have begun to provide health insurance for their employees , which mThe integration of genetics in health care leads to a significant transformation in the organization of general health services, as it facilitates a shift from curative to preventive services . The intFollowing the Human Genome Project's successful completion, healthcare technologies, especially the technologies related to medical genetic services, are developing faster than ever in the twenty-first century . It is nA comprehensive nationwide survey to understand the epidemiology of genetic diseases, followed by patient registration and disease-wise database development, would be an essential assignment to start genetic service. Planning and implementing new patient education and awareness programs to educate people about the importance of genetic services, changing from a single-patient perspective to a family, converging the experience of the disease itself with the experience of being at risk for the disease is also an essential elementary step , 141. ThExisting reports suggest that the high-throughput molecular diagnostics for thalassemias, which are rather costly in the context of LIC and LMICs, could be avoided in most cases if simpler and cost-effective methods could be utilized efficiently . AssessiSince the development of the polymerase chain reaction (PCR) technique in the early 1970s, its use in pathological experiments is continually increasing . Some vaOnce facilities for hematological testing and/or primary molecular analysis become accessible to most of the community, the clinicians can then prescribe panel screening for specific diseases like thalassemias. To achieve this goal, we propose a collaborative model within the existing facility in Bangladesh, described in the latter part of this manuscript. Also, we considered the critical impact of individual-, family-, cultural-, institutional-, or policy-level factors that may influence patient outcomes of genetic counseling , includiIn countries with limited and/or underutilized countries, like Bangladesh, the government needs to concentrate more on infrastructure and legal framework development. For example, the government can enforce laws for premarital screening for common recessive disorders, e.g., ß-thalassemic alleles, and advocate against consanguineous marriage. Indeed, the Bangladesh government has recently issued a rule for the premarital screening of hemoglobin variants . HoweverGenetic diagnosis, testing, and counseling are real challenges in Bangladesh due to the factors mentioned above. Additionally, professionals, including the clinical geneticists, laboratory geneticists, bioinformaticians, genetics counselors, who work together in the completely established medical genetics facilities in HIC and UMICs, have not developed in the country. To tackle these challenges, we propose a framework to initiate a diagnostic and counseling service in Bangladesh Fig. . We haveOur proposed framework is established on the concept that robust cooperation and collaboration, on both international and national levels, are key to success in implementing genetic services in the LICs and LMICs like Bangladesh. In this model, the primary care units, involving the primary care physicians , nurse, physician associates, and support professionals, plays the most prominent role in genetic service provision. In these units, primary care units will be provided training in human genetics. In Bangladesh, non-government organizations and the government organize many workshops, seminars, and training programs for doctors, nurses, and health associates. The government itself and pharmaceutical companies in Bangladesh usually sponsor these workshops and training programs. The primary care units can receive basic training through multiple comprehensive training programs, instructed by medical genetics and biotechnology experts from home and abroad. Once they complete the basic clinical genetics training, the primary care physicians will undertake an initial risk assessment using a standardized referral guideline. The primary care physicians will identify the patients with potential genetic conditions or individuals at-risk using their expertise and available diagnostic facilities. They will then refer the patients with potential genetic conditions and individuals at-risk for genetic services by experts in medical genetics for further consultation. The geneticist will assess the patients’ condition and, if required, will suggest genetic testing at a designated laboratory, perhaps at a university or a research center, where the tests can be performed. In case the patient faces difficulty visiting the laboratory by himself, they can provide the sample to a local diagnostic or hospital facility (under the supervision of the primary care physician), where the sample will be processed, and they will send the sample to the designated lab. Next, the designated lab will perform genetic testing and inform the outcomes to the primary care unit and consulting geneticist. Based on the genetic testing outcomes, the patient (and family members) will receive a suggestion about the possible management strategies and treatment options. The suggestion they would be given will comply fully with the country's socio-cultural, ethical, and legal standards. For complicated cases, further testing and expert opinion could be sought from home and abroad. The pathway associated with this model is as follows: patient → primary care units → geneticist → laboratory → geneticist/primary care unit/professional counselor → patient and family members. A central coordination directory maintained by the authority of health services and society of medical genetics will regulate this pathway's effective management.A major question that comes with this model is that who would pay for the genetic services? For the patients who have access to full coverage through social security or health insurance scheme, the service's cost could be covered by the scheme; however, it may require reviewing the coverage policies. A major challenge would be to finance the services for those who do not have access to such health coverage schemes. In such scenarios, the well-off households can finance the expenses from their savings or reduce on luxury stuffs of consumption , 151. HoIn the context of Bangladesh, medical doctors have direct access to handle patients, and most of the medical colleges/hospitals are equipped with different facilities, including radiology, biochemistry, hematology, histology, etc., for patient diagnosis. Nevertheless, within the existing situation, medical colleges/hospitals have insufficient facilities and lack of expert personnel for molecular diagnosis, especially for medical genetics work. On the other hand, many universities of Bangladesh have expert academics and molecular diagnosis facilities, but the academics depend on medical doctors for the subject cases. In this situation, if doctors at the medical colleges/hospitals make the primary or differential diagnosis with their facilities and collaborate with nearby university academics for molecular diagnosis, it can be a collaborative tire model for proper diagnosis. If medical doctors from all over the country, molecular biologists, geneticists, and bioinformaticians with training in genetic diseases can make a collaborative group, they can start genetic counseling programs. Most of the academics and many medical doctors also pursue their higher studies abroad with reputed medical colleges or universities, and most of them have a good network with international clinicians and expert groups. If necessary, for any complicated case of genetic disease diagnosis, treatment, or management, a foreign expert group's opinion can also be asked. In these collaborative ways, within the existing facilities, it is possible to mitigate many issues related to genetic diseases.One of the challenges in this model would be to distinguish between genetic and non-genetic conditions that are coexisting in the country. The general physicians, who will primarily report identify a possible genetic condition, would be relying on the routine clinical/non-genetic diagnostic techniques, e.g., complete blood count, liver function tests, kidney function tests, etc. Complete resources, like the GeneReviews®, on the clinical representations of all the genetic diseases available in the country, are required to be developed . HoweverThe subject has a positive family historyThe subject belongs to a consanguineous familyThe subject has developmental delays, which is, perhaps, due to an inherited or congenital issueThe subject him/herself or his/her parents have a reproductive complicationWhen this resource will be ready for clinical use, the clinicians will have free access to this resource. Subjects representing clinical conditions resembling any of the listings in the GDR and meeting any of the following criteria should be contemplated for a referral to genetic diagnosis:Until the geographic location-specific GDR comes prepared for clinical use, the clinicians can still start this type of program with having reference to the GeneReviews®. For novel and extremely rare genetic conditions, subject samples could be analyzed using an established gene-discovery strategy by an interested academic Table  6].Tabl.Tabl6].TFinally, genetic disease awareness and its pre-conceptional implications should be disseminated through leaflets, audio–video advertisements, TV programs, and by introducing it in the secondary level curriculum. Social activists, social workers, and religious and political leaders will also be provided with sufficient knowledge and asked to help disseminate scientifically factual information on genetic diseases among mass populations. For instance, the Imams (a Muslim leadership position) of each mosque could play a vital role in disseminating awareness about genetic conditions. Besides, in the last decade, Bangladesh has established more than 13,000 community clinics to provide primary healthcare in rural areas . These cThe incidence of genetic diseases in Bangladesh is exceedingly high, in part due to the high rate of consanguinity and chronic exposure to various environmental pollutants. Knowledge among the general population about premarital screening and genetic counseling programs is low and needs to be promoted, with educational campaigns in hospitals, schools, and universities. The provision of genetic screening with advanced technology and counseling in government-regulated facilities for all couples wishing to marry remains essential. To be effective, counseling should be provided by trained professionals who can provide accurate advice regarding the risks and childbearing options. This will require an additional focus on genetics and genetic disease in medical education but will also require implementation-specific courses for genetic counselors. Though time demanding, the start of a genetic counseling service at least in each of the district hospitals will help to better management of disease and will decrease the prevalence of genetic diseases in Bangladesh. If this framework proves efficient, the framework on which it is based can be used as a starting point for other LICs and LMICs to develop genetic services.


Eucalyptus salubris that unexpectedly detected two divergent lineages but was not sampled sufficiently to make clear interpretations. We undertook comprehensive sampling for an independent genomic analysis  to investigate whether the two purported lineages remain discrete genetic entities or if they intergrade throughout the species’ range. We also assessed morphological and ecological traits, and sequenced chloroplast DNA. SNP results showed strong genome‐wide divergence (FST = 0.252) between two discrete lineages: one dominated the north and one the southern regions of the species’ range. Within lineages, gene flow was high, with low differentiation (mean FST = 0.056) spanning hundreds of kilometers. In the central region, the lineages were interspersed but maintained their genomic distinctiveness: an indirect demonstration of reproductive isolation. Populations of the southern lineage exhibited significantly lower specific leaf area and occurred on soils with lower phosphorus relative to the northern lineage. Finally, two major chloroplast haplotypes were associated with each lineage but were shared between lineages in the central distribution. Together, these results suggest that these lineages have non‐contemporary origins and that ecotypic adaptive processes strengthened their divergence more recently. We conclude that these lineages warrant taxonomic recognition as separate species and provide fascinating insight into eucalypt speciation.Genetic studies are increasingly detecting cryptic taxa that likely represent a significant component of global biodiversity. However, cryptic taxa are often criticized because they are typically detected serendipitously and may not receive the follow‐up study required to verify their geographic or evolutionary limits. Here, we follow‐up a study of  Cryptic species are often detected serendipitously and don't receive thorough follow‐up investigation to verify their geographical, morphological or ecological limits. In this study, we undertake such a follow‐up with range‐wide sampling, high‐resolution population genomic (SNP) analysis, chloroplast sequencing, morphometrics and soil analysis to investigate two unexpected lineages found previously within Eucalyptus salubris. We confirm the presence of two highly divergent lineages that remain entirely genetically distinct in geographic proximity and exhibit minimal morphological separation. We conclude that historical isolation and ecotypic adaptation have likely contributed to their divergence. To provide an independent genomic analysis of the findings of Steane et al.  lane. Read assembly, quality control, and SNP calling were undertaken by DArT using DArTsoft14 software, aided by alignment to the E. grandis genome. This pipeline uses technical replicates for a measure of genotyping reproducibility and modelled Mendelian behavior of DArTseq markers to filter sequencing errors and paralogous regions. The DArTsoft14 pipeline produced 51,897 SNP loci with 23.75% missing data. We applied further quality control filtering using the ‘dartR’ package  for DArTseq™ analysis as per Sansaloni et al., . BrieflyXtX) to identify loci that are much more  or less  differentiated than expected under neutral expectations. The simulate.baypass function was applied in R to simulate allele count data and calibrate XtX to determine thresholds for identifying outlier loci in the empirical datasets. We extended the running parameters  and across three independent runs, 125 loci were identified as outliers at the 1% threshold, representing both directional and balancing selections. These loci were removed for a final dataset of 3,605 putatively neutral loci with 2.99% missing data. This dataset was converted to program‐specific input files using PGDSPIDER v.2.1.0.0  of K. Because discrete clustering patterns can be confounded with isolation by distance . The most likely value(s) of K was determined by stabilization in the plot of the deviance information criterion (DIC) against Kmax.To investigate genetic structuring at the individual level, we first used principal coordinates analysis (PCoA) because this multivariate analysis does not rely on any particular evolutionary model  was estimated using ARLEQUIN v.35.2.2  was also performed using ARLEQUIN to partition the total genomic variation within and among the major genetic groupings detected in the above clustering analyses. Genetic diversity was assessed using the ‘hierfstat’ package  and expected heterozygosity (HE), while GENALEX v.6.501 ) and geographic (ln(km)) distance across the whole sampled range, as well as within each of the major genetic clusters, we used mantel testing in IBDWS (available at http://ibdws.sdsu.edu/).Population differentiation  a three species model in which we split E. salubris into Lineages 1 and 2, while keeping E. ravida a separate species; and (c) a two species model in which we split E. salubris into Lineages 1 and 2 but combined Lineage 2 with E. ravida. The last two scenarios were chosen based on the results of our population genomic analyses (Results). Because only two populations of E. ravida were sampled, we retained all SNP loci but randomly reduced the number of Lineage 1 and Lineage 2 samples to four to provide even representation of each potential lineage. A path sampling analysis was performed with 24 steps for each model, using one million iterations, sampling every 5,000, after a burn‐in of 1,000. The allele frequencies in the dataset were used to calculate mutation rates and default settings were applied for the priors. Convergence was assessed using Tracer v.1.7.1  to compare the support for Models 2 and 3, relative to the current taxonomy (Model 1).Finally, Bayes factor (BF) delimitation (BFD*) was used to test alternate coalescent hypotheses regarding species delimitation in our data. The analysis was run using SNAPP  was determined in Matlab . Leaves were dried at 55°C and weighed. Specific leaf area was calculated as the ratio of leaf area to dry mass (cm2/g), averaged across leaves to give a single value per tree for all E. salubris populations. For soil phosphorus measurements, five soil cores (0–10 cm depth) were collected across the area spanning our tree sampling within each population. The cores for each population were pooled and analyzed for nutrient content by CSBP Analytical Laboratories  to obtain soil phosphorus levels per population.Of the suite of morphological, climatic, and soil traits measured by Steane et al. , only spt‐tests based on population means. Following Shapiro‐Wilk testing, the SLA dataset conformed to a normal distribution; however, the soil phosphorus data required log transformation to achieve normality. Levene's test was used to examine homogeneity of variance and the appropriate two‐tailed t‐test was performed for each dataset. If relevant, minor and major outliers in each dataset were identified by 1.5× interquartile range and 3× interquartile range, respectively.To test for differences in SLA and soil phosphorus between the major genetic clusters, we used 2.5E. salubris. We chose three regions for the full analysis based on sequence quality and nucleotide diversity: rpl16, trnG‐trnS, and psbD‐trnT. These regions were sequenced for all 176 DNA samples used in the DArTseq analysis. PCR amplifications were performed using the reaction details provided by Byrne and Hankinson  and sequenced by Macrogen Inc. The relevant regions were also extracted from the whole chloroplast genome sequences of E. salmonophloia and Corymbia gummifera (GenBank Accession numbers NC_022403 and NC_022407) for use as outgroups in phylogenetic reconstruction . Any indels arising from mononucleotide repeats were ignored, while more complex indels  were coded as single binary transitions for a total sequence length of 1,676 bp excluding the two outgroups and 1,742 bp including them.Thirteen non‐coding cpDNA regions that have been found to be useful for phylogeographic studies in Australian plants  and nucleotide (π) diversity were performed in DNASP v.5.10  using PERMUT v.2.0 , relative to minimal differentiation within lineages  or within Lineage 2 (p = .450); however, there was a weak signal of isolation by distance across the range of Lineage 1 .The distinction of the two lineages was further supported by high differentiation between lineages . Phosphorus content was more variable across the sampled range due to inflated values in some populations of both lineages, likely due to nutrient run‐off from nearby agricultural land , and this difference increased following the removal of major outliers  and the removal of both major and minor outliers .Both SLA and soil phosphorus content exhibited significant differences between the two lineages across the sampled range and in a pattern consistent with Steane et al. . Specifi3.3D and R2 statistics were non‐significant and thus chloroplast sequence variation is considered neutral  that associated with Lineage 2 in the SNP dataset but shared the common haplotypes, H3 and H4, found in the north. Sequence data for each haplotype have been deposited in GenBank for each chloroplast region separately .Population differentiation across the sampled range was high, and values of re Table . The maxre Table . The relre Table . The netre Table . There w4E. salubris , a wide range that may allow for two, non‐overlapping flowering seasons, one for each lineage. And while we found no genetic evidence of hybrids, our collections only sampled adult trees and post‐zygotic barriers in eucalypts often present at the juvenile stage . Alternatively, the only recorded morphological difference between both lineages of E. salubris and E. ravida is glaucousness ; Formal analysis (lead); Methodology (lead); Writing‐original draft (lead). Dorothy Steane: Conceptualization ; Data curation (supporting); Writing‐review & editing (supporting). Margaret Byrne: Conceptualization ; Funding acquisition (lead); Project administration (lead); Supervision (lead); Writing‐review & editing (supporting).We have no ethical considerations to declare.


The Lusitano Horse (LH) originates from Portugal, but is reared worldwide. Since 1994, the University of Milan has tested the LHs breed reared in Italy for parentage control using microsatellite markers. This study aims to assess the genetic variability of the LHs reared in Italy for more than four decades, in order to obtain information necessary for genetic management.is) were estimated. In the whole period, the total population showed Ho as high as 0.69, low Fis (0.057), and imbalance for HWE. When considering the unrelated horses, Ho was seen to increase over time (from 0.594 in 1975–1990 to 0.68 in 2010–2019) and frequencies were in HWE, again having low and decreasing values of Fis (from 0.208 in 1975–1990 to 0.019 in 2010–2019). Bottleneck analysis excluded a recent population decline. Principal Coordinate Analysis at the individual level defined two clusters, the major cluster including all the most recent horses. An increasing number of dams (156% more from 2001–2010 to 2011–2019) supports the good variability recorded in the population so far. However, the high number of foals (77.2%) sired by only four stallions in recent years suggests caution in the choice of the sires for the future.The Lusitano Horse (LH) originates from Portugal, but is reared worldwide. Since 1994, the University of Milan has routinely tested the LHs bred in Italy for parentage control. This study aims to assess the genetic variability of the LH reared in Italy using 16 microsatellites markers. Moreover, the genetic variability changes over the years in the total population (n.384) and in unrelated horses (n.47) were evaluated. Horses were grouped according to their date of birth . Standard genetic diversity parameters, including observed (Ho) and expected (He) heterozygosity, Hardy-Weinberg equilibrium , allelic richness, and inbreeding coefficient (F The Lusitano Horse (LH) is one of the most ancient equine breeds. It originates from Portugal, where it is economically the most important horse breed, with a registered population of about 5000 mares and 1000 stallions . Since bIn Italy, the Associazione Italiana Cavallo Puro Sangue Lusitano (AICL) was established in 2010 to promote the breed and its selection. A reciprocal partnership between the APSL and the AICL was signed in the same year. The agreement provides for all LHs born in Italy to be enrolled in the Portuguese studbook, after parentage verification.Currently, approximately 500 LHs are reared by 15 breeders in Italy and they are mainly trained as riding horses but are also used for other equestrian disciplines, such as carriage driving, working equitation, and dressage . Since 1The genetic variability of the LH using pedigree data and STR markers has been described in several studies ,2,3,5,6.The present study on 384 samples collected over the years in Italy and typed for a panel of STR markers aimed to analyze the genetic variability (i) on the 1975–2019 period considering the population as a whole and (ii) on the animals grouped in four subpopulations according to the decade of birth in order to verify the changes in genetic variability that occurred in this time.The Laboratory of Animal Genetics and Genomics at the University of Milan  maintains the blood specimens of all the LHs bred in Italy (433 horses), collected over the years (1994 to 2019), in its bio-repository . Specimens consist of EDTA whole peripheral blood kept at −20 °C. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. The stored samples were approved by the Ethics Committee of Università degli Studi di Milano Prot. OPBA_56_2016.® Genotyping Kit, . The commercial panel included the 12 STRs of the official ISAG panel for parentage testing   [® 310 Genetic Analyzer and fragments were analyzed by GeneScan® and Genotyper® software. Genotypes at the same loci of 87 LHs , parents of foals born in Italy and tested at Laboratório de Genética Molecular de Alter (PT), were also available. The ChrX marker LEX03 was excluded by the statistical analyses.Out of the LH stored, 384  were requested to be typed for parentage control in the period 1994–2019. Genomic DNA was extracted from EDTA blood samples using a commercial kit (Zymo Quick-gDNA MicroPrep). DNA samples were genotyped using 17 STR markers of StockMarks for the HorsesOut of the 384 genotyped horses, the analyses were performed on the following animal groups and periods, the generation interval periods defined according to Vicente 2015 [Allelic frequencies and Hardy-Weinberg equilibrium  were estimated using the GENEPOP v. 7 software ,19. The The observed number of alleles (Na) and the observed mean number of alleles value (MNa), the effective number of alleles (Ae) and the mean effective number of allele value (MAe), the Nei’s expected heterozygosity (N_He), and the observed (Ho) and expected (He) heterozygosity per locus and for all were calculated with POPGENE v.1.32 software .Allelic richness (Ar) per locus and in the population  was standardized for variations in sample size and was calculated using FSTAT v. 2.9.3 .The number of private alleles per population (N. PrA), the number of alleles with a frequency >5% (Na Freq. > 5%), and the unbiased expected heterozygosity (uHe) were estimated using GenAlex v. 6.503 . is) was estimated using the GENEPOP v. 7 software [Inbreeding coefficient  the “Two excess heterozygosity tests and Wilcoxon sign-rank test” . This meIT, and FST), according to Weir and Cockerham (1984) [FSTAT v. 2.9.3  was usedm (1984) .The dissimilarity matrix, the Principal Coordinates Analysis (PCoA), and the dispersion graph were performed using GenAlex v. 6.503 . The genetic variability parameters were calculated for each marker in both the total population  and the unrelated horses  . Among tThe total population showed significantly different Ho and He , resulting to significant deviation from HWE . The number of horses per group always gradually and progressively increased over time, approximately doubling from one period to the next .In the related and unrelated horses, Ho remained stable in time (range 0.688–0.690), constantly lower than He (ranging 0.765–0.688), apart from in the last period where their values came very close. The groups were always to deviation from HWE in each period .Several STR loci in every group deviated from HWE, HMS1, and HTG7 deviating in every period and HTG6 being in equilibrium only in the first period (1975–1990) . PrivateAdditionally, in the unrelated individuals , He showis values were quite low, in accordance with high values of heterozygosity . This sign of low inbreeding could be ascribed to the population structure regarding the number of dams and sire.Overall, Fzygosity . When coThe bottleneck test was applied to the total and unrelated animals in the four generation interval groups. The expected numbers of loci with heterozygosity excess for the three evolutionary models, the non-parametric Wilcoxon rank test, and the probability values calculated for the three models were reported in IT) showed values as low as 0.041 in the total population (n.384) and 0.064 in the unrelated horses (n. 47). Additionally, the proportion of genetic variance contained in the subpopulations relative to the total population variance (FST) were in low values (FST = 0.027) in the total population and very low values (FST = 0.019) in the unrelated horses. The FST analysis performed on the total population showed the highest diversity within the second-generation interval group , and the lowest in the third group . The last group (2011–2019) showed the highest differences with the previous ones when assessing the pairwise differences between populations , which is consistent with the results of the corrected average pairwise difference . Again, according to the chi-square test and permutation test of FST, the group 2011–2019 showed significant divergence from the others , is displayed in As shown by the parentage tests performed on the STR profiles, the composition of Italian LH breeders (related and unrelated) is reported in The number of mares progressively increased over time. In the period 2001–10 out of 32 mares, 84.4% delivered one single or two foals. In the last decade (2011–19), the number of dams more than doubled (156% increase) and the number of mares with only one or two foals decreased but remained high (to 68.3%).Similarly, males also increased their numerosity in time, but with a lower multiplication factor than mares in the last two periods (51.9% increase from 2001–10 to 2011–19).The number of foals consistently increased over time; it was three in 1975–1990, 21 in 1991–2000, 49 in 2001–2010, and 175 in 2011–2019. Additionally, an increase in the average number of foals per stallion was recorded. It started from 1.5 in 1975–1990 and reached an average of 8.3 in 2011–2019 . Despite the increased number of mothers of foals in this period, 83.1% of the full sibs belonged to only three sires .Management of genetic variability in domestic animals is a priority and it is essential in breeding programs. Changes in the genetic variability of a population over time can be assessed by evaluating the degree of inbreeding. In the present study, the picture of the genetic variability in LH bred in Italy was assessed using 16 microsatellite markers and analyzing the horses bred in the period 1975–2019. To evaluate the breed changes over time, analyses were also carried out that categorized the horses into four groups according to the date of birth . Moreover, both in the whole time and in the four periods, the total population (related and unrelated horses) or only the unrelated individuals were considered in turn.IS value (0.057) was similar to the values obtained in the LH bred in France (0.040), Brazil (0.038), and Spain (0.042) [The first two decades showed a low number of genotyped horses, due to the small size of the Italian population at the time and the scarce availability of STR profiles, due to those markers having been introduced only from the mid-1990s onwards in horse parentage control routines. Thus, horses bred in the 1970s and 1980s were genotyped later with STRs when requested and mainly abroad. LH is an autochthonous Portuguese breed. Italian breeders started importing individuals for breeding between the middle 1980s and 1990s and gradually started to use mainly local breeding stock. General variability parameter values, recorded in the examined Italian total population  that slightly exceeded the Ho value (0.69) in 2011–2019, a higher He in allele distribution always drove the populations in all the time groups always out of HWE in each period , which is consistent with the same individuals in the whole period 1975–2019 .is low or decreasing over time. The populations were always in HWE, as can be expected by a “not real” population, which is not subject to selection. Good heterozygosity was confirmed by the low values of Fis, which was particularly low in the last period (2010–2019), in accordance with the high values of Ho  including individuals from all the periods considered. This cluster included all the dams and sires of horses of the last decade . Data onThe low number of stallions used by breeders in all periods of analysis, and especially so in the last ten years , had an This study evaluated the genetic variability of the LH bred in Italy. The population recorded a good genetic variability with a low degree of inbreeding. Indeed, STR markers showed a level of heterozygosity comparable with those found in the LH populations reared in other countries. Nevertheless, considering all the horses, related and unrelated , a small trend towards some decrease in heterozygosity in the last ten years was recorded which was not strong enough to generate concern at this moment in time. The observed trend, however, suggests the need for careful genetic management of the population in the coming years, and more emphasis on the use of an increased number of different males as sires.


Myanmar is well known as a primary center of plant genetic resources for rice. A considerable number of genetic diversity studies have been conducted in Myanmar using various DNA markers. However, this is the first report using DArTseq technology for exploring the genetic diversity of Myanmar rice. In our study, two ultra-high-throughput diversity array technology markers were employed to investigate the genetic diversity and population structure of local rice varieties in the Ayeyarwady delta, the major region of rice cultivation. The study was performed using 117 rice genotypes with 7643 SNP and 4064 silicoDArT markers derived from the DArT platform. Genetic variance among the genotypes ranged from 0 to 0.753 in SNPs, and from 0.001 to 0.954 in silicoDArT. Two distinct population groups were identified from SNP data analysis. Cluster analysis with both markers clearly separated traditional Pawsan varieties and modern high-yielding varieties. A significant divergence was found between populations according to the Fst values (0.737) obtained from the analysis of molecular variance, which revealed 74% genetic variation at the population level. These findings support rice researchers in identifying useful DNA polymorphisms in genes and pinpointing specific genes conferring desirable phenotypic traits for further genome-wide association studies and parental selection for recombination breeding to enhance rice varietal development and release. Oryza sativa L.) is one of the most widely cultivated cereal crops distributed across the world and serves as a major food source for more than half of the global human population [Rice  are conserved in the seed bank at the Department of Agricultural Research, Ministry of Agriculture, Livestock and Irrigation (MoALI), Yezin, Myanmar . They reOryza sativa L. in Myanmar, SSR markers have been used most extensively to study the genetic diversity of more than 600 Myanmar rice landraces [Nowadays, molecular markers to analyze genetic relationships in crops have become increasingly popular since they are more reliable than other phenotypic or biochemical markers ,15. Mostandraces , while tandraces .Hordeum vulgare), rye , bean (Phaseolus vulgaris), macadamia (Macadamia integrifolia), etc. [DArT (diversity array technology) markers developed by Jaccoud et al.  are usefa), etc. .In view of this, we conducted genetic diversity and structure analyses involving a collection of currently cultivated rice germplasm accessions from farmers’ fields in addition to the collections from local gene banks using the most robust set of markers to obtain repeatable inferences. Thant et al.  extensivOut of 18,271 SNP markers, a total of 7643 markers cleared and passed all the quality parameters  . Among tThe genetic dissimilarities among the genotypes estimated through the SNP markers ranged from 0 to 0.753 . The PawSilicoDArT markers were also useful for the identification of genetic relationships among rice genotypes. The range of genetic dissimilarities identified through silicoDArT markers was broader than that observed through SNP markers. Among the 117 rice genotypes, dissimilarity ranged from 0.001 to 0.954 . The genA total of 7643 SNP markers were used for population structure analysis. The model-based Bayesian cluster analysis in STRUCTURE visualized the genetic structure of the population under examination. K value was used to estimate the number of clusters of the genotypes based on the genotypic data throughout the whole genome. In order to find the optimal K value, the number of clusters (K) was plotted against ΔK, which showed a sharp peak at K = 2 , and theA significant divergence was found among individuals within populations according to the Fst values of 0.832 for pop1 and 0.687 for pop2 obtained from STRUCTURE . PrincipResults from the analysis of molecular variance (AMOVA) revealed that 4% of the total variance was found within individuals, whereas maximum diversity was partitioned between the two populations (74%) and among individuals within populations (22%) . In addiThe average value for the number of different alleles (Na) and effective alleles (Ne) across the populations was 1.604 and 1.252, respectively , and theOur study highlights the suitability of DArT platforms that can be applied for genomic studies of rice genotypes. A total of 18,271 DArTseq SNPs were developed, of which 7643 markers provided robust information from the rice genome in the absence of sequence information, while silicoDArT markers provided 4064 informative markers. The average PIC values of both types of markers in rice were similar to the values identified in DArT markers in sorghum (0.41) , cassavaOryza sativa L.). Their study was performed using 59 rice genotypes with 525 SNPs derived from a DArTseq platform. Phung et al. [Of the different types of molecular markers, microsatellite markers (SSRs) have been used most extensively in Myanmar rice genotypes ,17,18. Mg et al.  also chag et al. . FurtherPopulation structure analysis is informative in understanding genetic diversity and facilitates subsequent association mapping studies . We expeIn total, 23 non-Pawsan traditional varieties and all 17 Pawsan varieties were clustered together into one genetically related population (pop1), which indicated that those non-Pawsan varieties were genetically close to Pawsan varieties. With regard to genetic dissimilarity indices, they revealed a smaller range (0 to 0.133) for SNP markers  and a laFurther, 72 non-Pawsan traditional varieties and all 5 HYVs were clustered together in the second genetically related population (pop2), probably reflecting the fact that breeding activities led to genetic similarities since new varieties were usually selected from local landraces. Most of the local landraces have unique taste and shape, which breeders want to keep intact, whereas breeding for traits such as short duration, short stem, low canopy type, etc. However, local varieties have some undesirable traits such as long duration, tall or plant architecture etc. Although these varieties fell into the same group, they had a broad range of dissimilarity: 0–0.281 and 0.001–0.688 for SNP and silicoDArT markers, respectively . MoreoveWe also observed the assignment of individuals to each population at K = 3 as our hypothesis b. The meIn addition, our study found that some duplicate genotypes, by their name (farmer-named varieties based on farmers’ taxonomies and nomenclature), were consistent with the naming and distinguishing through very close genetic relationships  . On the Fst (fixation index) is a measure of population differentiation due to genetic structure. A Fst value of 0.25 can be considered as significant in differentiating populations. The range 0.15–0.25 indicates moderate differentiation, whereas differentiation is negligible if the Fst value is 0.05 or less . A signiDitylenchus angustus) attacks, rice blast disease (Magnaporthe oryzae), and rice stem borer (Scirpophaga incertulas) [The allelic pattern and genetic diversity indices provided useful information on the genetic diversity in each population. The higher value for diversity indices is an indication of a higher level of genetic diversity ,45. Prevertulas) ; MKAUK, In our study, we used a total of 117 rice genotypes, which included 95 traditional varieties (other than Pawsan), 17 traditional Pawsan, and 5 HYVs . All genYoung healthy leaf tissue was collected from 3-week-old rice plants and stored on silica gel in plastic tubes for desiccation. DNA was isolated from dried leaves using the CTAB method developed by Doyle and Doyle  and modihttps://www.diversityarrays.com, accessed on 19 November 2021) for whole-genome scans using a combination of DArT complexity reduction method and next-generation sequencing platform. Whole-genome genotyping for the 117 rice genotypes was carried out using DArTseq technology as described by Barilli et al. [A total of 20 μL of each sample with a concentration of 100 ng/μL DNA were sent to Diversity Arrays Technology (DArT) Pty. Ltd., Bruce, Australia , call rate (%), polymorphism information content (PIC), and one ratio . A totalGenetic dissimilarity matrices were constructed in DARwin v. 6.0.21 to identify the genetic relationships among the genotypes . WeighteThe number of clusters determined with STRUCTURE using 7643 SNPs was used for AMOVA and the calculation of Nei’s genetic distance using GenAlEx v6.503 . From AMOur study used DArTseq technology (SNP and silicoDArT markers) to examine the genetic diversity and population structure of 117 Myanmar rice genotypes. SNP and silicoDArT markers developed a large number of highly polymorphic markers and were a robust and relatively inexpensive option for future allele/gene identification. To date, this study was the first to use DArT in genetic diversity analysis on Myanmar rice genotypes. Based on our findings, the rice panel was genetically diverse. This level of genetic diversity could be the basis for developing new rice varieties with desirable characteristics such as high yield potential, high eating quality, and tolerance of abiotic stresses while being adapted to diverse environments. Moreover, this study identified two populations that could be explained by regional adaptation and natural selection. The presence of structure in this studied rice panel did not meet our expectations, which were to have three populations. HYVs were not found as a separate group. Instead, they were clustered together with several non-Pawsan traditional varieties in pop2. This might be due to breeding activities, suggesting that some breeding activities led to genetic similarities since new varieties were selected from local/traditional varieties. We also investigated some non-Pawsan varieties that were closely related to Pawsan varieties, and they were grouped together in pop1. The pop2 has a larger number of genotypes and exhibited higher values for genetic diversity indices and was thus more diverse than pop1. These findings will be important for future genetic analyses, such as allele/gene identification using genome-wide association studies, which is an approach that can identify the most important alleles for grain quality. Additionally, this study is advantageous for international trade to avoid adulteration, which means that markers specific for identifying the sub-group of Pawsan varieties could be helpful in identifying true Pawsan, which are highly priced varieties.


Monodon monoceros) and beluga whale (Delphinapterus leucas), using very high-resolution (VHR) satellite imagery. We analyzed 12 VHR images acquired in August 2017 and 2019, collected by the WorldView-3 satellite, which has a maximum resolution of 0.31 m per pixel. The images covered Clearwater Fiord (138.8 km2), an area on eastern Baffin Island, Canada where belugas spend a large part of the summer, and Tremblay Sound (127.0 km2), a narrow water body located on the north shore of Baffin Island that is used by narwhals during the open water season. A total of 292 beluga whales and 109 narwhals were detected in the images. This study contributes to our understanding of Arctic cetacean distribution and highlights the capabilities of using satellite imagery to detect marine mammals.Emergence of new technologies in remote sensing give scientists a new way to detect and monitor wildlife populations. In this study we assess the ability to detect and classify two emblematic Arctic cetaceans, the narwhal ( Emergence of new technologies in remote sensing are pushing forward the fields of ecology and conservation by expanding the range and scale at which researchers can conduct their studies and monitor populations –4. RemotTo face this last challenge, researchers have been relying on aerial sampling methods, such as aircrafts or RPAS, employing predetermined survey transects to limit the probability of counting individuals multiple times when monitoring wildlife . HoweverRangifer tarandus granti) migration on the north slope of Alaska , southern right whales , hauled-out Weddell seals (Leptonychotes weddellii), and polar bears (Ursus marinus) [In recent years, imagery from very high-resolution (VHR) satellites have successfully been used as a non-invasive method to monitor remote wildlife species both on land and water such as the Central Arctic Caribou Herd (marinus) , 14. Themarinus) , 16.Balaena mysticetus), beluga (Delphinapterus leucas), and narwhal (Monodon monoceros) which are year-round residents [Monitoring Arctic cetaceans is particularly difficult due to the remoteness and vastness of the Arctic environment. The Arctic Ocean spreads over 14 million square kilometers and is home to many cetacean species including the bowhead whale , humpback whales (Megaptera novaeangliae) and gray whales (Eschrichtius robustus) from VHR satellite imagery [To date, researchers have been able to identify large whales (>10 m) such as right whales, fin whales ..Fig 1). The Baffin Bay narwhal population is the largest narwhal population in the world , whose Tremblay Sound  satellite is a VHR commercial Earth observing system owned by DigitalGlobe. Launched in 2014, WV3 images the Earth at an altitude of 670 km in a sun-synchronous orbit, passing over the equator at 13:30  in the descending path. The swath width of the imagery is 13.1 km at nadir. A single band panchromatic image from WV3 has a spatial resolution of 0.31 m at nadir; while multispectral imagery collected using eight different spectral bands from visible to near infrared has a resolution of 1.24 m.2 of Clearwater Fiord in August 2019. The date of satellite image acquisition was based on likelihood to observe large numbers of belugas in the study area with calm sea-state (maximum Beaufort Sea State 3) and <15% cloud cover . The date was carefully chosen based on the absence of sea ice, a calm sea state  file.Two observers, A and B, gained experience analyzing satellite images for beluga from 18 WV3 images of Clearwater Fiord in 2017, prior to this study.2 grid, the images were read independently by the two observers from left to right at a scale of 1:535. Each observer recorded beluga whales or narwhals as georeferenced point stored data layers. When an observation was considered uncertain by an observer this observation was recorded in a new georeferenced point layer named “uncertain”. In this study the term agreement signifies that both observers identified the same object of interest, at the same location, and gave the same classification of either a whale or uncertain. Disagreement is used when an object was only seen by one observer, or when the two observers disagreed on the classification of the object as a whale or uncertain.The panchromatic images from WV3 were interpreted using QGIS 3.10. Using a 2.5 km2 taken in Clearwater Fiord. The cropped images were selected to provide a range of number and quality of detections in the sections.To determine if results obtained by observer A and B could be repeated by other marine mammal observers with no prior experience in whale detection from satellite imagery a third observer, who has had substantial experience reading aerial photos of belugas in the same area but was new to satellite imagery analysis, examined 23 cropped satellite image sections of 6.5 kmAfter the full visual analysis of beluga whale imagery was completed, a subsection  of what both observers agreed were surface and submerged belugas were selected for pansharpening to determine whether this method could enhance animal detection. Three different pansharpening algorithms were visually assessed  on surfaWe were unable to test pan sharpening algorithms on narwhals due to corruption of the multispectral data for these images.Beluga and narwhal individuals were clearly detectable from satellite imagery taken from the WV3 satellite .From the satellite images acquired of Clearwater Fiord in 2019, 378 targets were detected combined between the two observers. Most targets were observed on 4 August with 276 belugas and 68 uncertain targets detected. Only 10 belugas and 18 uncertain were observed on 6 August, and six belugas on 9 August. Of the total targets recorded in all three days, 7.9% (n = 30) were undetected by observer B, and 14.5% (n = 55) were undetected by observer A. Of the detected targets by both observers the mean percentage classification disagreement was 7% (n = 27) where an observer classified a target as a whale and the other observer classified the target as uncertain. The whale agreement between both observers was 89.7% (n = 262).Total targets from narwhal images taken on 2 August, 2017 were determined using the same criteria as for the beluga imagery described above, yielding 226 total targets (including uncertain observations). Of this total, 14.6% (n = 33) were undetected by observer B, and 13.7% (n = 31) were undetected by observer A. The classification disagreement was 4.4% (n = 10) where an observer classified a target as a whale and the other observer classified it as uncertain. The whale agreement between both observers was 90.8% (n = 99).There was 100% agreement between observers for all narwhals and belugas at the surface. All disagreements were individual targets that were perceived to be submerged resulting in darker coloration and where it was impossible to observe the whole outline body shape. The resolution of the images did not permit to differentiate nuances in color pattern between individuals or age classes. While juveniles are smaller than adults, we did not believe we could accurately distinguish adults from juveniles based on this sole criterion. Therefore, targets classified as whales could not be assigned as adults or juveniles.In the 23 cropped images, the third satellite imagery observer detected 52 objects of interest with 45 categorized as belugas and seven as uncertain observations, whereas the experienced observers detected 50 objects of interest with 45 as a beluga whale and five as uncertain observations. The third observer detected two objects of interest that were not scored by the experienced observers and two observations were identified as uncertain by the third observer, but classified as whales by the experienced observers. Ultimately, the third observer had a 93% (n = 42) agreement with the experienced observers when detecting and categorizing an object as a beluga whale and 71% (n = 5) agreement categorizing an observation as uncertain.The BT algorithm performed the best of the three pansharenening methods tested on two subsections of the images; one subsection containing what observers perceived as mainly surface animals, and one subsection where animals were perceived to be submerged below the surface. Each subsection contained four individual belugas. Using the BT algorithm, all possibilities of three-band combinations were created. After visually inspecting all combinations, the three top performing combinations were selected. The band combinations a) 1, 2, 3 ; b) 1, 5, 8 ; as well as c) 2, 3, and 4   were mosThis study contributes to our understanding of remote sensing and highlights the capabilities of using satellite imagery to detect marine mammals. While great whales have been detected using VHR satellite , 14, we Identifying and counting beluga and narwhal from satellite imagery provides a promising mechanism for evaluating their distribution and abundance. The count estimates from this study are not representative of population estimates, as only a small portion of the Cumberland Sound beluga and Eclipse Sound narwhal summer ranges were covered. Moreover, to date, no availability bias correction factors exist for these species using VHR satellite imagery to account for whales that are unable to be detected since they are too deep in the water column. However, future studies could expand coverage and use satellite imagery for developing surface abundance estimates. Current abundance estimates for beluga and narwhal are developed from twin-otter aerial surveys , 37. TheIn this study we are confident that only one species was present in each study area as assessed by field programs in Tremblay Sound in 2017 and Cumberland Sound in 2019. Thus, we were able to avoid misclassification of species. However, the morphological similarity between the species make them indistinguishable to human observers using panchromatic satellite images. Use of pansharpened algorithms and tailored bands may provide more detail for differentiating the species from one another in the future, which could allow this method to be used in areas where the two species might overlap . As sateDespite success using satellite imagery to count individuals of different species, this method is still in its infancy in terms of using it as a monitoring tool. As any other monitoring or survey method, satellite imagery comes with its own limitations and challenges, primarily, clear environmental conditions are required . For ins2 at a scale of 1:1,500 whereas in our study it took ~2.5 mins to scan 1 km2 at a scale of 1:536. Therefore, the next step in the use of VHR imagery for cetacean monitoring is automated detection of whales from space. The spectral difference between whales and the environment from radiometrically corrected imagery provides an opportunity to automate whale identification. Hurdles in automation include; false positives where other features resemble whales such as ice, waves, or rocks; whales that are slightly below the surface changing the apparent colour of the whale compared to surface individuals; and varying environmental conditions Additional information, such as the size and shape of the object, as well as texture differences from the surrounding environment can be used to increase detection rates, while minimizing false positives [One of the many challenges of using satellite imagery for cetacean monitoring is the extensive time requirement for readers to process the images. Cubaynes et al.  reportedositives . A numbeositives .As the Arctic continues to experience unprecedented climatic changes, the probability of obtaining clear VHR imagery may be lower . HoweverIn recent years, space-based solutions have become more popular for monitoring wildlife populations . This neS1 TableAll images were acquired from L3Harris Geospatial.(TIF)Click here for additional data file.S1 Database(CSV)Click here for additional data file.S2 Database(CSV)Click here for additional data file.