Data on yield, material and energy consumption were based on pre- and pilot size production plants (TRL 5–6). Revenue optimization was accomplished by first analyzing the performance of single product value chains of the microalgal strains. Subsequently, a strategy was developed to exploit almost all biomass based on the most promising microalgal strains. The cultivation costs are most of the time the major costs of the value chains. For the single product value chains common process bottlenecks are low product yields, especially for soluble proteins where only a small fraction of the biomass is leading to economic value. The biorefinery costs (excluding cultivation) vary significantly for various species, due to the species-specific operating conditions as well as differences in product yields. For the evaluated single product value chain scenarios the costs for utilities and other inputs were in general the highest contributing expenses. A biorefinery approach significantly increases the biomass utilization potential to marketable products from 7–28% to more than 97%. Although the cascading approach increases the total production costs of the multiproduct value chains significantly, this is more than compensated by the increased overall biomass revenue. For all selected multiproduct chains there is a significant potential to become profitable at a relevant industrial scale of 10 kton per year. Additional insights in the product functionality, quality, and their market size are needed to narrow down the wide range of foreseen product revenues and resulting profits.The objective of this work was to identify industrial scenarios for the most promising microalgal biorefinery value chains on the basis of product selection, yields, and techno-economic performance, using biological characteristics of algae species. The development, value creation, and validation of several new processing routes with applications in food, aquafeeds and non-food products were particularly considered in this work. The techno-economic performance of various single product value chains (SP) and multiproduct value chains (MP) was evaluated for four industrial microalgal strains. Cost-revenue optimization was done for a 10 kton microalgal dry weight y Microalgae have a huge potential as bioresource for food, (aqua)feed, chemicals, and materials. Microalgae have the advantage of high growth yields, low land requirements, and the ability to grow on salt water and waste water te water . Furtherte water , rangingte water . Well-kn–1 biomass  depending on the production system and location  to 1 and 100 ha scale; (3) analysis of potential market for microalgal components based on a survey of current commercially substitute products, (4) generic processing efficiencies that do not take into account the effect of biological characteristics of algae species  on the processing performance .The objective of this work is to identify industrial product scenarios for the most promising microalgal biorefinery value chains on the basis of product selection, product yield and techno-economic performance using biological characteristics of algae species. In particular, the development, value creation, and validation of several new processing routes with product applications in food, aquafeeds and non-food products were considered in this work. The chains are designed using processing techniques developed or improved in the MIRACLES-project, complemented with data from best practice techniques (when required) and based on the marketable products that were validated in the project. The techno-economic performance of various single and multiproduct value chains was evaluated for four benchmark industrial microalgal strains.®. The processing chains integrate novel technologies with benchmark technologies, both developed and tested within the framework of the MIRACLES-project. A sensitivity analysis was applied to determine the range of processing cost. After economic evaluation of the single product value chains, four multiproduct value chains were designed and evaluated, taking advantage of a cascading principle and thereby valorizing almost all biomass.First single product value chains were selected and for each chain a processing chain was designed and implemented in SuperPro DesignerNannochloropsis gaditana (marine), Scenedesmus obliquus2 (freshwater), Phaeodactylum tricornutum (marine), and Isochrysis galbana (marine). A large variety of interesting products is reported for these strains: polyunsatured fatty acids , sterols, functional proteins, carotenoids/antioxidants, and specialty carbohydrates .–broken microalgae (SP-II).–water-soluble native proteins (SP-III).–pigments dissolved in lipids (SP-IV).–triacylglyceride-rich oil (SP-V).+-biomass’) and, (2) cultivation under nutrient limitation by nitrogen starvation to enhance the TAG content (‘N–-biomass’).A scheme of the five single product (SP) value chains is presented in Each process chain starts with harvesting by microfiltration and centrifugation. In SP-I the biomass is dried to obtain the main product of whole microalgae. For the other SP chains cell disruption by high pressure homogenization is applied, followed by a drying step for SP-II, SP-IV, and SP-V. SP-III continues after disruption with two–step centrifugation with resuspension in water for separating the supernatant from cell debris, followed by UF/DF at 300 kDa (separation of polysaccharides from soluble proteins) and at 8 kDa (separation of soluble proteins from monosaccharides and ashes). SP-IV applies high pressure pigment extraction, the procedure is species dependent:–I. galbana, spray drying, SFE→GXL→PLE–I. galbana, no pre-processing, directly the reverse process PLE→GXL→SFE–N. gaditana, disruption → spray drying → SFE→PLE–P. tricornutum, disruption → spray drying → PLE–S. obliquus, disruption → spray drying → SFE→GXL→PLESingle product SP-V uses solvent extraction by a mixture of hexane with ethanol or isopropanol. Details on each process step are given in the Supplementary Material.The multiproduct chains have multiple valuable marketable products. These multiproduct value chains (MP) were developed on basis of the most promising single product value chain scenarios. The biomass valorization was enhanced by applying a cascade approach to the multiproduct chains. All residue streams were either directly linked to a specific market application or were subjected to further refinery processing. Focus was on the main biomass components in terms of mass and/or product value. N. gaditana (‘N+-biomass’) and focusses on extraction of soluble proteins. The biorefinery processing consists of:The first multiproduct value chain (MP1) uses –Cell disruption by high pressure homogenization.–Two-step centrifugation with resuspension in water for separating the supernatant from cell debris.–UF/DF at 300 kDa (separation of polysaccharides from soluble proteins) and at 8 kDa (separation of soluble proteins from monosaccharides and ashes).–Cell debris is dried and high-pressure extraction is applied to yield a pigment containing fraction.–Alkaline extraction of proteins from the pigment extraction residue followed by enzymatically hydrolysis to obtain mainly peptides.I. galbana (‘N+-biomass’) and aims at extracting pigments as most valuable product. The process consists of:The second multiproduct value chain (MP2) uses –High pressure pigment extraction.–Alkaline extraction of proteins from the pigment extraction residue.N. gaditana cultivated under nutrient limitation to enhance the TAG content (‘N–-biomass’). The process chain is mainly based on the single product value chain V for oil extraction  considers tion see . The pro–Cell disruption by high pressure homogenization.–Drying.–Solvent extraction of oil by hexane/isopropanol.–Alkaline extraction of proteins from the pigment extraction residue followed by enzymatic hydrolysis to obtain mainly peptides.N. gaditana that was cultivated under nutrient limitation (‘N–-biomass’). This chain first aims at extraction of the lipids, then on pigment extraction, and separation of proteins for peptide production. The process consists of the steps:The fourth multiproduct value chain (MP4) considers again –Cell disruption by high pressure homogenization.–Drying.–Solvent extraction by hexane/isopropanol.–High pressure pigment extraction from the defatted biomass.–Alkaline extraction of proteins from the pigment extraction residue followed by enzymatically hydrolysis to obtain mainly peptides.–1 in a photobioreactor located in the South of Spain at a benchmark level of 2 g L–1 biomass concentration. The physico-chemical properties and composition of the biomass was dependent on species and cultivation conditions, (N+-biomass and ‘N–-biomass’). The biomass composition after cultivation is reported in +-growth conditions (20 ton ha–1 year–1) and N–-growth conditions (12 ton ha–1 year–1) at a required 500 and 840 ha scale, respectively. To place this area into perspective, it can be compared to the greenhouse horticulture sector in Netherlands with a total area of 9,300 ha in 2016 (–1 (‘N+-biomass’) and 7.5 € kg–1 (‘N–-biomass’). The latter correspond to an N starvation period up to the point where the further lipid accumulation becomes negligible.For each value chain, both single and multiproduct, a specific technical process model has been designed and linked to an economic evaluation. All scenario calculations are based on a microalgae production of 10 kton dry weight y in 2016 . Cultiva–1 was performed with the aid of the software SuperPro Designer v10.0b3® from Intelligen, Inc3. Data on yields, material and energy consumption were based on pre- and pilot size production plants (TRL 5–6). As far as possible, the processes have been modeled in the software adopting continuous operations. In case of batch operations (required for pigment extraction) a detailed schedule has been implemented according to the procedure carried out at lab and pilot-scale as reported in previous literature  and operating expenditures (OPEX). Biorefinery capital investments were calculated from the purchase costs of the main equipment (PC) increased by an overall Lang factor of 3.54 . More de4 and IndexMundi5. The costs for utilities and raw materials are specified in –3. For location-dependent costs, Spain was assumed as reference. The costs for CO2 adsorption and cultivation are provided and explained in the Operating expenditures are calculated as the sum of the costs of utilities , (raw) materials (like solvents), consumables (membranes), wastewater treatment, labor, laboratory costs like quality assurance (Lab/QC/QA), additional facility costs (based on Lang factor), and others . Free-on-board costs of raw materials were retrieved from the websites ICIS–1 are most of the time the major costs of the value chain. In general, every microalgal chains will benefit from lower cultivation costs. The biorefinery costs (excluding cultivation) per kg product are given in For each chain the cultivation costs of 4.50 and 7.50 € kg–1. The range in costs is caused by the difference in cell diameter between the microalgae species. S. obliquus is the cheapest to process as result of the large cell diameter. The cost for utilities account for about 50%, the facility costs  are another significant cost factor. For whole microalgae the majority of capital expenses is due to the membrane facilities. The steam use in the dryer contributes most to the operating expenses, followed by the electricity use of the membrane filtration.For whole microalgae, the biorefinery costs (excluding cultivation) are 0.30–0.35 € kg–1 broken microalgae, with a share for utilities above 50% of the costs. The total costs are thus strongly influenced by the ease of cell disruption. In all cases, the overall yield of broken microalgae on the whole process is around 93%, due to the combination of harvesting losses (∼2%) and losses due to incomplete cell disruption (∼5%).For broken microalgae the utility costs increase as homogenization is energy intense. This leads to biorefinery costs of 0.50–0.65 € kg–1 soluble purified protein. The lowest biorefinery costs are for processing I. galbana (high soluble protein content and easy to break) and the highest for S. obliquus (low protein content and low disruption and separation yield). The large difference in soluble protein yield and, in general, in cell disruption efficiency among the algal strains is also reported in literature (Processing biomass while aiming for soluble proteins results in biorefinery costs (excluding cultivation) of 4.50–12.80 € kgterature .The overall protein product yields with respect to the initial amount of biomass are for all microalgae species below 10%. As a result, the biorefinery processing costs per kg of product are significantly higher compared to whole and broken microalgae. The utilities contribute significantly to the costs. Furthermore, the relative contribution of labor to the total costs is higher due to the ultrafiltration and diafiltration processes.–1 pigment product. It mainly depends on the microalgal species that affects the design of the extraction process in terms of capacity and yield. Utility, materials and equipment (related) costs are most relevant in these chains. For the pigment chains all data are expressed per unit of product in which pigments are dissolved. The reason is twofold: (1) pigments are never extracted in pure form, and (2) pigments are frequently unstable and to ensure stability at long term storage they are often dissolved in oil. Drying of the biomass is needed when the first extraction step is supercritical fluid extraction (SFE). The product yields with respect to the initial amount of biomass vary from 9.1% for S. obliquus to 24.4% for N. gaditana. The steps starting with pressurized liquid extraction (PLE) do not require drying and have product yields around 20%. The capital expenses are mainly due to the investment costs for the extraction vessels. The extraction processes also contribute most to the utility and material costs.The range of pigment processing cost is quite broad: 5.80–68.95 € kg–1 extracted oil. The solvents and utilities used for lipid extraction contribute most to these costs. Equipment costs only contribute marginally to the costs. The overall product yields vary between 8.3% for S. obliquus and 29.1% for I. galbana. N. gaditana is also promising for lipid production with an overall yield of lipid extraction of 21.9% of the initial biomass.The biorefinery costs for oil are lower than for pigment, i.e., 3.80–14.45 € kg–1 throughput. For SP value chains I, II, and III cost savings of 50% are feasible. For the other chains the difference in costs is smaller, since their costs are for a large part determined by operating expenses. The cost reduction from 10 to 100 kton y–1 is less for all chains and negligible for pigments and lipids. Overall, the results indicate that biorefineries should have a throughput of 10 kton y–1 or more to benefit from economy of scale.The results above were based on a facility with 10 kton per year capacity. The biorefinery costs for facilities of 1, 10, and 100 kton capacity per year are shown in –1 scale. This was done by optimizing all the adjustable variables according to the ranges reported in A sensitivity analysis combined with process optimization was made for each single product value chain at the 10 kton y–1 whole microalgae. The potential revenue ranges between 1.5 and 3.0 € kg–1. For disrupted microalgae the costs can be lowered from 0.50–0.65 to 0.25–0.40 € kg–1 by optimizing the harvesting and disruption conditions. For soluble purified proteins the optimization reduces the biorefinery costs from 4.5–13 to 3.3–9.2 € kg–1. This reduction, however, does not necessarily lead to a profitable chain; the revenues for proteins are in the same order of magnitude. However, the value of insoluble components is estimated to be 3.00 € kg–1. In the SP protein chain for every kg of soluble protein also 9.68 kg insolubles are produced, thus potentially around 29 € can be gained from these insolubles per kg soluble protein. This is higher than the revenue of the proteins (4.75–8.25 € kg–1), thus the main product here is actually the insoluble fraction. For strains characterized by both high processing costs and low yields, such as Scenedesmus, the soluble proteins value chain has a small chance to become profitable in this scenario. Consequently, simplification and further exploitation of the other biomass components will increase the potential revenue.For both whole and disrupted microalgae the biorefinery cost are relatively low compared to the potential revenue, as a result of the high product yield and simple structure of harvesting and further biomass processing. By optimizing the operating conditions the biorefinery cost (excluding cultivation) for whole microalgae can be reduced slightly from 0.30–0.35 to 0.20 € kg–1 product. Compared to the range of potential revenue of pigments (14–31 € kg–1) the effect of the optimization is relevant to achieve a profitable process. In particular, the reverse extraction process applied to Isochrysis and the single PLE extraction process applied to Phaeodactylum . Again, the biorefinery process based on Scenedesmus shows the most critical situation due to the low extraction yield (8%). The exhausted biomass has, however, a residual value for feed or material applications and is potentially worth up to 2.00 € kg–1 residue, leading to 4.8 € additional revenue per kg oil extract. Isochrysis and Nannochloropsis are the most likely candidates for such a type of biorefinery, due to the high yield (29 and 22%). These yields are achieved with biomass cultivated under nitrogen replete conditions. Microalgal biomass obtained by growth under N-limitation or N-starvation can contain up to 50–65% lipids  applied to dry biomass has been selected as the most favorable approach. –-biomass and a breakdown of the composition and the extracted components. With the multiproduct cascading the biomass use increased from 33% till almost 98%. The biorefinery costs are 1.03 € kg–1 biomass, the lowest of all multiproduct chains  are 1.55 € kg–1 biomass . The center points of the profit ranges are used to evaluate the profitability of the value chains. A positive center point indicates that the estimated revenue is sufficient to balance the costs, a negative center point suggests that the estimated revenue is unlikely to be sufficient to balance the cost. No cash flow analysis was performed. Below the results are discussed together with other proposed value chains in literature.The potential profit ranges of the five single product and four multiproduct value chains at 10 kton y–1 for N+-biomass and 7.50 € kg–1 for N–-biomass) are most of the time the major production costs. One should realize that the used estimates of cultivation costs are already a projection compared to the current 1 ha production. In general, all SP and MP chains will benefit from lower cultivation costs, as also indicated previously  always lead to a negative potential profit at 10 kton y protein .–1 biofuel) is mainly related to the cultivation technology (open pond vs. closed photobioreactor) and on the lipid yield from cultivation (–1 pigment), they consider a small market for their product and assume higher pigment selling prices, i.e., for beta-carotene 1180 € kg–1 and for astaxanthin 5113 € kg–1.For pigments and oil, the profit of the process depends on: (I) the oil and pigments content of the microalgal strain; (II) the efficiency of high pressure extraction; (III) the selling price of the pigments or oil. These figures are also confirmed in literature. tivation . Thomasstivation . In contFor the multiproduct chains the profit range increases, with a positive center point (average) for MP1, 3, and 4. For these chains the worst-case estimate of revenue is thus not sufficient to balance the cultivation and biorefinery costs. For MP1 and MP2, large part of the revenue comes from pigments and peptides, while for MP3 and MP4 significant revenues are obtained from the oil and peptide products . TherefoTo develop a profitable micro-algae production chain a combination is required of (1) technological innovations enabling cost reductions, especially in micro-algae production, (2) developing multiproduct biorefinery concepts aimed at valorizing the full biomass through the cascading principle, and (3) deriving a range of new specialty products with applications in food, aquaculture and non-food. Cascading increases the biorefinery costs significantly, but this is compensated by the enhanced overall biomass revenue, provided that the sequence of operations does not affect the yield and properties of the final products. When these conditions are met the achieved overall biomass revenues of the integrated, multiproduct chains enable an economically competitive microalgae biorefinery.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PS and GO performed TE evaluation and interpretation, drafted the manuscript, and took the responsibility for the integrity of the work as a whole, i.e., from conception until the finished article. PS coordinated data acquisition. GO developed and optimized the flow sheets. PS, GO, EB, LS, and JR conceived and designed the study. ME, RW, and JR obtained funding. All authors contributed to the selection of the single value chains, have critically reviewed the proposed multiproduct chains and results, and have critically revised the manuscript, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The risk of emergence and/or re-emergence of arthropod-borne viral  infections is rapidly growing worldwide, particularly in Africa. The burden of arboviral infections and diseases is not well scrutinized because of the inefficient surveillance systems in endemic countries. Furthermore, the health systems are fully occupied by the burden of other co-existing febrile illnesses, especially malaria. In this review we summarize the epidemiology and risk factors associated with the major human arboviral diseases and highlight the gap in knowledge, research, and control in Sudan. Published data in English up to March 2019 were reviewed and are discussed to identify the risks and challenges for the control of arboviruses in the country. In addition, the lack of suitable diagnostic tools such as viral genome sequencing, and the urgent need for establishing a genomic database of the circulating viruses and potential sources of entry are discussed. Moreover, the research and healthcare gaps and global health threats are analyzed, and suggestions for developing strategic health policy for the prevention and control of arboviruses with focus on building the local diagnostic and research capacity and establishing an early warning surveillance system for the early detection and containment of arboviral epidemics are offered. Arthropod-borne viruses (arboviruses) have received much global attention recently due to the Zika virus epidemic in the Americas. The major human arboviruses belong to the Togaviridae, Flaviviridae, or Peribunyaviridae families ,2. TheseSeveral risk factors contribute to the emergence and re-emergence of arboviral diseases. These include climate change, international travel and trade, increased contact of humans and wild or domesticated animals, an explosion of the human population, and unplanned urbanization ,9,10,11.Yellow fever virus  is endemic in Sudan and infections occur throughout the country . Early sAedes aegypti, notorious as the major vector of this virus in urban areas, was the predominant mosquito species in the area. Later, in 2015, a novel outbreak of dengue fever occurred among refugees in the Darfur area, west Sudan, in which DENV-2 and 3 were co-circulating in the area [Dengue virus  is endemic in Sudan and its distribution was confined to the coastal and subcoastal regions of the country, where the disease was described for the first time by Saigh in 1906 and Balfour in 1907 on the Red Sea coast, Port Sudan city ,37,38,39the area . This wathe area . Additiothe area ,53. Recethe area . DENV trthe area ,53.West Nile virus  is endemic in Sudan with infections reported in different regions of the country since 1942 and 1953–1954  6,35,5555,56. SaThe first evidence of Zika virus  circulation in Sudan was reported in 1981 from El Gezira state, central Sudan . A recenChikungunya virus  is endemic with frequent upsurges in Sudan ,28,35,61The circulation of the only established Anopheles-mosquito-borne human arboviral pathogen, o’nyong-nyong virus (ONNV), has been reported in Central Sudan with seroprevalence of 13% among the population of Sennar State . PreviouAe. aegypti, Ae. vittatus, Ae. vexans, Anopheles arabiensis, An. coustani, and Culex quinquefasciatus) during an outbreak of RVFV in Sudan, suggesting vertical transmission, but the relative importance of each species in transmission was not determined [Rift Valley fever virus  is endemic with frequent outbreaks in Sudan, mainly in the central region and rich Savannah area, with continuous active transmission in both humans and animals ,6,66,67.termined ,84. A setermined . Human rtermined ,79,85. Utermined .Crimean-Congo hemorrhagic fever virus  is endemic in Sudan, and infections have been documented throughout the middle region of the country . The serArumowot virus  is a sandfly (Phlebotomus)-borne virus first isolated from a mosquito, Culex antennatus, from Sudan in 1963 . SeveralThe burden of arboviral diseases has devastating socioeconomic and health impacts by causing the loss of human and domesticated animals, and reductions in economic production in endemic countries, including Sudan . UnfortuThe major challenge for the prevention, control, and research of arboviruses in Sudan is the limited diagnostic capacity. The Ministry of Health is relying on external laboratories for confirmation of arboviral infections, commonly in Germany or Senegal  30,31,3,331,34, Although the risk of arboviral diseases in Sudan is high, it receives little attention, mainly due to limited resources, which heavily undermines building the capacity of healthcare providers and researchers with advanced training. Also, the lack of advanced diagnostic tools is a major challenge as detection of arboviruses typically requires molecular techniques and equipment such as simple polymerase chain reaction (PCR) machines that are available in very limited healthcare facilities. Additionally, the very inadequate funding for research limits the generation of evidence about the epidemiology and dynamics of arboviruses in Sudan; therefore, neither healthcare providers nor health policymakers are aware of these remarkably fast-growing public health threats and the need to develop a multisectoral approach to disease control and prevention. It is critically important to improve surveillance and investigate the circulating strains of these viruses and to establish viral genomic databases as a reference for current and future research. An arbovirology research center in Sudan, which would offer an extraordinary opportunity to advance the research and control of arboviral diseases in the region, is critically needed . This coWe highlight the urgent need for raising awareness among healthcare providers, researchers, and policymakers about the issue of arboviral infections, building the capacity of Sudan biomedical laboratories, and increasing the funding for research, particularly to investigate the burden of infectious diseases. Also, improving surveillance, diagnostics, and strengthening the health system overall by adopting a multisectoral approach to control will help in detecting the early emergence of these infections to take action to mediate massive epidemics. Particular attention needs to be paid to reducing the threat of arboviral diseases in the most vulnerable groups in the community, including people living in humanitarian crisis settings, informal settlements, refugees and nomads, and generally, impoverished people throughout Sudan.


N-carbamylglutamate (NCG), a structural analogue of N-acetylglutamate, may have the potential to improve the utilization of nitrogen by bulls. Therefore, the effects of adding different levels of NCG on the growth performance, nutrient digestibility, nitrogen metabolism and plasma metabolites of fattening Holstein bulls were investigated in this study. The addition of NCG increased concentrations of plasma Arg, Ile, Val, total essential amino acid and total nonessential amino acid, which in turn resulted in higher N utilization and CP digestibility for growth and, therefore, higher average daily gain in NCG-fed bulls.It is critical to find an effective and safe feed additive to improve the utilization of nitrogen by ruminants. N-carbamylglutamate (NCG), a structural analog of N-acetylglutamate, improves nitrogen utilization in dairy cows. However, the effects of NCG on bulls are unknown. The purpose of the current research was to investigate the effects of adding different amounts of NCG on growth performance, nutrient digestibility, nitrogen metabolism and plasma metabolites of fattening Holstein bulls. Twenty-four Holstein bulls with similar body weights  and ages  were selected for the feeding trial. After 2 weeks of adaptation, bulls were blocked by BW and age and subsequently randomly assigned to 1 of 4 groups: (1) CON group (control diet), (2) L group (supplementation with 20 mg/kg BW NCG), (3) M group (supplementation with 40 mg/kg BW NCG), or (4) H group (supplementation with 80 mg/kg BW NCG). The addition of NCG linearly and quadratically increased the average daily gain (CON vs. L vs. M vs. H = 1.03 vs. 1.19 vs. 1.40 vs. 1.26 kg/d) (p < 0.05), feed conversion ratio (CON vs. L vs. M vs. H = 11.92 vs. 9.22 vs. 7.76 vs. 8.62) (p < 0.05), crude protein digestibility (CON vs. L vs. M vs. H = 64.3 vs. 63.8 vs. 67.7 vs. 65.8%) (0.05 < p < 0.10), N retention (p < 0.05) and N utilization (p < 0.05) of bulls, whereas the contents of fecal N (0.05 < p < 0.10) and urinary N (0.05 < p < 0.10) in NCG-fed bulls linearly decreased compared with those in CON bulls. Bulls fed NCG showed a quadratic increased plasma nitric oxide (p < 0.05) concentration. Furthermore, Arg (p < 0.05), Ile (p < 0.05), Val (p < 0.05), Ala (p < 0.05), Glu (p < 0.05), Ser (p < 0.05), total essential amino acid (p < 0.05) and total nonessential amino acid (p < 0.05) concentrations linearly and quadratically increased with increasing doses of NCG. In contrast, plasma urea (p < 0.05) and ammonia (p < 0.05) concentration linearly and quadratically decreased with increasing doses of NCG. Overall, the addition of NCG increased plasma Arg, Ile, Val, TEAA and TNEAA concentration, which in turn resulted in a higher N utilization and, therefore, higher average daily gain in NCG-fed bulls, providing baseline data for the widespread application of NCG in beef cattle production. Nitrogen plays a critical role in the growth and development of animals . RoughagN-carbamylglutamate (NCG), a structural analog of N-acetylglutamate (NAG), can activate the rate-limiting carbamoyl-phosphate synthase in the urea cycle and arginine synthesis pathway and promote the synthesis of endogenous arginine -9).2) on floor bedding with a front metal gate allowing access to feed and water. After 2 weeks of adaptation, bulls were categorized by BW and age and subsequently randomly assigned to 1 of 4 groups: (1) CON group (Control diet), (2) L group (supplementation with 20 mg/kg BW NCG), (3) M group (supplementation with 40 mg/kg BW NCG), and (4) H group (supplementation with 80 mg/kg BW NCG). NCG was mixed with 500 g diets  and ages  were used for the feeding trial using a completely randomized block design was used in this trial . Bulls wAfter 2 weeks of adaptation, the feeding trial was performed and lasted for 7 weeks. Data measurement and sample collection were carried out at weeks 0, 4 and 7 of the trial. Body weights of bulls were the average of 3 consecutive weights at weeks 0, 4 and 7 of the trial, and thereafter, the average daily gain (ADG) was calculated. The diet offered and refused for individual bulls was weighed every day throughout the trial to calculate dry matter intake (DMI). Samples of individual feed ingredients, orts, and diets were collected weekly during the experimental period and stored at −20 °C . Thereaf2SO4 (0.036 mol/L) and stored at −20 °C for future determination of urine N output. Valadares et al. (1999) and Leonardi et al. (2003) have demonstrated that creatinine can be used as a marker to estimate urine volume /(% of CP in feces)/6.25 [N (g/d) = urine volume (L/d) × [CP in urine (g/L)]/6.25 [N intake-fecal N-urine N. Nitrogen utilization (%) = (N retention/N intake) × 100.The total tract apparent nutrient digestibility was estimated using the concentration of indigestible NDF (iNDF) in the diet and feces as an internal marker as reported by Lee and Hristov 2013) . The iND13 . The es)/6.25 . Urine NL)]/6.25 . NitrogePlasma total protein , albumin , glucose , urea (Method: two-point dynamic) and ammonia (Method: two-point dynamic) concentrations were measured using a fully automatic biochemical analyzer . Commercial acid kits from Nanjing Jiancheng Institute of Bioengineering  was used to analyze the plasma nitric oxide , total antioxidant capacity ; Kit number: HY-60021), catalase , total superoxide dismutase , malondialdehyde  and glutathione  concentrations. The concentration of plasma amino acids in plasma samples was measured using high-performance liquid chromatography-liquid chromatography-mass spectrometry .p ≤ 0.05, and trends were defined at 0.05 < p ≤ 0.10.Before analyses, the normality of all data was tested using Minitab 17  and all the data were in accordance with normal distribution. Thereafter, the data were analyzed using the mixed procedure of SAS 9.4  with repeated measures. The statistical model included initial variables (obtained at 0 weeks) as covariates and treatments, weeks and their interactions as fixed effects, as well as the random effects of blocks and bulls within blocks. Polynomial orthogonal contrasts were used to analyze the linear and quadratic effects of increasing NCG supplementation on each variable . Signifip < 0.05) and FCR (p < 0.05) were observed with increasing doses of NCG (p < 0.10). Moreover, M bulls had a highest ADG among treatments. No differences (p > 0.10) in feed intake of DM, CP, NDF and ADF were observed among treatments.A linear increase in ADG (s of NCG . Bulls fp > 0.10) was observed in the apparent digestibility of DM, NDF, and ADF. However, the CP digestibility (0.05 < p < 0.10) tended to show a linear increase with increasing doses of NCG. In addition, bulls fed NCG tended to show a linear decrease in the fecal N contents (0.05 < p < 0.10) and urinary N (0.05 < p < 0.10). A linear increase in N retention (p < 0.05) and N utilization (p < 0.05), a quadratic increase in N retention (p < 0.05) and N utilization (p < 0.05) were observed with increasing doses of NCG. Moreover, M bulls had a highest N utilization among treatments.p < 0.05) and ammonia (p < 0.05) were observed with increasing doses of NCG. Conversely, a quadratic increase in plasma NO concentrations (p < 0.05) was observed with increasing doses of NCG. Additionally, M bulls had highest plasma urea and ammonia concentrations among treatments. Regarding plasma antioxidant variables, T-AOC, CAT, T-SOD, MDA, and GSH concentrations were not different (p > 0.10) among treatments.As presented in p < 0.05) and Ile (p < 0.05) linearly and quadratically increased with increasing doses of NCG. Moreover, bulls fed NCG showed a linear increased Val (p < 0.05) and total essential amino acid (TEAA) (p < 0.05). Regarding the nonessential amino acid (NEAA) results, plasma Ala (p < 0.05), Glu (p < 0.05), Ser (p < 0.05) and total nonessential amino acid (TNEAA) (p < 0.05) concentrations linearly and quadratically increased with increasing doses of NCG. Nevertheless, no difference (p > 0.10) was observed in other amino acids among treatments.As shown in This study is the first to investigate the effect of dietary supplementation with NCG on growth performance, apparent digestibility, nitrogen metabolism and plasma metabolites in Holstein bulls. In the present study, no difference in DM, CP, NDF, and ADF intake was noted among treatments, indicating that NCG did not affect the bulls’ willingness to eat. This result is in agreement with the findings of Gu  and ChacIn the current study, CP digestibility increased in NCG-fed bulls, indicating that NCG may have the potential to improve CP digestion in the rumen and small intestine of bulls, thereby causing increased nitrogen utilization in NCG fed bulls. To the best of our knowledge, NCG fed bulls increased endogenous arginine production through efficient regulation of urea cycle and converted ammonia in blood into urea, thus reducing the content of fecal nitrogen and urine nitrogen, which in turn resulted in a higher CP digestion . Indeed,Blood ammonia is a metabolic waste product of various tissues and amino acids in the body, and its increase will affect the health of animals . It is aThe contents of free amino acids in blood can reflect the balance and metabolism of amino acids in animals. The free blood amino acid content will be correspondingly reduced when the amino acid content in the diet is insufficient . RegardiN utilization and CP digestibility for growth and, therefore, higher ADG in NCG-fed bulls. Furthermore, the 40 mg/kg supplemental NCG treatment was optimal to improve growth of fattening Holstein bulls.As hypothesized, the addition of NCG resulted in increased plasma Arg, Ile, Val, TEAA and TNEAA concentrations, which in turn resulted in a higher


Recognition of known shapes requires access to long-term memory, so it is not possible to know whether the experiment is assessing short-term encoding and working memory mechanisms, or has encountered limitations on retrieval from memory stores. Here we have used an inventory of unknown shapes, wherein a string of discrete dots forms the boundary of each shape. Each was displayed as a target only once to a given respondent, with recognition being tested using a matching task. Analysis based on signal detection theory was used to provide an unbiased estimate of the probability of correct decisions about whether comparison shapes matched target shapes. Four experiments were conducted, which found the following: a) Shapes were identified with a high probability of being correct with dot densities ranging from 20% to 4%. Performance dropped only about 10% across this density range. b) Shape identification levels remained very high with up to 500 milliseconds of target and comparison shape separation. c) With one-at-a-time display of target dots, varying the total time for a given display, the proportion of correct decisions dropped only about 10% even with a total display time of 500 milliseconds. d) With display of two complementary target subsets, also varying the total time of each display, there was a dramatic decline of proportion correct that reached chance levels by 500 milliseconds. The greater rate of decline for the two-pulse condition may be due to a mechanism that registers when the number of dots is sufficient to create a shape summary. Once a summary is produced, the temporal window that allows shape information to be added may be more limited.Many laboratories have studied persistence of shape information, the goal being to better understand how the visual system mediates recognition of objects. Most have asked for recognition of known shapes,  Vincent Di Lollo [1]It is well known that the human visual system can identify shapes using minimal cues. This can be demonstrated in various ways, including by displaying them out of focus i.e., providing only a spaced pattern of dots, precludes a ceiling effect that would likely occur if all the boundary dots were shown. One further avoids a contribution from long-term memory if each unknown shape is shown only once as a target. The respondent decides whether the two shapes that are displayed on a given trial are the “same” or are “different.” After completion of the full experiment with a given set of treatment conditions, the data are submitted to quantitative methods that are commonly known as signal detection analysis (or decision theory). This analysis provides an unbiased measure of the validity of the decisions. If the decisions are significantly above chance, one can assert that the task conditions influenced the shape encoding process and/or working memory. Using shapes that are not stored in long-term memory provides a better method for assessing early shape processing mechanisms.One can reduce the ambiguity by using shapes that are “unknown,” meaning that they are relatively dissimilar to any known shape or object. One can test recognition by providing two successive shape displays, the first being designated as the target shape and the second being a comparison shape that either matches the target or is a non-matching shape. Reducing the density of the boundary markers, Prior findings using this unknown-shape protocol found non-chance recognition of the target shape when the comparison shapes displayed extremely sparse boundary markers The Greene & Hautus 2.2.1.The experimental protocols were approved by the USC Institutional Review Board. Respondents were recruited from the USC Psychology Subject Pool. Thirty-two respondents provided the matching judgments, eight in each of the four experiments. Each respondent was told that participation was completely voluntary, and that they could end the test session at any time without any form of recourse or penalty.2.2.Shape boundary dots were displayed on a 64 × 64 array of light-emitting diodes (LEDs), designated as the “display board.” Respondents viewed the board from a distance of 3.5 m. At this distance, the visual angle of a given dot is 4.92 arc′, dot to dot spacing is 9.23 arc′, and the total span of the array (center-to-center) is 9.80 arc°. These LEDs emit at 630 nm, which appears as bright red. The dots forming the boundary of a given pattern can be displayed one at a time, in any order, or as simultaneous light emission from a specified subset of dots. For the present experiments, each dot to be displayed was flashed for 10 µs at an intensity of 1000 µW/sr. Ambient room illumination was 10 lx, which is mesopic, so the dots were quite salient.Control of the display board is provided by a Mac G4 Cube, running custom Tk/tcl instructions. The program code is further interpreted as machine language by a PropoxMMnet101 microcontroller that runs at 16 MHz. This allows for 1µs temporal resolution of display timing.2.3.There were 480 unknown shapes in the inventory, each providing a string of adjacent dots at the boundary of the shape. Across the inventory, the number of dots used to form this boundary ranged from 100 to 269, with the mean number of dots being 166. At the 3.5 m viewing distance, shape spans range from 2.0 to 3.5 arc°, with mean distance being 2.6 arc°.Each experiment displayed low-density versions of a given unknown shape. Which dots to include was based on a sampling protocol. First an address was chosen at random from the list, then successive addresses were included or not using an algorithm that maximized the net distance among dots. In other words, the protocol yielded a display wherein the dots were evenly spaced around the boundary at the density that had been specified.2.4.Each experiment randomly sampled the inventory to provide low-density “target shapes” that were always displayed first on a given trial. This was followed by a randomly selected “matching shape” that had the same pattern of boundary dots as the target shape, or was a low-density version of a different shape from the inventory, designated as a “non-matching shape” see . MatchinAcross experiments, each display trial was preceded by a fixation target, consisting of the four dots in the center of the LED array providing steady emission of light at 0.2 µW/sr. The fixation dots were extinguished 200 ms prior to display of the target shape.On a given trial, the target shape was positioned in a randomly selected corner of the display board, such that at least one boundary dot was in the outer row and column of the LED array. The comparison shape was then displayed in one of the other corners of the board, the location being chosen at random.Experiment 1 used 320 target shapes and 160 non-matching comparison shapes. This experiment displayed low-density versions of the shapes across five percentage levels, these being 4, 8, 12, 16 and 20%. All the boundary dots of a given target or comparison shape were displayed simultaneously, and the comparison shape followed the target display at an interstimulus interval of 300 ms. The total number of display trials was 320, half showing matching shapes and half showing non-matching shapes, which provided 32 replications at each density level.Experiment 2 again used 320 target shapes and 160 non-matching comparison shapes. All shapes were displayed at 12% density. Here the interstimulus interval was varied from 150 ms to 750 ms in 150 ms increments to determine whether the target shape information being held in working memory would decline. Here again, there were 32 replications at each of the five treatment levels.Experiment 3 used 300 target shapes and 150 non-matching comparison shapes, reducing the total number of trials to assure that the task conditions would not exceed the time allowed for the test period. All shapes were shown at 12% density. For this experiment one of the six treatment conditions provided for simultaneous display of all the dots in the target shape. The other five levels displayed the dots one at a time, adjusting the interval between successive dots to meet a specified total time for showing them all. To be specific, total display times were 0, 100, 200, 300, 400 and 500 ms. The comparison shape was displayed at an interstimulus interval of 750 ms. For display of 300 target shapes and six levels of total display time, there were 25 replications at each treatment level.Experiment 4 used 300 target shapes and 150 non-matching comparison shapes, displaying all shapes at 12% density. Here the dots in each low-density target shape were divided into two subsets using the following method. A dot was picked at random, and moving counterclockwise, successive dots were numbered. The odd numbered dots were assigned to one subset and the even numbered dots were assigned to the other. The dots in each subset were displayed simultaneously with a target interstimulus interval that varied from zero to 500 ms in 100 ms increments.2.5.Respondents were instructed to maintain their gaze on the fixation point prior to initiation of a given trial and to not move from that fixation as shape stimuli were being displayed. They were informed that the displays would briefly flash a pattern of dots that marked locations on the outer boundary of unknown shapes and that these shapes were unlikely to resemble recognizable objects. They were made aware that the dots would be spaced, but nonetheless would mark locations on the boundary of each shape. Respondents were also told that on each trial there would be two displays and the goal was to say whether the second shape was the same or different from the first. Respondents in Experiments 3 and 4 were further informed that the target dots could be shown as a sequence across an interval that could last up to half a second, and that the comparison shape would come after an interval that was even longer.The experimenter initiated a given trial by clicking an on-screen button, which displayed the target/comparison stimulus set. The task was not speeded, but respondents generally offered a judgment within a second or two, saying either “Same” or “Different” as an indication of whether they thought the comparison shape matched the target shape. The experimenter recorded this answer by clicking the appropriate on-screen button, which logged the stimulus treatments that were used for that trial along with the judgment of the respondent. The experimenter was not provided any information about which treatment condition was used for a given display, nor did the respondent receive any feedback about whether the judgment was correct. In each of the experiments, all the trials for a given respondent were typically completed within 45 minutes.2.6.d′, that is corrected both for response bias and the structure of the task. The most commonly adopted formula, d′ = z(H) − z(F), is appropriate for the standard detection-theory task, also called the yes-no task d′ = √2(z(H) − z(F)), where H was the proportion of “same” judgments to matching shapes, F was the proportion of “same” judgments to non-matching shapes, and z (•) was the inverse-normal transform F and H were adjusted  using the log-linear correction d′.Each respondent was able to complete the experiment for which he or she had volunteered, and no data was discarded. Evaluation of the data was based on signal detection theory, as described in detail in an earlier report d′ value was determined using similar calculations as in the tabulation method given by Miller d′ was employed to generate confidence intervals, e.g., d′ ± 1.96 √σ2, where σ2 is the estimated variance.The variance of each d′ for each individual respondent was averaged to provide the group estimate. The variance of group d′ was the sum of the N individual respondent variances divided by 2N. The group variance allowed confidence intervals to be established around group d′.When assessing performance across respondents, the data from different respondents should not be combined d′ with confidence intervals, we present a more familiar proportion measure called p(c)max. This measure, like d′, corrects for response bias, with 0.5 reflecting performance and chance level and with judgments that are always correct being scored at 1.0. For our matching task, p(c)max = (d′/(2√2)), where φ (•) was the cumulative normal probability distribution function. This formula facilitated transformation of estimates of d′ and their confidence intervals to estimates of p(c)max and their confidence intervals, which we report and illustrate in our figures. Finally, to assess systematic changes in p(c)max across conditions, linear contrasts consequent on repeated measures analysis of variance are reported.Rather than presenting 3.Experiment 1 varied the density (sparseness) of boundary dots in each display from 4% to 20%, with the target and comparison shapes being shown at the same density on a given trial see .p(c)max values, each being the average across the eight subjects, are plotted in Mean p(c)max. While means systematically increase, the linear contrast was not significant  = 2.586, p = 0.152).Degree of overlap in the confidence intervals can be used to assess the significance of differences in Experiment 2 examined whether effectiveness of judgments would decline as a function of length of the interstimulus interval, as illustrated in F  = 1.959, p = 0.204), but the quadratic contrast did reach significance  = 7.103, p = 0.032), giving further support to the p(c)max differential at 150 ms. Additional analysis done with this treatment level removed did not find the linear component to be significant  = 2.630, p = 0.149).Here, the linear contrast was not significant  = 18.34, p = 0.004). Nonetheless, one should note that p(c)max declined little more than 10%, and the probability of a correct decision was well above chance even when the total time for display of all dots was 500 ms.The linear contrast for Experiment 3 was significant  = 19.34, p = 0.003) supporting the linear decline apparent in For Experiment 4 the linear contrast is significant max. Further, the probability of being correct at 500 milliseconds was significantly below what was found in Experiment 2 for that interval.Experiment 3 displayed each dot of a given target shape one at a time, requiring that all the dots be provided within an interval that ranged from 0 to 500 milliseconds. Here the ability to correctly judge whether the comparison shape was the same or different depended on persistence of the information provided by each dot but also the ability of the visual system to integrate that information to yield a shape summary. It is possible that the duration of persistence for an individual dot would be shorter than for a full dot pattern. More likely, any decline that was manifested for intervals shorter than 750 milliseconds would be due to temporal restrictions on the integration process, e.g., animals, vehicles, furniture, tools, with the total time for display of all the dots varying between zero and 700 milliseconds. There was a linear decline in recognition, with the rate of decline depending on which shape was being judged. On average, shape recognition dropped by 50% across a total display time of 700 milliseconds. In Experiment 3 the recognition of unknown shapes declined by only 10% with a total display interval of 500 milliseconds. Finding that identification of unknown shapes declines much less than for known shapes suggests that the need to access long-term memory provides a substantial restriction on performance.Greene & Ogden Experiment 4 used a two-pulse protocol, wherein target dots were divided into two complementary subsets and these were displayed with six levels of temporal separation. Here the performance declined at a much steeper rate than in Experiment 3, and reached the chance level of 0.5 with subset separations of 500 milliseconds. The differential is pertinent to the earlier question of whether the persistence of information for individual dots might be less than for dot patterns. If that were the case, one would expect the decline in performance for the one-at-a-time protocol to be steeper than for a two-pulse protocol. Finding the reverse now puts the focus on the integration process, and raises the question of why the temporal window would be more restrictive for integrating two complementary patterns.Phillips With an interstimulus interval beyond 100 milliseconds, Phillips Several reports from this laboratory used the two-pulse protocol to evaluate recognition of known shapes. Greene Subsequent work with recognition of letters used low-density subsets in a two-pulse protocol and dim room illumination e.g., boundary markers, that are provided at a given moment. A one-at-a-time display sequence is open-ended, which apparently does not elicit closure of the encoding process. Alternatively, the very low-density subsets of Experiment 4 provided dot patterns that could be “packaged” as a shape summary, which then greatly restricted the interval wherein information could be added. A fourth mechanism would be the process by which a shape summary can access memories, thus allowing for identification of known shapes. Based on prior work, this final step also appears to place restrictions on the integration interval, but at least that constraint is not relevant for the present work wherein the shapes were displayed only once and therefore do not require retrieval from long-term memory.Any conceptual model of image analysis and shape recognition can be partitioned into a plethora of functional steps, but at this point it appears that there are four major operations that can be distinguished. The first is encoding, wherein the elemental shape cues are converted into a shape summary. Some conditions allow this to be completed within tens of milliseconds, or no more than a hundred milliseconds, as discussed in articles cited previously 5.i.e., sparseness of boundary markers, does not produce significant differentials in recognition of unknown shapes if both the target shape and the comparison shape have the same level of density. The shape information is encoded and stored in working memory with no significant decline of that information for at least 750 milliseconds. Boundary location cues of a target shape can be delivered sequentially, e.g., by displaying sparse dots one at a time, and shape recognition remains high for up to 500 milliseconds of total display time. However, if the same sparse pattern is divided into two subsets, with the dots in each being displayed simultaneously, there is a substantial drop in recognition across this same interval. This suggests that decay of information persistence is not the only reason that recognition would decline. There may be an active process of exclusion that protects the information that was gathered at one moment against disruption at a later moment. Apparently, this can occur even if the two sources of shape information are compatible.The present results demonstrate that manipulation of density,


Taraxacum officinale) consists of three developmental components: diplospory (apomeiosis), parthenogenesis, and autonomous endosperm development. The genetic basis of diplospory, which is inherited as a single dominant factor, has been previously elucidated. To uncover the genetic basis of the remaining components, a cross between a diploid sexual seed parent and a triploid apomictic pollen donor was made. The resulting 95 triploid progeny plants were genotyped with co-dominant simple-sequence repeat (SSR) markers and phenotyped for apomixis as a whole and for the individual apomixis components using Nomarski Differential Interference Contrast (DIC) microscopy of cleared ovules and seed flow cytometry. From this, a new SSR marker allele was discovered that was closely linked to parthenogenesis and unlinked to diplospory. The segregation of apomixis as a whole does not differ significantly from a three-locus model, with diplospory and parthenogenesis segregating as unlinked dominant loci. Autonomous endosperm is regularly present without parthenogenesis, suggesting that the parthenogenesis locus does not also control endosperm formation. However, the high recovery of autonomous endosperm is inconsistent with this phenotype segregating as the third dominant locus. These results highlight the genetic complexity underlying apomixis in the dandelion and underline the challenge of introducing autonomous apomixis into sexual crops.Apomixis in the common dandelion ( Apomixis is a form of reproduction in the flowering plants in which the seeds are clones of the mother plant ,2. ApomiTaraxacum officinale in the Asteraceae family, in which sexual diploid (2n = 2x = 16) and apomictic polyploid  cytotypes occur. In developmental terms, three main components of apomixis in Taraxacum can be distinguished: 1. diplospory—a modified form of meiosis in which chromosomal recombination and reduction are absent, resulting in non-haploid female gametophyte cells; 2. parthenogenesis—the direct development of an embryo from an egg cell without fertilization; and 3. the autonomous development of endosperm—endosperm arising from the dividing, hexaploid central cell of the female gametophyte. The need for an endosperm may at first appear non-obvious; however, the endosperm is a tissue that nourishes the developing embryo; without it, the embryo will abort [Asteraceae species produce autonomous endosperm [In the case of gametophytic apomixis, which may be the most straightforward approach to introducing apomixis into crops, a diploid egg cell is formed that is genetically identical to the mother plant and then transitions into an embryo without fertilization. Gametophytic apomixis is a rare reproductive system but widely distributed phylogenetically. One of the best-known apomicts is the common dandelion, ll abort . In diplndosperm .Asteraceae. Based on various crosses between sexuals and apomicts of Erigeron annuus (fleabane), Noyes and colleagues proposed that that apomixis was controlled by a diplospory locus (D), whereas parthenogenesis and autonomous endosperm were controlled by a single fertilization factor locus  identified loci containing two of the components of apomixis: the locus for apospory, termed Loss-of-Apospory (LOA), and a locus containing the genes for both parthenogenesis and autonomous endosperm, termed Loss-of-Parthenogenesis (LOP); [LOP locus uncovered rare recombination events between the parthenogenesis phenotype and the autonomous endosperm phenotype, consistent with two tightly linked genes . Unexpectedly, the genetic fine mapping of AutE in more crosses showed that this locus was on a different linkage group from the LOP locus, with a low penetrance (18%) and no additive effect [AutE phenotype in Hieracium is controlled by a more complex genetic mechanism than LOA or LOP.With an understanding of the three developmental components of apomixis, the genes responsible need to be cloned in order to enable the introduction of apomixis into sexual crops, for which genetic analysis is necessary so that the number and complexity of the genetic loci underlying the developmental components are understood ,9,10. Foermed F, ,12). Sims (LOP); ,14. Howee effect , suggestBABY BOOM-Like (PsBBL) from the monocot apomict Pennisetum squamulatum [PsBBL is a universal mechanism for parthenogenesis in apomicts. By analysis of mutations in Arabidopsis thaliana, parthenogenesis was reported in mutants in the Polycomb Group 2 (PcG2) complex member MULTICOPY SUPPRESSOR OF IRA1 (MSI1) [Parthenogenesis has had extensive research focus in an attempt to understand the genes that drive the phenotype, both in natural apomicts as well as in model sexual systems (extensively reviewed in ), culminamulatum . No othe1 (MSI1)  and in l1 (MSI1) .Erigeron and Hieracium. This is in part because many well-studied apomicts, such as Pennisetum, use pseudogamy and thus lack this component. Additionally, if present, the penetrance of the phenotype can be low, with Boechera holboelli showing a maximum penetrance of just 15% [Taraxacum is complete, making this system ideal for uncovering the basis of this component of apomixis, in combination with the wealth of information known about endosperm formation in general, as well as specific mutations that can induce its autonomous formation.Unlike parthenogenesis, the genetic control of the autonomous endosperm component of apomixis has not been thoroughly investigated outside of the apomicts in just 15% . In contArabidopsis, mutations that give a Fertilization Independent Seed (FIS)-formation phenotype are found in genes encoding the PcG2 complex and act by initiating autonomous endosperm development, although the endosperm fails to cellularize and, eventually, the seeds abort development [FIS orthologs would appear to be clear candidate genes for autonomous endosperm development in autonomous apomicts and potentially more, as MSI1 affects parthenogenesis as well. However, while the silencing of FIS-genes by RNAi in Hieracium affected endosperm development in sexuals, the transgene by itself did not induce autonomous endosperm [FIS genes may not be the causative lesions behind the autonomous endosperm formation in apomicts of the Asteraceae. In addition, the Hieracium MSI1 gene did not map to the LOP locus, eliminating this gene as a candidate for parthenogenesis and autonomous endosperm [Taraxacum.In elopment ,22,23. Tndosperm , suggestndosperm . Thus, oTaraxacum have focused on the inheritance of diplospory by using a tetraploid diplospory pollen donor [DIP locus showed that this dominant component of apomixis was tetrasomically inherited, and the genotype had the Dddd simplex constitution [DIP locus was weakly linked to the 18S–25S rDNA locus, which was later confirmed by the fluorescent in situ hybridization of Bacterial Artificial Chromosome (BAC) probes on one of the Nucleolus Organizer Region (NOR) chromosomes [DIP locus and estimated the distance between the SSR MSTA78-a allele and the DIP locus to be 3.5 centimorgans (cM). Positional information of the DIP locus was used to identify the DIP gene as a Vacuolar Protein Sorting-associated 13 (VPS13)-like gene [Earlier studies in en donor . The teten donor . Co-domititution . Additioomosomes . Vijverbomosomes  used theike gene . The molPAR) in Taraxacum were controlled by at least two separate genetic loci. Van Dijk and colleagues [Taraxacum could be controlled by two or three major loci. However, the numbers of offspring in these crosses were too small for detailed segregation analysis.The fact that the tetraploid pollen donor plant above was diplosporous, but lacked parthenogenesis, suggested that diplospory and the parthenogenesis-encoding locus ; in the three-locus-model, each apomictic component is determined by a separate unlinked locus. Assuming that the loci are uncoupled and have the same genotypic constitution as the diplospory locus, 44 percent of the offspring from the two-locus model are expected to be apomictic, while in the three-locus model, 30 percent of the offspring are expected to be apomictic.To find the genes controlling parthenogenesis and autonomous endosperm in MSTA78-a allele was tightly linked to diplospory. This allele was used as a molecular marker for diplospory so that the phenotyping could focus on elucidating parthenogenesis and autonomous endosperm formation in the diplosporous progeny. It is critical to focus on the diplosporous progeny because both parthenogenesis and autonomous endosperm are gametophytic traits, which will segregate on a plant with normal meiosis. Unfortunately, meiosis will be highly disturbed due to the chromosomal imbalance inherent in the triploid F1 plants, causing the seed phenotypes to show segregation distortion, which impedes clear analysis. When focusing the analysis on the diplosporous progeny, the problem of segregation distortion is avoided because these F1 plants do not undergo meiosis. Analysis of the F1 showed that the SSR MSTA44B-d allele is tightly linked to parthenogenesis: the first molecular marker for parthenogenesis in Taraxacum. Additionally, while 29% of the diplosporous offspring contained all three of the components of apomixis, which is consistent with the three-locus model, nearly 95% of the diplosporous offspring showed some degree of autonomous endosperm formation, independent of whether or not the egg cell underwent parthenogenesis. The absence of the clear segregation of autonomous endosperm development in Taraxacum is different from the coupling between autonomous endosperm and parthenogenesis previously reported in other apomictic Asteraceae.In this article, the inheritance of the individual components of apomixis in a cross between a diploid sexual plant and an apomictic triploid plant is described. Although pollen fertility due to triploidy was very low, by making a large number of crosses, it was possible to generate sufficient triploid offspring, allowing the quantification of the segregation of different elements of apomixis. As previously found, the SSR T. officinale line triploid A68 was crossed with stigmas from the pollen-sterile sexual diploid T. officinale line TJX3-20 [1 seeds were sown, and the resulting F1 plants were screened by flow cytometry to determine ploidy.Pollen from a pollen-fertile apomictic  TJX3-20 . TJX3-20 TJX3-20 . The see TJX3-20 ). From a1 plants were vernalized for nine weeks in a cold room at 4 °C. Like the seed parent TJX3-20, all the F1 plants were male sterile and physically isolated from other wild-type Taraxacum, eliminating the possibility of sexual seed setting. Under these conditions, the development of a large seed head is a sign of apomictic seed setting. To determine the degree of apomictic seed setting, for each F1 plant, 50 randomly chosen brown seeds were germinated, and the numbers of seedlings germinating were counted. Germinating seeds implied that the mother was apomictic and had all components of apomixis.To induce flowering, eight-week-old FMSTA31, MSTA44B, MSTA53, MSTA64, MSTA67, MSTA73, MSTA74, MSTA78, MSTA85, MSTA101, MSTA105, and MSTA131 (Twelve codominant SSR loci were screened (Micro Satellite TAraxacum (MSTA) ,38: MSTA MSTA131 . DNA extMSTA78-a positive but did not show seed setting  were investigated by Nomarski DIC microscopy, as described in [Methyl-salicylate cleared ovules from plants that were ribed in . At leasTaraxacum apomictic seeds can be detected with a flow cytometer as a 6 × peak, derived from the unfertilized central cell, which contains two fused 3 × polar nuclei . Matzk and colleagues also reported that endopolyploidization peaks were absent in the autonomous apomictic seeds of T. officinale and Hieracium pilosella, suggesting that the presence of 6 × peaks in isolated seeds would be due to autonomous endosperm formation. To assess the ploidy of the endosperm, developing seeds from the apomictic F1 plants were collected five days after anthesis and directly analyzed by flow cytometry. Ten developing seeds were homogenized in Otto I buffer [Matzk and colleagues  have shoI buffer  by choppI buffer .1 seeds were produced in total.As previously described by Van Dijk and colleagues , stigmas1 plants were diploid (50%), 95 were triploid (49.5%), and one (0.5%) was tetraploid. These plants were the products of the fertilization of a haploid egg cell by a haploid, diploid, or triploid pollen grain, respectively. A68 produced many collapsed and small pollen grains, reflecting the high frequency of inviable aneuploid pollen grains produced because of unbalanced triploid pollen meiosis. However, no viable aneuploid offspring was recovered, which was fortunate, as the lack of aneuploidy in the F1 simplifies the interpretation of the genetic analysis of apomixis. None of the diploid F1 plants were apomictic, while the triploid F1 plants segregated for apomixis. The only tetraploid plant recovered was also capable of apomictic reproduction.The average seed set per seed head from this cross was only 2.1 seeds, while, in the diploid × diploid sexual crosses, an average of ~100 seeds per seed head were obtained. Flow cytometry showed that 96 of the 192 FMSTA78-a to diploid and triploid hybrids of this cross. While the segregation ratio of the three alleles in the triploid progeny was not significantly different from the Mendelian 1:1:1 ratio, the ratio was highly distorted in the diploid hybrids (0.01:0.47:0.52). Assuming that the single case of a MSTA78-a allele transmitted to a diploid hybrid was a recombination event between the a-allele and the DIP allele, it was postulated that haploid pollen grains carrying the diplospory allele were lethal [To gain further insights into the genetics of apomixis, Van Dijk and colleagues  (2009) ae lethal . To expa1 plant died before its reproductive system could be assessed, the remaining 94 3 × plants were used for further analysis. Apomictic seed setting was variable, as can be seen in the examples shown in 1 plants produced no germinating seeds , seed setting was not quantified). By contrast, 24 percent of the triploid F1 plants produced more than 80% germinating seeds (22/93). The remaining six plants produced seeds with a germination rate between 6 and 78 percent, which may be due to incomplete penetration of apomixis factors. The two-locus model outlined in 2 = 0.05, degrees of freedom (d.f.) = 1; the p-value is 0.82, not significant at p < 0.05) and contradicting the two-locus model .As one triploid F1 triploids and the 66 non-apomictic F1 triploids are listed. MSTA31 could not be scored due to the presence of null alleles in the parental genotypes. As expected, all 29 F1 triploids that reproduced via apomixis carried the paternal MSTA78-a , supporting the previously reported tight linkage between the MSTA78-a allele and the DIP locus [MSTA78-a SSR was used as a molecular marker for the presence of diplospory induced by the DIP locus (independent of the phenotypic assessment of apomixis) and will be referred to as the “Dip-marker” below. Molecular analysis showed that 63 of the 94 3 × F1 plants carried the MSTA78-a allele and were considered to be diplosporous. The observed frequency of 65 percent of F1 plants carrying the Dip-marker is also consistent with the expected two-thirds of the diploid A68 pollen grains carrying the D allele . Due to the function of the DIP locus blocking meiosis, no gametophytic segregation of the parthenogenesis or autonomous endosperm loci is expected in these plants. To see if molecular markers for either parthenogenesis or autonomous endosperm formation could be uncovered, these F1 plants were genotyped for additional markers for linkage to the apomictic phenotype /d (171 bp)/e (183 bp).To further understand the segregation of the components of apomixis, DNA from the progeny was screened with additional molecular markers . In IP locus ,29. Due 1 plants, 27 had the d allele and two did not . To determine if the MSTA44B-d allele was linked to either parthenogenesis or autonomous endosperm formation, Nomarski DIC microscopy phenotyping of developing seeds was performed.Of the 29 apomictic F1 plants that produced viable seeds, it is reasonable to assume that all apomixis components are present  carrying the MSTA44B-d allele, in which parthenogenetic embryos were observed with DIC microscopy but that had no observed autonomous endosperm formation and no viable seed set ; χ2 = 12.20; d.f. = 2; the p-value is 0.002, significant at p < 0.05). Additionally, in two diploid male-sterile progeny-plants carrying the MSTA44B-d allele, embryo-like structures, and autonomous endosperm-like tissues were visible with Nomarski DIC microscopy , althoucopy see . These d2 seeds of 24 3 × F1 plants were screened via flow cytometry (FCSS) to determine their ploidy  along with the non-apomictic 3 × F1 that had the DIP-marker allele and in which autonomous endosperm was directly observed , a total of 53 diplosporous 3 × F1 plants demonstrated autonomous endosperm formation. Only three did not show any sign of autonomous endosperm formation, and the remaining seven could not be scored (due to either failure to induce flowering or premature death). According to the three-locus model with a single dominant autonomous endosperm formation locus . Additionally, for the 20 diplosporous (Dip-marker present) 3 × F1 plants that lacked the parthenogenesis-linked Par-marker allele, 19 3 × F1 plants showed autonomous endosperm formation (95 percent). Thus, significantly more plants with autonomous endosperm were found than were expected, which suggests that the developmental function of autonomous endosperm formation in apomixis is more complex in Taraxacum than can be determined by a single dominant genetic locus.Due to the previously mentioned asynchronous initiation of autonomous endosperm, in order to perform a more robust assessment of the autonomous endosperm phenotype, the Fr ploidy . By combon locus , only 38Hieracium, Pennisetum, and Taraxacum, starting with a fulsome understanding of the underlying genetic system [Taraxacum cross examined here segregated for apomixis as a whole trait as well as for the individual components of apomixis. When considered as a single trait, apomixis was consistent with a three-locus model controlling apomixis. In addition to the already-known SSR marker for diplospory , a co-dominant marker tightly linked to the parthenogenesis allele was found  that was not genetically linked to the Dip-marker. Thus, the genotype for parthenogenesis is dominant simplex Ppp, and the parthenogenesis and diplospory loci are not genetically linked. A dominant simplex parthenogenesis genotype has also been reported in other members of the Asteraceae, such as Erigeron [Hieracium [Taraxacum. The presence of a single, major parthenogenesis locus significantly enables the cloning of the parthenogenesis gene in Taraxacum, as was done for the VPS13-like gene in the DIPLOSPOROUS locus [Apomixis has since long ago been a holy grail of agriculture ,4; now, c system ,43. The Erigeron ,12 and Hieracium ,14, perhUS locus . Reducin1 through a combination of cytological observations, seed flow cytometry, and molecular marker analysis. That autonomous endosperm did not appear to segregate was unexpected, because, in an earlier cytological study, phenotypically complementary recombinants between parthenogenesis and autonomous endosperm had been found [T. officinale. In addition, Cooper and Brink [T. officinale, while development was very synchronous in the sexual diploid related species T. koksaghyz. As extreme examples for T. officinale, Cooper and Brink [Diplosporous locus/gene, because at a distance of 3.5 cM [1 lines used for analysis or, at most, ~5 cM away. However, there is no sign of the suppression of recombination in the diplospory locus [While having the Par-marker will aid in finding the causal gene, the introduction of apomixis into crop plants also requires endosperm formation: either pseudogamy or autonomous formation. In this study, the segregation of autonomous endosperm was investigated in the segregating 3 × Fen found . An explen found  found a nd Brink  describend Brink  describef 3.5 cM , 2–3 meiry locus ,29, makiT. officinale. This is supported by the study of Mártonfiová and colleagues [Taraxacum are clearly different from what was seen in Erigeron and Hieracium. In these other apomictic Asteraceae species, autonomous endosperm co-segregated with parthenogenesis, either due to genetically linked genes in a single locus or due to the pleiotropic effects of a single gene. Since the activation of the egg to develop into an embryo and activation of the central cell to develop into an autonomous endosperm are in some ways similar processes, pleiotropy is a possibility, consistent with the observations of Guitton and Berger [Arabidopsis. It is also possible that in some cases, at least, the parthenogenetic embryo triggers autonomous endosperm development, as has been reported in Arabidopsis for a mutant with a single sperm cell [Our results strongly suggest an independent control of autonomous endosperm and parthenogenesis in lleagues . They ind Berger  in Arabierm cell .Taraxacum and other members of the Asteraceae.To aid the isolation of apomixis components, Van Dijk and colleagues  generateTaraxacum, each of which is controlled by a single dominant locus. However, apomixis as a whole does not fit a two-factor model, suggesting the presence of a third, critical dominant locus. Based on the observations presented here, this locus does not appear to be the single dominant autonomous endosperm formation locus that might have been expected. Apomixis may be co-controlled by a third unknown major locus of unknown developmental function or by several smaller background or modifier genes that jointly appear as a third main factor in this cross. In order to distinguish between these various hypotheses, further research is needed, which is critical if there is to be any progress in cloning the genes involved in the autonomous endosperm formation component of apomixis. While autonomous endosperm is a major component of apomixis in the Asteraceae, it is still the least understood.Diplospory and parthenogenesis are clearly major loci of apomixis in


Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field. Plant viruses are a class of obligate intracellular parasites, with minimal coding capacity, which heavily rely on their host to complete their infection/life cycle. Unlike in the case of other pathogens, there are barely any efficient antibiotics or other chemicals that can eliminate an infecting plant virus without perturbing host cells. Therefore, molecular plant breeding plays a pivotal role in generating virus immunity, virus resistance, or virus-tolerant plants in agricultural production to prevent and control plant viruses.Plant diseases caused by plant pathogens, including bacteria, fungi, nematodes, and viruses in different crops, lead to enormous economic losses worldwide. Plant viruses cause almost half of plant diseases, and the annual global cost of viral infection of cultivated crops is estimated to be more than US$ 30 billion . About 1CRISPR /CRISPR-associated (Cas) systems, derived from bacterial and archaeal sources, serve as an important adaptive immunity against invading nucleic acids. With the fast development of our understanding of CRISPR/Cas systems, they have been converted into convenient tools to edit endogenous and exogenous DNA or RNA sequences in different organisms. To date, a simplified CRISPR system only contains an easily engineered guide RNA (gRNA) and a Cas effector protein. Based on the different characteristics of Cas proteins in sequence, structure, and function, CRISPR/Cas systems are separated into two distinct classes , 2017a,bGeminiviridae and Caulimoviridae are two major destructive plant DNA virus families that contain 485 species with single-stranded DNA (ssDNA) genome and 85 species with double-stranded DNA (dsDNA) genome, respectively, see text foot note 1. Multiple independent works have aimed to directly target and destroy the genomic DNA of geminiviruses or caulimoviruses by plant gene editing technologies. Before the emergence of the CRISPR/Cas systems, the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been applied to manipulate the host and viral DNAs in plants. ZFN- and TALEN-mediated resistance to several geminiviruses, including tomato yellow leaf curl China virus (TYLCCNV) and tobacco curly shoot virus (TbCSV), by targeting the viral genomic replication-associated region has been reported . CompareNicotiana benthamiana or Arabidopsis thaliana plants, respectively (N. benthamiana plants (CRISPR/Cas9 constructs with single guide RNAs (sgRNAs) targeting the viral replication-associated region, or intergenic region (IR), have exhibited effective DNA interference and conferred viral resistance against beet severe curly top virus (BSCTV), cotton leaf curl Multan virus (CLCuMuV), and bean yellow dwarf virus (BeYDV) in transgenic ectively . Targetia plants . The sgRa plants . These fa plants .With the fast development of CRISPR/Cas9 system-based technologies, more and more successful examples show the promising resistance to plant DNA viruses. For example, a CRISPR/Cas9 system duplexed with sgRNAs targeting the movement protein (MP) or CP region established resistance to wheat dwarf virus (WDV) or banana streak virus (BSV), respectively . This apA. thaliana transgenic plants consistently expressing both Cas9 protein and sgRNAs targeting the CP region of cauliflower mosaic virus (CaMV) in the Caulimoviridae family conferred effective resistance to this species  and Cas13a  from Leptotrichia shahii (LshCas13a)  . Among thCas13a) . The LshhCas13a) , which ohCas13a) . A plantectively .N. benthamiana or A. thaliana plants, respectively , wherein sgRNA was driven by a pea early browning virus (PEBV) promoter in a tobacco rattle virus (TRV)-based vector in LshCas13a transgenic ectively ,b. Similectively . By targectively .Ruminococcus flavefaciens was characterized and classified into the type VI-D effector, named Cas13d (CasRx). This Cas13d effector with two higher eukaryotic and prokaryotic nucleotide-binding domains (HEPN) that dictate CRISPR RNA maturation and target cleavage also possesses RNase activity to target ssRNA , papaya ringspot mosaic virus-W (PRSV-W), and cucumber vein yellowing virus (CVYV)  (eIF(iso)4E in cassava and A. thaliana conferred full resistance to cassava brown streak virus (CBSV) or TuMV-GFP, respectively -resistant plants  . Also, as (RTSV) . The indectively . Cassavaectively . The newt plants . Recentlt plants .However, the current establishment of efficient, recessive resistance-type antiviral strategies was mainly dependent on eIF4s and their homologs against potyviruses and some related plant viruses. Therefore, more host susceptibility genes need to be identified and applied to obtain effective genetic resources against many other economically important plant viruses.Arabidopsis ssi2 mutant, which accumulates high levels of the plant defense hormone salicylic acid (SA), confers resistance to CMV, but shows an abnormal growth phenotype  The negative regulators of plant defenses, such as rgs-CaM that inhibited RNA silencing through repressing the transcription level of RDR6 and degrading the SGS3 protein utophagy , and homutophagy .(2) The susceptible factors involved in viral replication/translation/movement or other life cycles, such as tobamovirus multiplication 1 (TOM1) and its homologs involved in the replication of tobamoviruses , eEF1A a(3) The host factors participating in the modification of viral proteins to achieve their effective function in the infection, such as shaggy-related protein kinases  which could interact with and phosphorylate geminiviral C4 proteins, and are required to trigger disease symptoms . The CP (4) The factors involved in the cellular processes beneficial for virus behaviors, such as phenylpropanoid metabolism factor  or secondary cell wall synthesis factor , whose silencing in phloem tissue delays geminivirus infection .N. benthamiana and A. thaliana) and crops   and transgenic  assays without off-target defect. A recent report showed that the sonchus yellow net virus (SYNV)-delivered transgene-free CRISPR/Cas9 system could generate the genetic resources only by one step  and RNA viruses  have demonstrated efficient gene targeting frequencies in model plants (d maize) . In orded maize)  developeone step , which wone step . HoweverThe CRISPR/Cas systems provide many valuable tools and new thoughts in creating gene loss-of-function and gain-of-function mutants of plants, especially for economic crops. In the plant virus-combating arsenal, the interaction and battle between host plants and viruses never end. When plant virologists are determined to control the dissemination of plant DNA virus by using CRISPR/Cas9 technology to interfere with and destroy viral genome DNA, some accompanying challenges also come out.N. benthamiana  failing to confer any resistance to ACMV infection. The depth analysis of the full-length viral genomes in transgenic plants revealed about 33–48% of inserted geminiviruses. Although these new variants of ACMV generated by CRISPR/Cas9 could not proliferate themselves alone, they are able to rely on wild-type ACMV to accumulate in thamiana . Ali et thamiana . Howeverthamiana . Also, tle risks . In addiTherefore, the cases and risks that viruses escaped from the CRISPR/Cas system-mediated antiviral immunity in plants warn that the CRISPR/Cas system may be considered as a double-edged sword in antiviral engineering. Although it can target, destroy, or interfere with the viral genome to inhibit viral infection, it raises an important issue that the new viral variants or species may be generated as genome editing by-products, indicating that it would speed up the virus evolution or that the obtained transgenic crops may lose their specific resistance to these viruses.Molecular breeding has played a pivotal role in the prevention and control of plant viruses causing diseases, and the emergence and development of CRISPR/Cas technologies would speed up the generation and obtainment of new resistance resources. With the development and understanding of CRISPR/Cas systems and the exploration and function identification of recessive resistance genes, the resistance materials with the mutation or deficiency in host susceptibility factors generated by CRISPR/Cas systems would better benefit viral prevention and control in the field. Compared to targeting, destroying, or interfering with viral genomes, which might increase the risks of viral evolution, the editing of recessive resistance genes would have a promising perspective in engineering-resistant plants against plant viruses. Additionally, host susceptibility genes for insect vectors can be important targets by CRISPR/Cas systems to prevent the viral spread and dissemination. Of note is that it is also useful for antiviral engineering by utilizing newly developed CRISPR/Cas systems. For instance, the transcriptional activation of many plant-dominant resistance genes by CRISPR-Act2.0 would enhance the plant immunity to confer a broad range of resistance to different kinds of pathogens , and theFL, HZ, and XZ conceived the project. YC, FL, and XZ wrote the manuscript with contributions from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.