Eimeria oocysts counting per gram of feces were performed for each animal within 84 days of experiment. The following serum analytes were determined: total protein, albumin, globulin, cholesterol, haptoglobin, and 10 oxidative stress biomarkers: cupric reducing antioxidant capacity, ferric reducing ability of plasma, trolox equivalent antioxidant capacity, thiol, uric acid, paraoxonase-1, total oxidant status, ferric-xylenol orange, advanced oxidation protein products, and reactive oxygen metabolites derived compounds. The inclusion of WCS suggested the benefit in controlling infection as well as inducing an increase in antioxidants and a decrease in oxidants in lambs naturally infected by gastrointestinal parasites. The combination of WCS and ICLS could be a useful tool in controlling gastrointestinal parasite infection without affecting the production performance.The purpose of this trial was to evaluate serum levels of oxidative stress biomarkers and biochemical analytes in crossbred lambs during the rearing phase in an integrated crop-livestock system (ICLS) to control gastrointestinal parasites. The experiment used 36 crossbred lambs (cross: Ile de France × White Dorper × Texel) divided into two groups. The WCS group was supplemented with whole cottonseed (WCS), and controls had no supplementation. Body weight, blood collection, and fecal analysis of nematode eggs and  Eimeria spp. infections, which leads to considerable economic losses. Haemonchus contortus followed by Trichostrongylus colubriformis are the main GIN infecting sheep raised in Brazil [Eimeria spp.; they can have clinical and subclinical disease [Sheep farming faces a constant battle against gastrointestinal nematodes (GIN) and n Brazil ,2 and as disease . Lambs a disease . Thus, aGossypium L.). It combines high energy with high proportion of protein and effective fiber for ruminant supplementation [The production of sheep in an integrated crop-livestock system (ICLS) enables strategies that increase the income of those who produce milk, meat, and grains; it decreases the seasonality of animals for slaughter . Furtherentation ,8. It haentation  and can entation .The imbalance between oxidants and antioxidants either in cells or in the body are defined as oxidative stress ,12. OxidEimeria spp. with and without dietary inclusion of WCS in an ICLS.In animal health, biomarkers are less widespread and less developed than in human health. However, when applied from the One Health perspective, animal biomonitoring could provide important information, thus supporting the prevention and management of health risks . AntioxiThis experiment was conducted between December 2018 and July 2019 at Lageado Experimental Farm  belonging to FMVZ-UNESP, Botucatu, São Paulo, Brazil. According to the Köppen climate classification system, the region’s climate is Cfa type, which covers 6.5% of the Brazilian territory and 33.4% of the São Paulo state territory. The Cfa type is a warm temperate (mesothermic) humid climate ,15. The Zea mays L.) or soybean (Glycine max (L.) Merr.) silage in summer/autumn and black oat (Avena strigosa Schreb) oversown in winter/spring with different treatments made with intercropped marandu palisade grass (Urochloa (syn. Brachiaria) brizantha cv. Marandu), black oat, pigeon pea (Cajanus cajan (L.) Millsp.), and aruana guinea grass (Megathirsus (syn. Panicum) maximum cv. Aruana). The black oat pastures were grazed by lambs during the winter [The experimental area is part of an ICLS established since 2010. Before the present study, the area was used to produce maize  was sown in December 2018. The aruana guinea grass was intercropped with soybean as recommended [The study population comprised 36 crossbred (Ile de France × White Dorper × Texel) female lambs during the rearing phase with a mean age of 105 days, mean body weight of 25.6 ± 5.4 kg acquired from a commercial farm located approximately 160 km from Botucatu, São Paulo, Brazil. This study was approved by the Faculty’s Animal Experimentation Ethics Committee of the São Paulo State University—FMVZ, UNESP, Botucatu (0099/2019—CEUA).Clostridium spp. . Fecal samples were collected for counting of nematode eggs and Eimeria spp. oocysts per gram of feces  [®, Zoetis, São Paulo, Brazil) as a single application at 0.2 mg/kg on 6 May 2019. There was oral treatment with 5% toltrazuril suspension  due to an outbreak of eimeriosis on 26 June 2019, as a single application at 20 mg/kg.Before starting the experimental trial, the animals were tagged and vaccinated against ctively) . All ani2). The animals always had free access to water. The two groups were balanced as closely as possible by body weight, and the lambs were adapted to the diets for 14 days prior the sampling. At the paddocks, the grazing of the aruana guinea grass was rotational with a fixed stocking rate. This was distributed in groups as previously determined for pens in the covered shed. The area was divided by an electric fence into 24 paddocks 16 m × 30 m (480 m2); 12 paddocks for each group were rotated in the same three paddocks during the trial where they grazed for seven days each.Eighteen lambs were housed in four pens . They were fed concentrate, maize silage, and WCS. Another eighteen lambs were also housed in four pens . They were fed similarly but without WCS. Details about the diet ingredients are presented in The diets were formulated using the Small Ruminant Nutrition System (SRNS), computer program for sheep . The estFeces and blood samples were obtained from each animal every 14 days. Body weight was recorded at the same time. The samplings were: 0 days (8 May 2019), 14 days (22 May 2019), 28 days (5 June 2019), 42 days (19 June 2019), 56 days (3 July 2019), 70 days (17 July 2019), and 84 days (31 July 2019). The fecal samples, blood samples, and body weights at 0, 14, 28, 42, 56, 70, and 84 days were obtained at 6:00 a.m. with the animals fasting in the shed.Eimeria spp. oocysts per gram (g) of feces.Fecal samples were collected directly from the rectum of each lamb, stored in a labeled plastic bag, and transported in thermal boxes. The fecal counting of EPG and OPG were determined using the modified McMaster technique with a sensitivity of 100 eggs per gram of feces (EPG) ,22. The ® Blood Collection Tube; Becton, Dickinson and Company, Holdrege, NE, USA). After centrifugation (1500× g for 5 min) sera were stored in Eppendorf microtubes  at −80 °C until laboratory analysis of the biochemical analytes and biomarkers of oxidative stress. Serum haptoglobin (Hp) concentrations were measured via a hemoglobin binding assay using a commercially available colorimetric method . Total serum protein and albumin were measured using an automated analyzer  following the instructions of the manufacturer using Olympus commercial kits. The estimated concentrations of globulins were calculated by the difference between total proteins and albumin concentrations. Total serum cholesterol was determined using a commercial kit .Blood samples were collected from the jugular vein in plain tubes with gel separators  in serum was determined with three different assays : Trolox Oxidant biomarkers such as advanced oxidation protein products (AOPP), a marker of oxidative damage to proteins, was based on di-tyrosine containing cross-linked proteins and oxidatively modified albumin . Total o® Studio  with the General Linear Model (GLM). Tukey’s test at a 5% significance level was used to compare means. The EPG and OPG transformed results were reported in tables with arithmetic means (±standard deviation). The graphs used raw data with medians (interquartile range). The results of the variables that had normal distributions were reported in tables with arithmetic means (±standard deviation); the graphs plotted the arithmetic means (±standard error of the mean). A p-value of < 0.05 was considered statistically significant for all statistical analysis.All variables were first assessed for normality with graphic analysis of histogram, QQ plot, and a Shapiro-Wilkes test. The data that did not present a normal distribution (EPG and OPG) were transformed to log10 (x + 1). All data were analyzed by analysis of variance via the Statistical Analysis System-SASEimeria spp. infections were detected in lambs during the trial. Significant days effects for EPG and OPG counting were observed during the trial for both groups  for ADG, however, there were significant group versus days interactions (p = 0.028) and days effects (p < 0.0001)  .Biochemistry analytes for control and WCS lambs are presented in Antioxidant serum biomarkers for control and WCS lambs are presented in Serum oxidant biomarkers for control and WCS lambs are presented in Eimeria spp. with a low number of Eimeria spp. oocysts at the beginning of the trial. However, there was a peak of OPG counts at 42 days for both treatments. Lambs raised in high densities contribute to the transmission of this protozoan, thus causing a significant elimination of oocysts in the feces [At the beginning of the experiment, a higher general mean of EPG counting was observed which reduced along the experimental period. High means of EPG counting were observed at the beginning of an ICLS trial with crossbred male lambs with a subsequent decrease in the EPG counting throughout the experiment . The lamTwo factors that could have played major roles in decreasing the EPG and OPG counts were the good quality of the diet and the inclusion of WCS because immunity possibly developed efficiently to control the GI parasites infection, thus again increasing the body weight gain ,6. The sEimeria spp. infection. This effect could be attributed to the presence of gossypol in the WCS, which is an antinutrient polyphenolic compound secreted by pigment glands distributed throughout the cotton plant with higher concentrations in the seeds [Trypanosoma cruzi [Entamoeba histolytica [There was a significantly lower overall mean of OPG counts for the WCS treatment in this trial, thus suggesting a positive effect for WCS supplementation to control he seeds ,36. In ama cruzi  as well tolytica . Althougtolytica ,36.Eimeria spp. and GIN co-infection. There could be changes in serum proteins in animals with parasitic infections caused by GIN [Eimeria spp. in sheep [In our study, a higher albumin concentration was observed for the control group with a significant decrease in albumin and total protein concentrations in both groups at 56 days, thus demonstrating the side effects and the body late recovery reaction to d by GIN ,39,40 anin sheep ,42. Albuin sheep . In the An impairment of the intestinal absorption of lipids could lead to diminished appetite and low digestibility of food, which are negative effects of GI parasite infection . No chanBiomarkers of oxidative stress provide data regarding the monitoring of different procedures to reduce the parasite load and GIN in sheep ,47,48,49The TEAC, FRAP, and CUPRAC assays involve several antioxidant molecules and were used to evaluate TAC. Although FRAP and CUPRAC concentrations did not change, the higher TEAC concentration in lambs supplemented with WCS could reflect an increase in the antioxidants related to a decrease in GI parasites . IncreasAt the end of the trial TOS and FOX concentrations showed a significant decrease in the WCS group. There are literature reports demonstrating increased oxidants concentrations in sheep infected with GIN ,48,49, wEimeria spp. led to limitations for the lambs including an initial poor performance of the animals. Furthermore, the Eimeria spp. infection played a role in this scenario although the lambs reached the expected daily weight gain at the end of the trial. Our current scenario aims for sustainable sheep production including animal welfare, thus decreasing the use of unnecessary anthelmintic treatments that could contribute to the development of parasite resistance while diminishing the environmental impacts caused by animal production. According to the findings of this experimental trial, the WCS inclusion in lambs’ diets could be considered an additional alternative aiming to control GI parasite infections. Further studies should be conducted with different percentages of WCS inclusion in the diet.In summary, the sum of the co-infection by GIN and The WCS inclusion in this trial suggested a benefit to controlling the natural infection by GIN as well as inducing a decrease in oxidants such as TOS and FOX and an increase in the antioxidant TEAC in lambs. The combination of WCS and ICLS could be a useful tool in controlling gastrointestinal parasite infection without affecting the production performance.


The global burden of critical illness falls disproportionately outside high-income countries. Despite younger patient populations with similar or lower disease severity, critical illness outcomes are poor outside high-income countries. A lack of data limits attempts to understand and address the drivers of critical care outcomes outside high-income countries.We aim to characterize the organization, available resources, and service capacity of public sector critical care units in Malawi and identify barriers to improving care.We conducted a secondary analysis of the Malawi Emergency and Critical Care Survey, a cross-sectional study performed from January to February 2020 at all four central hospitals and a simple random sample of nine out of 24 public sector district hospitals in Malawi, a predominantly rural, low-income country of 19.6 million in southern Africa. Data from critical care units were used to characterize resources, processes, and barriers to care.There were four HDUs and four ICUs across the 13 hospitals in the Malawi Emergency and Critical Care Survey sample. The median critical care beds per 1,000,000 catchment was 1.4 (IQR: 0.9 to 6.7). Absent equipment was the most common barrier in HDUs (46% [95% CI: 32% to 60%]). Stockouts was the most common barriers in ICUs (48% [CI: 38% to 58%]). ICUs had a median 3.0 (range: 2 to 8) functional ventilators per unit and reported an ability to perform several quality mechanical ventilation interventions.Although significant gaps exist, Malawian critical care units report the ability to perform several complex clinical processes. Our results highlight regional inequalities in access to care and support the use of process-oriented questions to assess critical care capacity. Future efforts should focus on basic critical care capacity outside of urban areas and quantify the impact of context-specific variables on critical care mortality. Realizing the goal of Universal Health Coverage (UHC)—that all people receive the full range of quality health services without exposure to financial hardship —requiresCritical care—the recognition, monitoring, and treatment of patients with organ dysfunction or high risk of rapid deterioration and associated morbidity and mortality —is a sig45656Malawi is a landlocked LIC in southern Africa with a predominantly rural population of 19.6 million.  DistrictFacilities in Malawi lack many of the medications, equipment, and guidelines essential for the management of severe illnesses . There iA lack of data limits attempts to understand why critically ill patients outside of HICs have worse outcomes. This is particularly true for LICs, which are often left out of large international critical care studies. The few critical care studies with data from LICs are limited by an emphasis on urban academic and central referral hospitals and the use of convenience samples 131415.1314Improving critical care outcomes and progressing towards UHC requires additional data on LIC critical care resources, processes, and barriers . In the We conducted a secondary analysis of the MECC Survey, a cross-sectional study conducted from January to February 2020 assessing emergency and critical care (ECC) service readiness at all four central hospitals, in addition to a simple random sample of nine out of 24 public sector district hospitals in Malawi. The design and primary analysis of the MECC Survey are described elsewhere . This seEthical approval for the MECC Survey protocol was granted by the Partners Healthcare Institutional Review Board in Boston, USA (2019P003457) and the National Health Science Research Committee in Malawi .The MECC Survey instrument is designed to generate estimates of critical care service readiness at the level of the hospital or hospital unit  using data collected from clinical staff informants working at each targeted hospital unit. The instrument consists of the WHO Hospital Emergency Unit Assessment Tool (HEAT)  with theThe instrument uses several response structures. Signal function questions asked informants to describe the availability of a given resource or intervention on a scale of 1 to 3, with 1 indicating “generally unavailable”, 2 “some availability”, and 3 “adequate availability”. Availability was defined as how often patients in the target unit are able receive a resource or intervention within the timeframe needed for critical care.Signal function responses of “generally unavailable” or “some availability” triggered a follow-up probe on the barriers encountered within the unit. Barriers were coded by informants and study staff into WHO HEAT categories of infrastructure, absent equipment, broken equipment, stockout, personnel, training, user fees, and opening hours. Informants were permitted to identify multiple barriers for each signal function. For frequency questions, informants were asked to respond on a scale of 1 to 5, with 1 indicating “almost never”, 2 “infrequently”, 3 “sometimes”, 4 “frequently”, and 5 “almost always”.Between January 20 and February 18, 2020, the MECC Survey research team visited all 13 sampled hospitals. At each hospital, study staff administered the instrument to one administrator and 3 clinicians from each targeted unit . To minimize the time burden, questions on staffing, protocols, cardiac monitoring, crash trolleys, social work, security, dieticians, physiotherapists, and spiritual support were only asked to one designated clinical lead at each unit. Data was collected and managed using REDCap electronic data capture tools.Informants for the MECC Survey were clinical staff aged 18 years or older, who self-reported working for at least one month at the hospital unit of interest. All informants provided written informed consent.The MECC defined critical care units  as discrete physical spaces within a hospital, dedicated to the care of critically ill patients. In Malawi, ICUs are typically in central hospitals and have the most resources and capacity; critical care units at district hospitals are generally designated as HDUs. In central hospitals, HDUs provide care for critically ill patients that are not prioritized for admission to ICUs . The MECVariables were analyzed and reported at the level of the hospital unit . We defined “adequate availability” at the unit level as a mean informant response >2.5 (out of 3). For yes/no questions, an item with at least two “yes” responses in a unit was considered available. For frequency questions, an item was considered present at the unit level if the mean was >4 out of 5. For barriers, participant level data was first calculated by taking the number of times each barrier was identified by the informant divided by the number of times the informant was probed for barriers .Participant-level data for each barrier category  was calculated using the following formula:n is the total number of signal function questions; y represents signal function questions to which the participant responded, “generally unavailable” or “somewhat available” and identified the barrier category  when probed; and x represents signal function questions to which the participant responded, “generally unavailable” or “somewhat available.”where We defined a level 1 ICU as any unit meeting nine criteria adapted from the World Federation of Societies of Intensive and Critical Care Medicine (WFSICCM): physicians with some experience in critical care available at least during the day, higher nurse to patient ratios, at least a twice daily reassessment of patients, pulse oximetry, cardiac monitoring, oxygen therapy, non-invasive support, basic quality improvement, and transfer policies .The Society of Critical Care Medicine’s ABCDEF bundle is a tool for the implementation of quality mechanical ventilation . The MECBecause resources or interventions cannot be provided in a timely manner if staff are unaware of their availability, responses of “don’t know” were coded as “generally unavailable” for signal functions. Responses of “don’t know” were coded as “missing/incomplete” for frequency questions because the frequency of an event is not dependent on clinician awareness. These same approaches were used for unit variables with one “missing/incomplete” response . If data was missing from two or more informants in a unit, we considered unit data for the variable as “missing/incomplete”.Data was analyzed using Stata (Release 16). The sample size of nine district and four central hospitals was determined for the broader MECC Survey and reported separately . We repoDespite their overlapping functions, there are substantial differences between ICUs and HDUs . Therefore, we reported separate data for HDUs and ICUs. Comparisons of barriers between ICUs and HDUs were made with Fisher’s exact test, using a nominal level of 5% for statistical significance (two-tailed).This manuscript adheres to the reporting standards of the STROBE Statement for cross-sectional studies.The funders had no role in study design; collection, analysis and interpretation of data; decision to publish; or preparation of the manuscript.A total of four HDUs and four ICUs were included in MECC Survey sample of nine district hospitals and four central hospitals. All four HDUs and four ICUs were included in this analysis . Three cHDUs had a median 4.0 (range: 2 to 4) beds per unit, while ICUs had 4.0 (4 to 8). The median number of critical care beds  per 1,000,000 catchment population was 1.4 (interquartile range [IQR]: 0.9 to 6.7). Most HDUs and ICUs provided care to medical, surgical, obstetric, and trauma patients. No unit met all level 1 ICU criteria (Table S1).All critical care units had nurses and providers present 24 hours a day . In ICUsAll HDUs and two (50%) of the ICUs had written policies outlining criteria for admission to the unit (Table S3). Staff reported checking vital signs at regular intervals every 1.3 (1 to 2) hours at HDUs and 2.0 (2 to 2) hours at ICUs. The availability of condition-specific treatment protocols was variable. Half of all critical care units had protocols for asthma and pneumonia treatment. Although a sepsis protocol was available at three (75%) HDUs and three (75%) ICUs, a volume resuscitation protocol was available at one (25%) HDU and three (75%) ICUs. Protocols for end-of-life care were available at two (50%) HDUs and no ICUs.Aside from peripheral intravenous cannulas, which all critical care units could place, options for venous access were limited. Only one (25%) HDU and two (50%) ICUs could establish central venous access, and no units were able to place an intraosseous device or perform venous cutdown. . One (25%) HDU and three (75%) ICUs were able to administer intravenous vasopressors .Except for one HDU, all critical care units could perform bag-valve-mask ventilation . All ICUICUs could perform most signal functions from the ABCDEF mechanical ventilation bundle . All ICUFor critical care signal functions in This study of Malawi’s public sector ICUs and HDUs identified several strengths, including nurse staffing and the ability to perform a variety of core critical care processes. We also found a need for an increased availability of critical care specialists, clinical protocols and policies, and adequate equipment and supplies, particularly in HDUs. This information has the potential to shape policymaking, care delivery, and the critical care research agenda outside of HICs.Critical care unit capacity in Malawi is limited with only 20 ICU and 14 HDU beds across the MECC Survey sample of all four full-service public sector central hospitals and nine of 24 district hospitals. Despite reported capabilities—such as cardiac monitoring, administration of vasopressors, regular clinician reassessment, and routine patient repositioning—no unit met all the criteria for a level 1 ICU. Although the WFSICCM ICU criteria is designed to be adjusted based on context-dependent factors [Resources and capacity were disproportionately lower in HDUs; several lacked essentials, such as electricity, running water, and oxygen. While it may be unsurprising that ICUs have greater capabilities than HDUs, the degree of disparity raises concerns about access to care. The urban distribution of ICUs is misaligned, with 82% of Malawians residing rural areas .In terms of human resources, critical care units in Malawi were relatively well-staffed with nurses. The daytime bed-to-nurse ratio of 1.6 in Malawian ICUs is in line with other national and international assessments 2324, alt231525The presence of condition-specific protocols, including for common conditions, such as asthma, pneumonia, and diabetic ketoacidosis was inconsistent. ICU-specific protocols were also variably present, with only 50% of ICUs with formal admission criteria. Similar findings have also been reported in Tanzania (50%)  and UganAlthough barriers were identified across all categories, absent equipment and stockouts were the most common barriers identified in HDUs and ICUs, respectively. This diverges from prior LIC critical care data, which identified equipment and commodity availability as relative strengths 14. WhileCritical care often includes caring for dying patients. This includes ensuring patient comfort and dignity and discontinuing aggressive interventions  when inconsistent with the expressed goals of the patient or family. None of the ICUs included in the MECC Survey had a written policy guiding the withdrawal of mechanical ventilation, and only one reported the ability to de-escalate care in these situations. This is striking, considering that in one Malawian ICU, nearly 10% of all ICU admissions are diagnosed with brain death, yet all receive intensive care until cardiac death .Mechanical ventilation is one of the most complex interventions provided in critical care units, requiring detailed quality processes reliant on the availability of equipment, reliable infrastructure, meticulous expert nursing care, and well-trained clinicians. ICUs in this study had 0.9 ventilators per 1,000,000 catchment population. By comparison, Ghana has 1.7 , The GamDespite a limited numbers of ventilators, Malawian ICUs reported the ability to perform several processes central to high quality mechanical ventilation, such as spontaneous breathing and awakening trials. However, one ICU study from Malawi estimated a 29.8% mortality for mechanically ventilated patients . SimilarThis study provides additional evidence of the global disparity in access to critical care and highlights the need for a comprehensive approach to strengthening basic critical care services across all levels of the health system. For LICs with predominantly rural populations, this must include an emphasis on equipping district hospitals so they can rapidly identify and stabilize critically ill patients and provide access to safe transfer services to higher level facilities, as appropriate. Opportunities for postgraduate critical care physician training and ongoing nursing training are a priority. Yet, even with specialized training, critical care staff are unable to effectively treat patients without the necessary equipment and resources. HDUs may need upfront investment and procurement, and ICUs would benefit from an ongoing strengthening of the supply chain and logistics.A research agenda should focus on identifying and measuring factors that determine critical care outcomes. Commentators have advocated for a multifaceted approach  30, whichThis study has several limitations. Data from ICUs in Malawi may not be generalizable to other LICs. The facility sample, calculated for a broader study, included all public central hospital ICUs, but was relatively small. This study aimed to characterize publicly accessible critical care in Malawi and therefore, only sampled government facilities. Although scarce, there are at least two non-governmental ICUs in Malawi , and theThese findings provide a detailed description of critical care, including the resources, processes, and barriers in public sector HDUs and ICUs in Malawi. Although many gaps are present, HDUs and ICUs reported the ability to perform multiple complex critical care processes. Our results highlight both international and regional disparities and raise the possibility that process-oriented questions could provide more accurate information about critical care service availability. Future efforts should be directed at strengthening essential critical care capacity outside of urban areas and quantifying the impact of context-specific variables on mortality from critical illness.The additional file for this article can be found as follows:10.5334/aogh.4053.s1Supplemental Tables.Tables S1 to S3.


Treponema pallidum subspecies pallidum. Since 2012, syphilis rates have risen dramatically in many high-income countries, including England. Although this increase in syphilis prevalence is known to be associated with high-risk sexual activity in gay, bisexual, and other men who have sex with men (GBMSM), cases are rising in heterosexual men and women. The transmission dynamics within and between sexual networks of GBMSM and heterosexual people are not well understood. We aimed to investigate if whole genome sequencing could be used to supplement or enhance epidemiological insights around syphilis transmission.Syphilis is a sexually transmitted bacterial infection caused by T pallidum genome sequences previously generated from patient samples collected from across England between Jan 1, 2012, and Oct 31, 2018, and performed detailed phylogenomic analyses.We linked national patient demographic, geospatial, and behavioural metadata to whole T pallidum sublineages in England. Sublineage 1 was found throughout England and across all patient groups, whereas sublineage 14 occurred predominantly in GBMSM older than 34 years and was absent from samples sequenced from the north of England. These different spatiotemporal trends, linked to demography or behaviour in the dominant sublineages, suggest they represent different sexual networks. By focusing on different regions of England we were able to distinguish a local heterosexual transmission cluster from a background of transmission in GBMSM.Of 497 English samples submitted for sequencing, we recovered 240 genomes . Three duplicate samples (same patient and collection date) were included in the main phylogenies, but removed from further analyses of English populations, leaving 237 genomes. 220 (92·8%) of 237 samples were from men, nine (3·8%) were from women, and eight (3·4%) were of unknown gender. Samples were mostly from London (n=118 [49·8%]), followed by southeast England (n=29 [12·2%]), northeast England (n=24 [10·1%]), and southwest England (n=15 [6·3%]). 180 (76·0%) of 237 genomes came from GBMSM, compared with 25 (10·5%) from those identifying as men who have sex with women, 15 (6·3%) from men with unrecorded sexual orientation, nine (3·8%) from those identifying as women who have sex with men, and eight (3·4%) from people of unknown gender and sexual orientation. Phylogenomic analysis and clustering revealed two dominant T pallidum genomes globally, genomics can still be used to identify putative transmission clusters for epidemiological follow-up. This could be of value for deconvoluting putative outbreaks and for informing public health interventions.These findings show that, despite extremely close genetic relationships between Wellcome funding to the Sanger Institute, UK Research and Innovation, National Institute for Health and Care Research, European and Developing Countries Clinical Trials Partnership, and UK Health Security Agency. Treponema pallidum subspecies pallidum . Syphilis rates have been rising in many high-income countries since the beginning of the 21st century.Syphilis is a sexually transmitted infection (STI) caused by the bacterium Evidence before this studyTreponema pallidum are challenging due to low bacterial loads in clinical specimens, and difficulty in culturing the bacteria. We searched PubMed from Jan 1, 1998, to Aug 9, 2022, using the search terms “Syphilis” or “Treponema pallidum” and “genomic” or “genome(s)” or “sequencing”. We found 23 studies describing whole genome sequencing of the T pallidum subspecies pallidum, of which two used whole genome phylogenies to investigate sexual network epidemiology, with one large study of sexual networks done primarily in Victoria, Australia, which characterised two major circulating sublineages in that setting and putative sexual transmission networks with distinct sexual behavioural characteristics and potential bridging between networks.Detailed phylogenomic analyses investigating the epidemiology and transmission dynamics of Added value of this studyT pallidum genomes, and characterised the transmission dynamics of syphilis using samples from across the whole of England. Integration of national-level sociodemographic, spatiotemporal, and genomic data allowed the delineation of putative sexual networks at both the national and region levels, and revealed patterns not previously detected using epidemiological or genomic data alone.In this study, we linked national surveillance data to Implications of all the available evidenceOur findings are consistent with findings in Australia that indicate that genomics can identify putative sociodemographic transmission clusters. However, in that study, genomic clusters included samples separated by multiple single nucleotide polymorphisms, which could represent several years of evolution. Our study explored the value of linking identical genomes, and highlights that, despite technical constraints, whole genome sequencing can be used to enable outbreak exclusion and identify putative local transmission clusters for epidemiological follow-up.T pallidum strains. However, these methods might not accurately reflect recent evolutionary relationships between strains,Although epidemiological surveillance provides insights into the rise in syphilis rates, this is not always sufficient. For example, a group of spatiotemporally clustered cases could represent a single outbreak and chain of transmission, but could also be the result of separate or unrelated transmission networks. Molecular typing methodsT pallidum lineages globally (Nichols and SS14),T pallidum genomes accumulate single nucleotide polymorphisms (SNPs) very slowly, with a median molecular clock rate equivalent to one substitution per genome every 6·9 years (95% highest posterior density 5·9–8·2 years),T pallidum within the past 30 years, driven by a small number of multi-country sublineages,Whole genome sequencing (WGS) has shown there are two co-circulating T pallidum, with studies from JapanT pallidum samples from England to gain insights into the different spatiotemporal and genomic transmission patterns of syphilis affecting GBMSM and heterosexual people.Few studies have combined WGS with patient demographic and sexual behaviour metadata to explore epidemiological trends of T pallidum-positive genomic DNA samples were retrieved from historical archives (2012–17) held at the UK Health Security Agency  STI Reference Laboratory , and prospectively collected (2016–18) from five laboratories with high syphilis caseloads who do in-house molecular T pallidum diagnostic testing .A detailed description of sample collection and patient metadata linkage in this population-based study is provided in GUMCAD]). For samples prospectively collected from the five non-referring laboratories, patient metadata available from local laboratory information systems was linked to GUMCAD data and integrated into the larger dataset after deduplication  Health Research Authority and Health and Care Research Wales  and the London School of Hygiene & Tropical Medicine Observational Research Ethics Committee (16014). Diagnostic samples were identified at UKHSA using internal laboratory information systems. UKHSA has permission to process confidential patient data under regulation 3 (control of patient information) of the UK Health Service Regulations 2002. Information governance advice and ethics approval for this study were granted by the UKHSA Research Ethics and Governance Group. Full details of approvals and pseudonymisation of samples and patient metadata are described in T pallidum samples used in this study has been previously described,WGS of all clinical T pallidum samples and distinguish transmission clusters by location or sexual orientation. A secondary outcome was to investigate rates and distribution of macrolide resistance-conferring alleles among T pallidum sublineages.The primary outcome was to identify local lineages of All statistical analyses were done in R, version 4.1.2 .The funders of the study had no role in study design, data collection, data analysis, data interpretation, writing of the report, or decision to submit.Between Jan 1, 2012, and Oct 31, 2018, we identified and submitted 497 samples considered suitable for sequencing, from which we recovered 240 genomes  of 237 genomes came from GBMSM, compared with 25 (10·5%) from MSW, 15 (6·3%) from men with unrecorded sexual orientation, nine (3·8%) from WSM, and eight (3·4%) from people of unknown gender and sexual orientation . Notablyvs non-London), and HIV status (GBMSM only). However, a greater proportion of cases in the genome collection were GBMSM , with fewer women (nine [3·8%] vs 6467 [12·7%]) and MSW (25 [10·5%] vs 8978 [17·7%]). The genome collection also had a much higher proportion of primary syphilis cases compared with GUMCAD (114 [81·4%] in the genome collection compared with 14 178 [27·9%] from GUMCAD), largely reflecting the clinical presentation of primary syphilis with ulcers that permit swabbing.To establish how representative our national genome collection was of syphilis cases in England, we compared the distributions of sociodemographic characteristics of cases in the national genome collection with those from GUMCAD . OverallT pallidum genomes carried a wild type ribosomal 23S gene and were therefore predicted to be sensitive to macrolide antimicrobials.We inferred the presence of macrolide resistance-conferring SNPs in the ribosomal 23S as previously described,T pallidum phylogeny  were largely representative of syphilis patients overall, with 130 (74·3%) of 175 classified as GBMSM . 11 (55·0%) of these 20 patients were part of a cluster containing only other heterosexual individuals. Of the four genome clusters containing WSM, three contained only other heterosexual people and each of these clusters was detected in only a single region. However, the fourth cluster containing a WSM was the largest cluster of identical genomes, comprising 42 samples from sublineage 1, of which 35 patients (83·3%) were GBMSM, compared with three MSW, one WSM, and three with unknown sexual orientation. Therefore, this primarily GBMSM cluster also includes four patients who identify as heterosexual, indicating that there might be some bridging between the populations.To explore sociodemographic patterns of T pallidum from England, we used a Bayesian time-scaled phylogeny of the global dataset  was present in all regions (and represented the only sublineage present in the three northern regions), while sublineage 14 (Nichols lineage) was co-circulating with sublineage 1 in London, the south, and central England but not in northern England , all other regions contained at least three sublineages (range 3–7). 118 (49·7%) of 237 samples were from London, and we detected six sublineages  and one sublineage previously labelled as a singleton. As elsewhere, London was dominated by sublineages 1  and sublineage 14 . The rare sublineages 15 (n=2) and 16 (n=1) were each found in a single region (sublineage 15 in the east of England and sublineage 16 in London), but all other sublineages were found in multiple regions .Our geospatial analysis showed that all samples from the three most northerly regions of England were from SS14 sublineage 1 , contrasCluster 2 comprised two samples collected in 2012 and 2013 from GBMSM with identical core genomes and, similarly to cluster 1, formed a clade with eight other samples, of which five were from elsewhere in England (four of five GBMSM and one of five unknown) and three were from PortugalT pallidum genomes from 237 patients diagnosed with syphilis in England between 2012 and 2018. Our analysis shows a variety of English sublineages, dominated by global sublineages 1 and 14,T pallidum population structures co-circulating in Melbourne and the Northern Territory. Both common sublineages (1 and 14) contained people living with and without HIV, and there was no phylogenetic delineation by HIV status, suggesting either that HIV status might not be strongly associated with transmission patterns, or that such patterns are beyond the ability of WGS-based analyses to detect.In this study, we linked patient demographic, spatiotemporal, and behavioural metadata to previously generated We were also able to examine whether the data could be used to define GBMSM and heterosexual transmission networks based on the proportion of individuals identified as GBMSM or heterosexual for each genomic cluster.T pallidum genomes occupying positions across the sublineage 1 phylogeny and interspersed with samples from around the world, in the northeast of England we identified a genetically distinct cluster of identical core genomes found exclusively in heterosexual people, consistent with reports of a syphilis outbreak in heterosexual people at that time.T pallidum,T pallidum. This could offer opportunities to intervene or educate sexual networks, and to determine or exclude outbreak membership.As in most countries, samples from England were dominated by sublineage 1.Neisseria gonorrhoeae accumulates SNPs at a rate of eight substitutions per genome per year,T pallidum. Therefore, even T pallidum isolates with identical genomes do not necessarily indicate recent direct patient-to-patient transmission. Conversely, samples separated by even a very small number of SNPs are unlikely to share a recent common ancestor. Furthermore, because the potential transmission window of T pallidum might be as high as 2 years,In other STIs, such as gonorrhoea, SNP cutoffs of either five or ten SNPs have been used to infer transmission chains.T pallidum transmission dynamics, and enable the fuller usefulness of sequence data to inform public health interventions.Our study has several limitations, including the small number of samples compared to the total number of syphilis cases during the time period, and overrepresentation of samples from GBMSM in the national genome collection. The genomes represented 0·5% of the recorded number of syphilis cases in England during the study period and all samples referred to the National Reference Laboratory with sufficient treponemal DNA for sequencing. Although the available referral population might not be fully representative of syphilis in England due to regional variation in molecular testing and referral practices, all samples were collected and sequenced in the absence of any genetic relatedness information, so our genomic observations provide a snapshot of circulating English lineages. Future studies that focus on the systematic collection of samples from a higher proportion of cases, combined with improved sequence quality, will enable further insights into https://www.ebi.ac.uk/ena) in BioProjects PRJEB28546, PRJEB33181, and PRJNA701499. All accessions, corresponding sample identifiers, and related metadata are available in appendix 2. Patient metadata for the UK genomes is available in pseudonymised form in appendix 3. UK shape files for Public Health region boundaries were downloaded from the UK Office for National Statistics, available at https://geoportal.statistics.gov.uk. The R code for all phylogenetic and statistical analysis and plotting is available in an Rnotebook along with all sample metadata (pseudonymised) and intermediate analysis files at https://doi.org/10.6084/m9.figshare.21543333.v1 and https://github.com/matbeale/Syphilis_Genomic_Epi_England_2022-23.Sequencing reads for all genomes used in this study have been previously published and described, and are available at the European Nucleotide Archive (We declare no competing interests.


Zebu cattle can produce food in high quantity and quality under adverse farming conditions due to their greater tolerance to heat stress and resilience to parasites compared to taurine breeds. With advances in genetic improvement programs, Gyr dairy cows currently have production rates comparable to those of some taurine herds. The present study aimed to characterize the metabolic profile  of Gyr cows during the transition period, which comprises the 21 days before and after parturition. This period constitutes a fundamental phase in the life of a dairy cow, as significant hormonal and metabolic changes occur, which can predispose the cow to diseases, in addition to affecting its productive indices. This study provides relevant information by showing that high-yielding Gyr cows are metabolically balanced and less predisposed to disease during the transition period compared to taurine dairy breeds. The possibility of rearing this Zebu breed in the tropical regions of the world with excellent production rates highlights the importance of this subject.This study aimed to evaluate the effects of parity, body condition score (BCS) at calving, and milk yield on the metabolic profile of Gyr (Zebu) cows. Healthy cows in late pregnancy were grouped according to parity ; to BCS scale at calving ; and to milk yield (high—HP and moderate—MP production). BCS was assessed, and blood samples were collected on −21, −7, 0, 7, 21, and 42 days relative to parturition. The concentrations of non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), cholesterol, glucose, total protein (TP), albumin, total calcium (Ca), phosphorus (P), and magnesium (Mg); and activities of aspartate aminotransferase and gamma-glutamyltransferase were measured. Data were analyzed by two-way repeated measures ANOVA. The frequencies of high lipomobilization, subclinical ketosis, subclinical hypocalcemia (SCH), and the occurrence of diseases during early lactation were established. Regardless of grouping, NEFA, BHB, and cholesterol increased during early lactation; glucose showed higher values at calving; TP and albumin were higher at 21 and 42 DIM; and Ca, P, and Mg were lower at calving. Parity had little effect on the metabolic profile, HBCS did not differ from NBCS cows, and HP did not differ from MP cows in most metabolites. High lipomobilization in early lactation and SCH at calving were the most common imbalances but were not related to postpartum diseases. High-yielding Gyr cows have a balanced metabolic profile during the transition period, with few biologically relevant effects of parity, BCS at parturition, or milk yielded. Bos taurus indicus) are characterized by their ability to produce milk in pasture systems under adverse environmental conditions due to their tolerance to heat stress and parasites [Gyr cattle  guidelines. The project was previously approved by the Ethics Committee on the Use of Animals (CEUA) of the Universidade Estadual de Londrina (protocol number: CEUA-UEL 9250.2018.90).This study was carried out between June and December 2019 in a commercial dairy farm located in the state of Minas Gerais, Brazil . The rainy season (November to March) is humid and cloudy with an average monthly minimum temperature of 18 °C and maximum of 31 °C. The dry season (May to September) is generally clear, with a minimum temperature of 12 °C and a maximum of 26 °C . The herd comprised 154 lactating Gyr cows with mean body weights varying between 370 kg (primiparous cows) and 520 kg (multiparous cows), and an average individual daily milk yield ranging from 19 to 33 kg. Total daily production varied between 3000 and 3700 kg of milk.n = 26), biparous , and multiparous  groups, all with BCS at calving ranging from 3.0 to 3.5, according to the traditional scale [n = 21) and normal BCS . The HBCS group was composed exclusively of multiparous cows. For the NBCS group, all cows from the M group were included, in addition to 4 cows drawn from the B group. It was not possible to include cows that calved with a low BCS because this condition was not observed during the study period. Finally, most of the cows included in the study, except the P cows, were redistributed into the following two other groups according to milk yield at 60 days in milk (DIM): a high producing group  and a moderate producing group . The HP group consisted of 5 biparous cows and 17 multiparous cows, while the MP group consisted of 13 biparous cows and 17 multiparous cows. Cows with dystocia or twin pregnancies were not included in the study.Eighty-five healthy cows at approximately 240 days of gestation were included based on parity and BCS. They were divided into primiparous  calculated for the cows in the prepartum transition period and during lactation  and lactating cows. Each batch was housed in uncovered paddocks, with an available space of 10–15 mactation . The TMRThe calves were separated from the cows immediately after calving, and the colostrum was milked within one hour postpartum using a mechanical milking system. Throughout lactation, the cows were manually milked twice daily (at 5 a.m. and 3 p.m.). Individual milk yields were measured by means of weighing every 15 days.The BCS was always evaluated by the same trained examiner at −21 (±3), −7 (±2), 0, 7, 21, and 42 days relative to calving, using the traditional scale .g for 10 min). The serum and plasma obtained were stored in 2 mL aliquots and frozen (−20 °C) until processing six months later.At the same time points, blood samples were collected by coccygeal venipuncture using a 30 × 0.8 mm needle and vacuum tubes without an anticoagulant and with EDTA and sodium fluoride. On the day of calving, blood samples were collected immediately after milking the colostrum. At other time points during lactation, samples were collected after the first milking of the day. After a 60 min rest, the samples were centrifuged (2000× Serum concentrations of non-esterified fatty acids (NEFA) ; beta hydroxybutyrate (BHB) ; cholesterol ; total protein (TP) ; albumin ; total calcium (Ca) ; phosphorus (P) ; and magnesium (Mg) ; plasma glucose concentration ; and serum aspartate aminotransferase (AST)  and gamma-glutamyltransferase (GGT)  activities were measured in an automated spectrophotometer  using specific commercial reagents.® at 7, 21, and 42 DIM for the diagnosis of endometritis or metritis according to the established classification [The cows were monitored daily, and the diseases that occurred during early lactation (up to 60 DIM) were evaluated. Uterine secretions were evaluated by MetrichekThe cutoff points adopted to define high lipomobilization were as follows: prepartum NEFA concentrations >0.3 mmol/L  or >0.4 The Shapiro–Wilk and Brown–Forsythe tests were used to verify the Gaussian distribution and equality of variance, respectively. Two-way repeated measure ANOVA was used to test the effects of time relative to calving (−21 × −7 × 0 × 7 × 21 × 42 days), the effect of parity (P × B × M), and the interaction between these two factors. The same analysis was applied to the cases of BCS at calving (HBCS × NBCS) and milk yield (HP × MP). All measured metabolites and BCS were included in the analyses, and the variables are presented as means and standard error or standard deviations. The milk yield at 60 DIM, average daily milk yield, total DIM, and total milk yield were compared between groups using one-way ANOVA. Tukey’s test was used for multiple comparisons. The probability of error for all the tests was 5%. The analyses were performed using SigmaPlot for Windows 13.1 .p = 0.500), but P cows had a lower milk yield at 60 DIM (p = 0.007) and lower average daily production throughout lactation (p = 0.001) than B and M cows. The total milk yielded during lactation was lower in P cows than in M cows (p = 0.005). Milk yield did not differ between HBCS and NBCS cows (p > 0.05) but was higher in HP cows than in MP cows (p < 0.001). On average, HP cows had 1 month longer lactation time than MP cows (p = 0.020).The milk yield results for all groups are presented in The BCS differed between groups related to parity and BCS at calving but showed no difference when the cows were grouped according to milk yield . RegardlThe metabolites analyzed in the serum and plasma showed variations over time relative to calving . RegardlWhen the cows were grouped according to BCS at calving, there were no differences between the groups and no interactions between factors . In cowsHP cows did not differ from MP cows in most serum metabolites . HP cowsRegarding metabolic imbalances, the frequency of high lipomobilization in the prepartum period was 27.0% (23/85) and 23.5% (20/85), assuming the cutoff points of >0.3 mmol/L and >0.4 mmol/L of NEFA, respectively. During early lactation, high lipomobilization was observed in 35.3% (30/85) of cows, whereas SK was present in only 11.7% (10/85) of cows. The frequency of SCH was 32.9% (28/85) at calving and only 4.7% (4/85) of cows maintained SCH at 7 DIM. Among the hypocalcemic cows, 5 were primiparous, 5 were biparous, and 18 were multiparous.Few diseases were observed in the studied cows up to 60 DIM, including clinical mastitis  and metritis . Retained placenta, endometritis, hypocalcemic paresis, clinical ketosis, or abomasal displacement were not observed.Information involving the metabolism of dairy Zebu cows is scarce in the literature, with the vast majority of studies carried out on cows of taurine breeds raised in temperate climate regions. To the authors’ knowledge, this is the first study to characterize the metabolic profile of high-yielding Gyr dairy cows. Milk production of the cows studied (20–40 kg/day), including MP cows, was higher than those previously described. Values ranging from 6 to 15 kg/day have been reported in Gyr cows ,28,29,30Even with high production, the values found in the studied cows were still below those described for high-producing Holstein cows, ranging from 40 to 50 kg/day ,32,33, bThe parity influenced the milk production in the studied cows, being higher in M than in P cows. Lower production levels are reported in first lactation cows due to differences in tissue mobilization and nutrient partition for growth and milk production . FurtherThe BCS slightly varied between the groups according to parity during the transition period, showing a difference at 7 and 42 DIM when the P cows had lower values, indicating that they lost more BCS during early lactation. This corroborates the results in Holstein cows . RegardiIn the study presented here, BCS at calving had no effect on milk production, and HBCS cows had total and mean milk production similar to that of NBCS cows. Consistent with these results, no influence of BCS at calving on milk production was found in Holstein × Gyr cows . The lacBased on the increased NEFA and BHB concentrations in early lactation, our results corroborate those previously described in Holstein cows, demonstrating no relationship between parity and NEFA and BHB concentrations ,49. HoweDifferences in NEFA and BHB concentrations were observed between the HBCS and NBCS cows at 42 DIM. The HBCS cows showed higher BCS values and a greater loss of BCS after calving. Similar results were previously reported in Holstein cows; those with high BCS during the prepartum period and at calving showed a greater loss of body condition and energy imbalance during early lactation ,54,55. HThe observed variation in NEFA and BHB values over time in Gyr cows agrees with previous reports on Holstein cows ,54,61 anElevated NEFA concentrations are indicative of pre and postpartum NEB and high lipomobilization, and are associated with several diseases that occur during this period, such as clinical and subclinical ketosis, abomasal displacement, retained placenta, and metritis ,64. In tThe higher concentration of glucose at calving is consistent with the results of Holstein  and HolsThe elevated serum cholesterol levels found at 21 and 42 DIM were similar to those reported in Holstein ,67, HolsThe TP concentrations showed differences related to parity and were lower in P cows. Similar results have been reported in Holstein cows . AccordiSerum Ca concentrations differed between parity groups at calving, with lower values in M cows than in P cows. SCH at calving occurred more frequently in multiparous cows than in primiparous or biparous cows. Cows with three or more parturitions are more prone to this imbalance and have greater difficulties in maintaining calcemia within the physiological range at the beginning of lactation . In highCa concentrations varied over time in all groups, especially at calving. Loss of this mineral through colostrum and milk explains the occurrence of lower levels on the day of calving , which cSerum P and Mg levels varied over time; however, few differences were observed between the groups during early lactation. According to a previous report , P is exTwo points must be considered as limitations of the present study. The absence of cows with low BCS at calving compromised a more comprehensive comparison of the effects of this factor on the studied blood metabolites, preventing comparison between groups according to BCS. The small number of cows studied prevented confirmation of the existence of a relationship between high NEFA values at the beginning of lactation or low Ca values at calving and the occurrence of diseases in early lactation. Future studies with Gyr cows may clarify these doubts.Gyr cows, selected for high milk yields and maintained under good nutritional conditions, had a balanced metabolic profile during the transition period. Parity, BCS at parturition, and level of milk production had few biologically relevant effects on the metabolic profile of these animals. High lipomobilization in the postpartum period and SCH at calving were the most common imbalances found; however, they were not related to the presence of diseases during early lactation.


Sedum spectabile ‘Stardust’ and woody shrub Hydrangea arborescens ‘Annabelle’. A total of five substrate mixes composed of peatmoss and miscanthus straw were used: 100%P, 70%P:30%M, 50%P:50%M, 70%P:30%M, 100%M. Each substrate was subjected to three fertilizer treatments: Basacote, Basacote + YaraMila, and YaraMila. The growth response of both tested species was very similar. In general, plants performed best in 100%P, and the quality decreased with increasing miscanthus straw amendment; however, differences in height and dry weight at the level of ~9% suggest that Sedum plants obtained market value with up to 50% miscanthus amendment and Hydrangea plants with up to 30% miscanthus mixed in media. The most favorable effect on the tested parameters was a combination of Basacote + YaraMila, which delivered more soluble salts, and in higher rates than Basacote and YaraMila used separately. Decrease in EC and nutrients in the substrate with increase in miscanthus straw amendment suggest that uniform irrigation among all the treatments contributed to nutrients leaching from miscanthus media due to its lower water holding capacity.The aim of this research was to assess the influence of fresh miscanthus straw shreds as a component of growing media in nursery production of perennial  Greenhouse and nursery production depends on peatmoss as the basic growing medium. Increasing plant production requires progressively greater use of soilless substrates in the horticultural industry. Block et al.  predictsThe search for new materials suitable for container production is occurring in two ways: finding components that reduce the quantity of peatmoss used in the mix, or completely replacing peatmoss. Two raw materials that were profitably and successfully used as peatmoss substitutes are pine bark and coir, used separately or in mixes ,7,8,9. PMiscanthus × giganteus), a tall hybrid C4 grass genotype with proven utility as a biomass crop, was described by Heaton et al. [As highlighted by , to achin et al.  as one on et al. , who shon et al. . This len et al. ,32,33,34Sedum spectabile ‘Stardust’ and woody shrub Hydrangea arborescens ‘Annabelle’, followed by the chemical analysis of leaf tissues of the tested species and substrate analyses.As there are few studies on the use of miscanthus straw in ornamental plant production, the authors decided to assess plant performance in miscanthus straw amended media on two selected ornamental species: perennial Sedum .Sedum spectabile ‘Stardust’ and woody shrub Hydrangea arborescens ‘Annabelle’. Both species are commonly grown ornamental plants with high nutritional requirements. Plants were propagated at the Research Development Station of Wroclaw University of Environmental and Life Sciences in plugs and used in the form of rooted cuttings as the starting material for this study.For the purpose of this research study, two plant species from different groups were selected: perennial The two main factors in the research were substrate mix and fertilizer type.100% peatmoss (control)70% peatmoss + 30% miscanthus50% peatmoss + 50% miscanthus30% peatmoss + 70% miscanthus100% miscanthusThe first factor was a substrate mixture composed of different proportions of peatmoss and shredded miscanthus straw:® Plus 6M 16-8-12(+2+TE); Compo) and water soluble YaraMila Complex , used separately and in a mix with different fertilization schemes:−3 of Basacote premixed with each substrate mix3 g.dm−3 of Basacote premixed with each substrate mix with YaraMila Complex top dressing, 3 times during vegetation period. at a dose of 1 g.dm−33 g.dm−3 of YaraMila Complex premixed with each substrate mix with YaraMila Complex top dressing, 3 times during vegetation period, at a dose of 1 g.dm−31 g.dmThe second factor was the two different fertilizer types: controlled released fertilizer Basacote (BasacoteMiscanthus × giganteus Greef et Deu) was delivered from the experimental station of the Wroclaw University of Environmental and Life Sciences in Pawlowice, shredded in a hammermill and then screened to a particle size not exceeding 4 × 2 × 0.5 cm. To decrease the high carbon to nitrogen ratio in miscanthus close to the optimal level (24:1), shredded straw was premixed with YaraMila Complex . Peatmoss  was mixed with miscanthus straw in proper ratios, samples of each substrate were taken to determine pH (Elmetron (CPI-501)), and then amended with the proper amount of lime to establish pH at the level of 6.2–6.5 based on the neutralization curve (data not shown). All five media were split into three piles and mixed with fertilizers: Basacote, Basacote with YaraMila, and YaraMila. Rooted cuttings of Aster and Spiraea were transplanted into 3 L pots filled with the proper substrate × fertilizer mix and placed in the outdoor nursery on black nursery fabric. Plants were trimmed by 1/3 height to stimulate shoot growth, and pots were fully saturated with water. Watering continued throughout the entire vegetation period until plants were ready to be measured. Irrigation was performed using overhead irrigation as needed, on average 3 times a week, with 300 mL of water per 1 dm−3 of substrate.Fresh miscanthus straw plant diameter (measured at the widest and narrowest axis and averaged)For Hydrangea measurements included:main shoot numbermain shoot length Additional leaf number leaf blade lengthleaf blade widthleaf blade areaLeaf performance of both species was tested on 8 randomly selected plants within the treatment and included:Leaf blade length, width and area were tested on 15 leaves per plant, selecting mature, fully developed leaves in the central area of the plant, using a field portable leaf meter AM 300 Sedum, the inflorescence number per plant, average inflorescence height and diameter were assessed. Hydrangea did not flower in the first year after planting.Additionally, for Fresh shoot and root weight for both species was measured immediately after performing the above mentioned measurements. Dry weights were measured after drying above ground, and root biomass in the oven at 70 °C for 72 h.in fresh material, chlorophyll a,b and total using spectrophotometry by Arnon From each of the two tested species 20–25 leaves per plants within the substrate × fertilizer treatment  were collected and unified. Three subsamples from each treatment were subjected to testing:https://www.nixsensor.com/free-color-converter/ (accessed on 13 February 2023). Fresh leaf blades were spread flat on a white sheet, a reading head was placed on the leaf blade ensuring that the leaf surface covered the reading area, and measurements were taken and downloaded from the deviceleaf brightness and two color tones, using HunterLab MiniScan EZ working in CIE L * a * b * scale that was recalculated to RGB scale using color converter P and Mg by the colorimetric method of King   measurements were made with a conductivity meter (Orion model 142) and pH of the soil with Elmetron (CPI-501) at a soil:distilled water ratio of 1:2. Total N was measured by the Kjedahl method, P and Mg by the colorimetric method (Spectrophotometer S106 WPA), K and Ca by flame photometry (Carl Zeiss Jena flame photometer), and NOF-test was used to identify the treatments’ main effects and interactions, followed by Fisher’s range test at the 0.05 significance level using Statistica 13.3.721.0. As the research was conducted over three consecutive years and statistical analyses did not show significant differences between years, averaged data from 2014, 2015 and 2016 were analyzed and shown together.The data were subjected to analysis of variance (ANOVA). The Sedum and Hydrangea performed best in 100%P media and plant height decreased along with increase in miscanthus amendment. However, Sedum height in 50% miscanthus amended media was ~9% lower than in peatmoss and, for Hydrangea with 30% miscanthus amendment, this was ~9% lower than in peatmoss. A similar tendency was observed in both species with dry weights. Based on these results, it can be considered that, for Sedum, miscanthus amendment up to 50% and, for Hydrangea, up to 30% still produced plants with market value. EC and nutrient content in substrates decreased along with increase in miscanthus straw amendment in media. The lower water holding capacity of miscanthus straw suggests that the same irrigation frequency and amount could cause nutrient leaching, and further investigation is needed to develop more suitable practices for this media component.Both


FST value of 0.08. We also reported QTL regions associated with forage biomass yield based on field phenotype data measured on a subset of the Napier grass collections. The findings of this study offer useful information for Napier grass breeding strategies, enhancement of genetic diversity, and provide a guide for the management and conservation of the collections.Genetic diversity amongst genotypes of several Napier grass collections was analyzed and compared with the diversity in a set of open pollinated progeny plants. A total of 114,881 SNP and 46,293 SilicoDArT genome-wide markers were generated on 574 Napier grass genotypes. Of these, 86% of the SNP and 66% of the SilicoDArT markers were mapped onto the fourteen chromosomes of the Napier grass genome. For genetic diversity analysis, a subset of highly polymorphic and informative SNP markers was filtered using genomic position information, a maximum of 10% missing values, a minimum minor allele frequency of 5%, and a maximum linkage-disequilibrium value of 0.5. Extensive genetic variation, with an average Nei’s genetic distance value of 0.23, was identified in the material. The genotypes clustered into three major and eleven sub-clusters with high levels of genetic variation contained both within (54%) and between (46%) clusters. However, we found that there was low to moderate genetic differentiation among the collections and that some overlap and redundancy occurred between collections. The progeny plants were genetically diverse and divergent from the germplasm collections, with an average  Cenchrus purpureus (Schumach.) Morrone syn. Pennisetum purpureum Schumach.), also known as elephant grass, is a perennial C4 grass belonging to the genus Cenchrus, one of the most diverse genera, consisting of about 140 species including important cultivated species such as Napier grass, pearl millet and kikuyu grass3. Napier grass is native to Sub-Saharan Africa and has been distributed to other tropical and subtropical regions around the world, adapting to a wide range of agro-ecologies, from sea level to 2500 m above sea level (m.a.s.l.). It has been acclimatized in areas of Central and South America, tropical parts of Asia, Australia, the Middle East and the Pacific islands4.Napier grass  source11. It has many other beneficial characteristics, including resistance to most pests and diseases, ease of establishment and fast regrowth capacity, withstanding repeated cuttings. It is also resistant to high temperatures, and has low water and nutrient requirements12. However, its productivity is primarily checked by the unpredictable weather arising from changes in climatic conditions14 and biotic stressors, such as the diseases head smut and Napier stunt, particularly in Central and Eastern African countries17.Napier grass is mainly cultivated as a forage crop for animal feed due to its high palatability and feed quality, high dry matter production (up to 78 tons of dry matter/ha/year), and year-round availability under supplementary irrigation18. Napier grass is a cross-pollinating allotetraploid species with a chromosome number of 2n = 4x = 28 (genome A’A’BB)20. The Napier grass A’A’ genome has been reported to be homologous to the AA genome of pearl millet 21. The two species are cross-compatible, producing triploid (AA’B genome) sterile hybrids that can only be propagated by vegetative means23. The hybrids are favored as a forage as they combine the superior forage quality of pearl millet with the high yielding ability of Napier grass1. Consequently, these interspecific triploid hybrids have become a crucial part of the improved forage crop value chain in Africa, Asia and South America4.The availability of genetic diversity is a prerequisite for breeding and genetic improvement programs. It serves to identify novel alleles for agronomically important traits, such as disease and pest resistance, drought tolerance, and withstanding challenges associated with the changing climatehttps://www.genesys-pgr.org) have collections containing a considerable amount of germplasm. Although Napier grass exhibits high genetic variation due to its strict out crossing and self-incompatibility nature, the global diversity is low due to its vegetative propagation and the common use of cuttings as planting material. This would likely have limited the exchange of genes between genotypes. However, limited information exists about the overall available genetic diversity and germplasm exchange among the global collections.Several national and international genebanks have conserved a substantial number of Napier grass genotypes collected from various geographical regions. For example, the International Livestock Research Institute (ILRI); the Brazilian Agricultural Research Corporation, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), United States Department of Agriculture (USDA-ARS), International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and the Millennium Seed Bank Kew  , and other genotypes with unknown or different names, were included. We also included the 60 ILRI and 47 EMBRAPA genotypes, which have been studied previously32 to assess and visualize the overall diversity and identify new and unique genotypes. Out of these initially selected 581 Napier grass genotypes, genotypes with high missing values for the genome wide SNP markers were removed and a final set of 574 , 86 from EMBRAPA, and 23 from the USDA-ARS collections. The genotypes from the USDA-ARS were selected, based on a previous genetic diversity study on the collection19. The highest number of markers mapped onto chromosome CpA01 (8057 SNPs and 2947 SilicoDArTs) while the lowest number mapped onto CpB07 (4411 SNPs and 1245 SilicoDArTs).Genotyping generated 114,881 SNP and 46,293 SilicoDArT markers, distributed over the Napier grass genome. Approximately 86%  of the SNP and 66%  of the SilicoDArT markers were aligned onto the fourteen chromosomes of the Napier grass genome and about 2% (2653 SNPs and 918 SilicoDArTs) of the markers were mapped onto different contigsThe percentages of the markers missing values ranged from 0 to 22% for the SilicoDArT markers and 0 to 62% for the SNP markers. Heterozygosity (He) values for the SNP markers ranged from 0 to 0.5 with an average value of 0.15 while polymorphic information content (PIC) values ranged from 0 to 0.38 with an average value of 0.13 , missing values (≤ 10%), independence from each other (linkage disequilibrium-LD ≤ 0.5), and their distribution across the Napier grass genome. The PIC and He values of the selected markers ranged from 0.08 to 0.38 and 0.09 to 0.50 with an average value of 0.26 and 0.32, respectively, and about 60% of the markers had PIC and He values above 0.25  to 0.47 (between sub-clusters III and VI) in the eleven sub-clusters (Table P < 0.01) genetic variation among and within the major as well as the sub-clusters (Supplementary Table The pairwise 33 and fixation index (FST)34. The pairwise Nei’s genetic distance ranged from 0.018, between ILRI and KALRO/EMBRAPA, to 0.05, between the KALRO collection and progeny plants. Similarly, the estimated FST values ranged from 0.029, between ILRI and ICRISAT collections, to 0.101, between the KALRO collection and progeny plants (Table P < 0.001) both between and within collections, however, higher variation was detected within (90%) than between (10%) collections  and progeny plants were compared using Nei’s genetic distancePotential duplicate genotypes in each collection were identified using Nei’s genetic distance at a threshold of 0.025, which was equivalent to the identity-by-descent (IBD) kinship threshold (k ≥ 0.45) for self, or duplicate identification. A greater number of potential duplicates was detected in the KALRO collection, while all genotypes from ICRISAT were distinct Fig. . From th35 and Mendelian-inconsistent errors37 analyses. First, we examined the genetic relationship between the recorded seed parents and progeny plants by the IBD method of the maximum likelihood (ML) relatedness estimator35, and these were corrected based on the IBD result whenever there was a discrepancy between the recorded seed parent and the IBD result.We estimated the parentage of the 219 progeny plants, raised from 13 open pollinated seed-setting ILRI genotypes, using identity-by-descent (IBD)37. The Mendelian error counts ranged from 2 to 316 for PC1 , 1 to 401 for PC2 , and 37 to 937 for PPC , respectively. In general, we identified or confirmed seed parents, potential pollen parents, and possible crosses as complete parents for 189 (86%) of the progeny plants , using quality filtered DArTseq markers  and field phenotype data measured on 85 Napier grass genotypes, identified seven markers (6 SNPs and 1 SilicoDArT) significantly associated with fresh biomass yield. The associated markers mapped on chromosomes CpA01, CpA02, CpA03, CpA07, CpB01, CpB02, and CpB06 and explained 0.21–21% of phenotypic variation , Africa (mainly from Kenya), and Tifton (genotypes derived from the cultivar “Merkeron” in a breeding program)42.It is thanks to the diversity of plant genetic resources serving as a source of new alleles which support the development of new cultivars with desirable qualities, including breeder’ and farmers’ preferred traits such as high biomass yield, high nutritional quality, larger seeds, and pest and disease resistance43, an efficient and cost-effective sequencing technology that concurrently generates dominant SilicoDArT and co-dominant SNP markers (https://www.diversityarrays.com/services/dartseq/) with a high density and genome coverage. Similar to our previous reports44, we obtained a large number of markers, 114,881 SNPs and 46,293 SilicoDArTs, distributed over the fourteen chromosomes of the Napier grass genome. More than 80% of the markers (from both marker types) were able to be mapped onto the genome, approximately equally onto the A' and B sub genomes19. We applied strong SNP filtering criteria by setting a maximum of 10% missing values, a minimum of 5% minor allele frequency, a maximum of 0.5 linkage-disequilibrium (LD) value to ensure independent markers and, markers that were able to be mapped across the genome. The filtered polymorphic markers, with average PIC and He values of 0.26 and 0.32, respectively, were used in the genetic diversity analysis.We employed the GBS method of the DArTseq technology33. These genetic distance values were greater than the range that Wanjala et al.45 reported for 281 Napier grass genotypes. The high genetic variability detected could be attributed to the high levels of heterozygosity that exists in Napier grass due to its self-incompatibility and obligate outcrossing nature26. Napier grass is a highly heterozygous tetraploid species with a broad parental diversity and rich gene pool40.The Napier grass genotypes from the worldwide collections and progeny plants examined in this study demonstrated substantial genetic variation with an average value of 0.23 (0 to 0.41 range) Nei’s genetic diversity coefficientsFST values ranged from 0.14 to 0.15 between the three clusters and 0.17 to 0.47 between the 11 sub-clusters. This substantial genetic variation was detected both within and between clusters, while within-cluster variation was slightly larger (54%) than between-cluster variation (46%). Generally, outcrossing species show higher levels of genetic variation among genotypes and less population divergence, compared to self-pollinating species46. Similar results were reported in other outcrossing species such as Rhodes grass (Chloris gayana)47 and perennial ryegrass (Lolium perenne)48. Nevertheless, about 46% of the genetic variation was detected between the eleven sub-clusters. This higher divergence could be explained by the clonal propagation of Napier grass through stem cuttings, which reduces genetic drift and gene flow among genotypes within a population49. Genotype adaptation to specific environmental conditions might have also contributed to the high genetic diversity among the clusters, given that the genotypes were collected from different parts of the world.Furthermore, we detected three major clusters and eleven sub-clusters that were significantly different from each other, based on the admixture model in STRUCTURE, hierarchical clustering, PCA, and AMOVA analyses. The FST value of about 0.47, cluster III  and cluster VI (mainly from the ILRI and KALRO collections) had the highest genetic distance, followed by between cluster I  and cluster VI, and cluster III and IX . The results indicated a divergence between the collections and progenies, showing the potential of the progeny plants, that were produced from open pollination, for enhancement of genetic diversity in Napier grass collections and potential for future use in breeding programs, such as heterosis breeding. Heterotic contributions to increased biomass production in the F1 hybrids of Napier grass have previously been reported51.With an 18. The genotypes in cluster II are low to medium biomass yielders, whereas the genotypes in clusters VI and VII are medium to high biomass yielders5. From the EMBRAPA collection, the majority of the elite lines , which have been identified for their superior biomass yields, drought tolerance, and comparatively higher protein content5.The ILRI genotypes were distributed across sub-clusters, mainly in sub-clusters II, VI, VII, IX, and the admixture groups, and their clustering was mostly consistent with our previous report28, genotypes derived from the cultivar ‘Merkeron’ were clustered together in this group. ‘Merkeron’ is a well-known cultivar in the USA, released by a breeding program in Tifton, Georgia50 and was derived from an intraspecific cross between a high yielding clone and a dwarf leafy clone, with improved yield and disease resistance29. Among these, the well-known variety “Mott” (Tift N75), a dwarf leafy type selected from progeny of selfed Merkeron30, is included in this cluster. The variety "Mott" was introduced to ILRI, has been preserved in the genebank with accession number ILRI_15743 and distributed to farmers and national research institutes in Ethiopia. However, none of the samples collected from farmers’ fields and national research institutes, as well as the genotype labeled as ILRI_15743, clustered in this group. Similar findings were reported by Harris et al.28, who found that none of the "Merkeron" samples taken from the various countries where "Merkeron" was distributed were genetically identical, which possibly happened due to genetic mixing up through outcrossing.Cluster XI was primarily comprised of USDA-ARS genotypes, with a few EMBRAPA genotypes. In line with the report by Harris et al.C. purpureus × C. americanus hybrids including the commercial varieties ‘Maralfalfa’53, ‘Super-Napier’, and the hybrids from the ILRI collection ILRI_16840, ILRI_16837, ILRI_1535718. ILRI_15357 showed very high genetic similarity to ‘Maralfalfa’, with more than 0.47 IBD value. Maralfalfa has been reported for its adaptability to higher altitudes (3000 m asl) and for high biomass yield (60 tn/ha) with higher protein content53. Many genotypes from Kiambu under the KALRO collection were also found in this cluster, perhaps suggesting that there are also hybrids held in this collection.Cluster VII consisted of most FST values of 0.20 to 0.33 and an average value of 0.25. The progenies in cluster I and III were particularly distinct from the other clusters with an average FST value of 0.32, showing their divergence from genotypes and other progenies. Napier grass's high heterozygosity within genotypes, self-incompatibility, and obligate outcrossing nature54, which can increase the likelihood of developing new genetic make-up through recombination, may be responsible for this high level of diversity and population stratification in progeny plants.The progeny plants were almost exclusively distributed in clusters I, III, IV, and X, which were significantly different from each other with a range of FST range of 0.024 between the ILRI and USDA-ARS collections to 0.072 between the KALRO and ICRISAT collections. The genetic variation between collections explained only about 10% of the variation as compared to 90% of the variation explained by genotypes within collections, indicating a degree of overlap and redundancy that may have resulted from sharing and exchanging materials among the genebanks. Although Napier grass genotypes are primarily maintained via vegetative multiplication using cuttings, there is still some degree of hybridization existing in nature and also controlled crossings in some collections26. Such redundancy of genotypes between genebanks could also have arisen because each genebank or breeding program gives its own different accession numbers, as there is no standardized labelling and registration procedure. Facilitated by the International Treaty on Plant Genetic Resources for Food and Agriculture, the ILRI forage genebank has assigned a digital object identifier (DOI) to each genotype held in its genebank47, which could be a promising solution to this issue but should then be adopted by more national and international genebanks and research institutions.The collections we studied generally exhibited low to moderate genetic differentiation with an 32.The presence of genotype overlaps and redundancy among the collections was further confirmed by Nei’s genetic distance and identity-by-descent (IBD) analysis; the largest overlap was between the ILRI and KALRO collections, followed by between the KALRO and EMBRAPA, and the ILRI and EMBRAPA collections. The substantial overlap between the ILRI and KALRO collections could be due to the close proximity of the two institutes and historical sharing of genotypes, as indicated in the genotypes name, a large number of KALRO's genotypes were sourced from the ILRI collection. This suggests that the introduction and incorporation of new genotypes to a collection should be carried out strategically to minimize duplications and capture only new genetic diversity that is distinct from the existing one, as conservation is a costly businessWithin collection genotype redundancy analysis, also showed a greater number of potential duplicates in the KALRO collection whereas all genotypes from ICRISAT were distinct. While genotype redundancies and overlaps between collections from various institutions, especially those from different continents like Africa, South America, Asia, and North America, can be taken into consideration and should be encouraged to preserve genotypes with specific adaptations to a wide range of farming systems and agroecological conditions, overlap within a collection should be reduced as it imposes a conservation cost. However, it is crucial to carefully remove overlaps using molecular marker analysis supported by field phenotype and passport data whenever possible in order to preserve unique alleles in closely related genotypes.FST values ranging from 0.067 between the progenies and ILRI collection to 0.101 between the progenies and KALRO collection. This finding further supports the potential of progeny plants to increase genetic diversity with the potential to be used in breeding programs in the future. Open-pollinated progeny selection has been one of the breeding approaches in Napier grass and has resulted in the generation of improved cultivars1. Progeny plants from an open-pollinated population of cultivars ‘A146’, ‘A148’ and ‘A149’ were developed in Taiwan, and the progenies produced 20% more dry matter than the seed parents, had very few hairs on the leaves, flowered late and grew more erect55. However, the use of open pollination breeding schemes allows the control of only one-half of the genome of the resulting progeny material because contributions from pollen parents are not controlled, and the transfer of undesirable traits from unimproved lines can limit or slow the successful release of new cultivars. Therefore, Napier grass breeding programs should focus on targeted crossings that have the potential to yield higher rates of success50 in rapidly generating desirable genotypes and eventually culminating in hybrid breeding wherein a superior individual can be maintained by vegetative propagation after being identified.When compared to the variation among the five collections, the progeny plants were genetically separated, with 37. The progenies in clusters I and III were produced primarily from the ILRI_P9_16821 and ILRI_P6_16835 genomes, while the progenies in clusters IV and X were dominantly from ILRI_P7_16837 and ILRI_P13_16790 and ILRI_P1_1026 and ILRI_P8_16803 genomes, respectively. These results could offer a stronger basis for selecting superior progeny plants for further selection and improvement through heterosis breeding in Napier grass.In this study, we were able to identify potential seed and pollen parents for more than 80% of the progeny plants using identity-by-descent (IBD) analysis based on the maximum likelihood (ML) relatedness estimator and Mendelian-inconsistent errors44.A genome-wide association study identified seven QTL regions associated with forage biomass yield, which represents the above-ground fresh biomass production. The QTLs on chromosome CpB06 and CpA07 showed a strong association and were detected by five different statistical models, suggesting that they can potentially be exploited in marker-assisted selection to develop high biomass producing Napier grass varieties. A marker on chromosome CpB06 that was strongly associated with fresh and dry biomass yields and water use efficiency has previously been reportedThis study represents the first comprehensive genetic diversity study in global Napier grass collections and revealed the presence of extensive genetic variation among Napier grass genotypes. The analysis detected major clusters and sub-clusters that were significantly different from each other with higher within cluster/sub-cluster genetic variation. The study provides a global picture of the available genetic diversity of this important tropical perennial crop and the findings could be used to guide targeted use and conservation of the diversity by breeders and genebanks. Additionally, the information can be used to determine heterotic groups for future Napier grass breeding and improvement through heterosis breeding. Given that the genotypes were collected from different parts of the world, which shows their potential adaptation to a wide range of environmental conditions, studies on genotype by environment interactions are necessary in the future.The genetic variation amongst the different genebank collections studied here was generally low and various levels of genotype redundancy were detected both within and across collections. Genotype overlaps between genebanks, particularly those from different continents, should be encouraged in order to preserve genotypes with specific adaptations to a wide range of agroecological conditions. To reduce the cost of conservation, overlaps within a genebank should be minimized. However, overlaps within a genebank must be carefully removed to maintain unique alleles in closely related genotypes.Higher genetic variation and divergence was detected in the progeny plants produced from open pollination, which creates new genetic makeup and diversity. Incorporating some of the distinctive progeny plants into the genebank collections will be advantageous to enhance the genetic diversity and population size of the collections. However, a few genotypes predominated as a pollen source, as suggested by the parentage analysis, which invites further research on the mechanisms of pollination in Napier grass genotypes maintained in the ILRI forage genebank.53 and Super-Napier (Pakchong 1)—both reported as hybrids of Napier grass and pearl millet with high biomass yield and protein content, were included. An additional 19 genotypes, collected from different places, with the name ‘Mott” or with unknown names were also included , Nairobi, Kenya for genotyping.Young leaf samples from each individual plant were collected into 2 ml Eppendorf tubes, stored in an icebox containing frozen ice, and transferred to a − 80 freezer as quickly as possible. The samples were freeze-dried for about 48 h and ground into fine powder using a tissue lyser. Genomic DNA was extracted from the leaf powder using a DNeasy® Plant Mini Kit (250)  following the manufacturer’s procedures. The concentration and quality of the extracted DNA was assessed using a Nano-drop spectrometer (DeNovix DS-11 FX spectrophotometer) and 0.8% agarose gel electrophoresis. The DNA concentration was adjusted to 50 to 100 ng/μl in 96 well semi-skirted plates and sent to SEQART-AFRICA  markers, combining the DArT complexity reduction with sequencing on the Next Generation Sequencing (NGS) platforms43. The markers were aligned with the Napier grass genome19 using the short sequence fragments associated with each marker to generate information on map position of the sequences and markers across the genome. A heatmap showing the density and distribution of markers across the fourteen Napier grass chromosomes was generated using a free online tool at https://www.bioinformatics.com.cn/en. For genetic diversity analysis, polymorphic markers were filtered based on the marker’s minor allele frequency (MAF), missing values, and independence from each other  using the dartR56 and SNPRelate57 R-packages in R statistical software .The genotyping was conducted using the GBS (Genotyping By Sequencing) method of the DArTseq platform58 using the filtered polymorphic markers. The burn-in time and number of iterations were both set to 100,000 with 10 repetitions, assuming hypothetical subpopulations (K) ranging from 1 to 20 in an admixture model with correlated allele frequencies. The results of the run were uploaded to the software “Structure Harvester” (http://taylor0.biology.ucla.edu/structureHarvester/) and the most likely number of subpopulations was determined by the Evanno method59. Genotypes with less than 60% membership probability were considered admixed. Population structure was further examined using hierarchical clustering and principal component analysis (PCA) using Poppr60 and adegenet61 R-packages. Genetic diversity was estimated using pairwise Nei’s33 genetic distance.Population structure and sub clustering among the Napier grass genotypes was assessed based on the Bayesian algorithm implemented in STRUCTURE (ver. 2.3.4) software62 and fixation index (FST)34 in the R-packages Poppr60, adegenet61, and APE63. AMOVA was used for partitioning the total variance in allele frequencies within and among clusters, sub-clusters, and collections and to assess the significance level of the variations. FST, which ranges from 0 to 1, assesses the degree of genetic differentiation between clusters, sub-clusters, and collections. A value of 0 indicates no differentiation while a value of 1 indicates complete differentiation. In general, an FST value of 0:00 to 0:0.05 indicates low differentiation, 0:05 to 0:0.15 indicates moderate differentiation, 0.15 to 0.25 high differentiation, while FST > 0.25 are considered to represent very high levels of differentiation64.Pairwise estimates of population differentiation and divergence between clusters, sub-clusters, and collections were analysed by Analysis of MOlecular VAriance (AMOVA)35 using the R package SNPRelate57 and by counting the Mendelian-inconsistent errors using a custom R script reported in Vanderzande et al.36. The IBD analysis was mainly used to confirm the genetic relationship between the recorded seed parents and progeny plants, whereas Mendelian-inconsistent errors count was used to determine any putative set of parent–child (PC) and parent-parent–child (PPC) relationships. In the Mendelian-inconsistent errors count, a low percentage of errors was used as an indication of putative parent–offspring relationships while higher percentages were indicative of more distant relationships65.Parentage and genetic relationship analyses were conducted using identity-by-descent (IBD) analysis based on the maximum likelihood (ML) relatedness estimatorA subset of 85 Napier grass genotypes were planted in the main rain season (mid-June to mid-September 2018) using an augmented p-rep design with two replications at Addis Ababa, Ethiopia. Six stem cuttings per genotype were planted in a single row with a distance of 750 mm. After establishment, the plants were clean cut every 12 weeks of regrowth to a standard height of 50 mm above ground. Total fresh biomass yield per plant (TFW) was taken by weighing and calculating the average from three randomly selected plants per row. The average value per genotype was calculated based on 24 plants, taken from three plants per row, in two replications, across four harvests from the 2018 to 2021 cropping seasons.66. The models used include General Linear Model (GLM), Mixed Linear Model (MLM), Multiple Loci Mixed Model (MLMM), Fixed and random model Circulating Probability Unification (FarmCPU) and Bayesian information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK). A total of 16,083 markers  were retained after filtering. The markers were filtered based on call rate (≥ 0.90), minor allele frequency (MAF ≥ 5%), missing values (NA < 10%), and their distribution across the genome.Marker-trait association analysis was conducted using five statistical models implemented in Genomic Association and Prediction Integrated Tool version 3 (GAPIT3)Supplementary Tables.