Interestingly, when SU5402 was used to inhibit FGF signaling, neuromast cells expressed much lower levels of the FGF receptor gene, fgfr1, but produced increased levels of Wnt target genes, including ctnnb1, ctnnb2, and tcf7l2, while bFGF treatment produced no alterations in the expression of those genes, suggesting that fgfr1 might restrict Wnt signaling in neuromasts during proliferation. In summary, our analysis demonstrates that both the Wnt and FGF pathways are tightly integrated to modulate the proliferation of progenitor cells during early neuromast development and regenerative cell proliferation after neomycin-induced injury in the zebrafish neuromast.Wnt and FGF are highly conserved signaling pathways found in various organs and have been identified as important regulators of auditory organ development. In this study, we used the zebrafish lateral line system to study the cooperative roles of the Wnt and FGF pathways in regulating progenitor cell proliferation and regenerative cell proliferation. We found that activation of Wnt signaling induced cell proliferation and increased the number of hair cells in both developing and regenerating neuromasts. We further demonstrated that FGF signaling was critically involved in Wnt-regulated proliferation, and inhibition of FGF abolished the Wnt stimulation-mediated effects on cell proliferation, while activating FGF signaling with basic fibroblast growth factor (bFGF) led to a partial rescue of the proliferative failure and hair cell defects in the absence of Wnt activity. Whole-mount in situ hybridization analysis showed that the expression of several FGF pathway genes, including  Studying sensory organs on the skin of zebrafish is revealing details of molecular signaling pathways that may be relevant to our own sensory systems, especially the hair cells of the ear. These cells have fine hair-like structures that move in response to sound waves and help generate electrical signals to the brain that result in perception of sound. Huawei Li and colleagues at Fudan University, Shanghai, China, studied the roles of two well-known cellular signaling pathways in regulating the proliferation of similar sensory hair cells in zebrafish, a commonly used model organism. These pathways involve cell surface proteins that interact with small extracellular molecules to stimulate molecular changes within cells. Learning how the pathways control hair cell generation and multiplication may assist modification of similar systems in humans to study and treat hearing loss. Cell proliferation is a critical process guiding many aspects of auditory organ specification and morphogenesis during embryonic development, and its misregulation is associated with various malformations. However, the precise molecular signaling pathways that control proliferation and their coordination remain unclear.2. Mechanosensory HCs share many structural, molecular, and functional similarities with vertebrate inner ear HCs and thus are an excellent model system for studying HC biology related to hearing and balance and for unraveling their genetic control4. However, unlike mammals, zebrafish retain the ability to quickly regenerate HCs after damage8. Thus, the molecular signaling pathways that guide HC regeneration in the zebrafish lateral line are of great biological and clinical interest.The zebrafish lateral line is a powerful vertebrate model system for the in vivo study of sensory organ developmental biology due to its well-established genetics and ease of in vivo visualization and manipulation. The posterior lateral line (pLL) comprises a series of mechanosensory organs called neuromasts, which lie on the surface of both the head and body. Neuromasts are composed of sensory hair cells (HCs) in the center that are surrounded by nonsensory supporting cells (SCs)11, FGF signaling13, and Wnt/β-catenin signaling11. Most of our current understanding about these pathways is related to early embryonic pLL formation, which entails primordium formation, migration, and deposition stages; however, the precise molecular signaling that initiates and guides cell proliferation and HC regeneration in zebrafish neuromasts remains poorly defined. The Wnt/β-catenin signaling pathway is a well-known signaling cascade that plays central roles in embryonic development and organogenesis by modulating cell migration, proliferation, and specification14. During the early stages of zebrafish pLL development, Wnt/β-catenin signaling is active in the leading part of the primordium and is necessary for cell proliferation and proneuromast formation as the primordium migrates along the body of the organism17. Constitutive activation of canonical Wnt signaling broadly promotes cellular proliferation throughout the primordium but stalls migration during developmental patterning, whereas pharmacological or genetic inhibition of Wnt reduces cellular proliferation, increases cell death, and results in a dramatic truncation of the pLL18. Recently, activation of the Wnt/β-catenin pathway was reported to promote proliferation and increase HC generation in the developing and regenerating zebrafish lateral line19. Conversely, inhibition of canonical Wnt signaling decreases proliferation and arrests neuromast morphogenesis at an early stage of neuromast development. Little is known, however, about the molecular mechanisms underlying this signal transduction during the periods of proliferation and HC regeneration in neuromasts.Previous studies have revealed that the development of the pLL requires complex coordination among diverse molecular signaling pathways, including chemokine signaling20, and FGF is active in the trailing region of the zebrafish pLL primordium, which contains two or three rosette-shaped proneuromasts. FGF acts as a key molecule in promoting the epithelialization and formation of the proneuromasts in the course of the migration of the primordium13, and FGF inhibition leads to a loss of apically constricted rosettes and aberrant migration of the primordium. FGF activation not only induces pro-neuromast formation by initiating the organization of center-oriented epithelial rosettes but also initiates the specification of HC precursors by regulating atoh1a expression13. Although the function of FGF signaling in facilitating pLL morphogenesis has been reasonably well studied, its contribution to proliferation during the establishment of the pLL and to regenerative cell proliferation has not been well addressed.FGF signaling has been shown to regulate cell migration and cell fate changes during embryonic developmentfgf3 and fgf10 genes were Wnt targets during neuromast cell proliferation and acted to modulate FGF activity and that fgfr1, an FGF target gene, acted to repress Wnt activity. Taken together, our findings suggest that the Wnt and FGF signaling pathways are tightly connected to regulate developmental cell proliferation and regenerative cell proliferation in the zebrafish pLL neuromast.In this study, we used pharmacological agonists and antagonists and transgenic zebrafish to regulate Wnt and FGF signaling, and we found significantly more proliferating cells and more HCs and SCs after activation of Wnt or FGF signaling. To further understand whether the Wnt and FGF pathways act synergistically to regulate proliferation, we performed epistasis experiments to verify the mechanisms underlying proliferation in neuromasts. We stimulated Wnt signaling first and then inhibited FGF activity and found that the proliferation of the progenitors induced by Wnt activation disappeared after blocking FGF in Wnt-activated embryos. Conversely, we found that inhibition of Wnt signaling using the DKK1 conditional knockout transgenic line or IWR-1 treatment followed by treatment with basic fibroblast growth factor (bFGF) resulted in significantly more proliferating cells than in the group treated with IWR-1 alone, suggesting that activation of FGF could partly rescue proliferation failure caused by Wnt signaling inhibition. Similar results were observed in HC regeneration experiments. To better understand the interactions of both signaling pathways during cell proliferation in the larval zebrafish neuromast, we performed whole-mount in situ hybridization analysis. We showed that the 21, and embryo ages were marked as hours post fertilization (hpf). The wild-type strain was derived from the AB line, and the Tg(brn3c:mGFP)s356t line was used to visualize HCs. The apcmcr line was a generous gift from Professor Xu Wang. APC genotyping primers were as follows: APCR—CAT GGC TCA CTC TGC ACA; APCWTF—ATA ATG TTG CAG CTG ACC; and APCMTF—ATA ATG TTG CAG CTG ACT. Tg(hsp70l:dkk1b-GFP) offspring were incubated at 42 °C for 5 min at 48 hpf and allowed to recover at 28.5 °C to inhibit Wnt signaling. Approximately 50% of the embryos did not turn green, and these served as controls. To prevent pigment formation, embryos were treated with 0.003% 1-phenyl-2-thiourea  in E3 water from 14 hpf onwards. The larvae were anesthetized in 0.02% MS-222  before fixation. All zebrafish experiments were performed following the institutional guidelines approved by the Institutional Animal Care and Use Committee of Fudan University, Shanghai.Embryos were obtained by natural spawning and developed at 28.5 °C in E3 medium. They were staged according to standard protocolsWe used 1 μM BIO to activate Wnt signaling and 10 μM IWR-1 (Sigma-Aldrich) to inhibit Wnt signaling, and we used 5 μM SU5402  to inhibit FGF signaling and 20 ng/ml bFGF (Invitrogen) to activate FGF signaling. Neomycin sulfate (Sigma-Aldrich) was added to a final concentration of 400 μM, and the 5 dpf larvae were incubated for one hour, followed by three rinses in fresh egg water. The larvae were then allowed to recover at 28.5 °C.For immunofluorescence experiments, 10 mM BrdU (Sigma-Aldrich) was coincubated with the pharmacological treatments described above to label the proliferating cells. Zebrafish larvae were fixed with 4% PFA for 2 h at room temperature and washed three times with PBT-2 (PBS containing 1% Triton X-100). For DNA denaturing, the fixed larvae were treated with 2 N HCl for 30 min at 37 °C followed by three rinses with PBT-2. Before antibody staining, the larvae were incubated in blocking solution (10% donkey serum in PBT-2) for 1 h at 37 °C. The primary antibodies were anti-Sox2 , anti-BrdU , anti-GFP , and anti-Myosin VI . After incubation with primary antibodies overnight at 4 °C, the larvae were washed three times with PBT-2 and then incubated with Alexa Fluor 488-, 594-, and/or 647-conjugated secondary antibodies  for 1 h at 37 °C. After the larvae were washed several times in PBT-2, they were incubated with DAPI  for 20 min to label the nuclei.Fluorescent specimens were examined using a Leica confocal microscope . All images were edited using Adobe Photoshop CS6. The labeled cells in the pLL neuromasts (pLL2–pLL5) were counted in the confocal and fluorescence images using the counting and measuring tools in Adobe Photoshop CS6.22. After the color reaction, the embryos were mounted with 100% glycerol and observed under a bright field microscope (Nikon Instruments). Binding sites were identified as blue-black dots.Digoxigenin-labeled probes were prepared as recommended by the manufacturer . Primers for cloning the examined genes are listed in Table Apoptotic cells in the trunk of the zebrafish were detected by TUNEL assay . After the larvae were rinsed three times with PBT-2, they were incubated with the TUNEL reaction mixture in a humid dark chamber at 37 °C for 1 h and then labeled with DAPI to visualize the nuclei.t-tests and one-way ANOVA. In all figures, error bars represent the mean ± SEM. p < 0.05 was considered statistically significant, and p < 0.001 was considered highly significant. *Shows the comparison with controls, and # is used when the Wnt and FGF signaling pathways were shown to interact.All statistical analyses were performed with GraphPad Prism 6 Software . Cell counts were analyzed using Student’s ctnnb1, ctnnb2, and tcf7l2. After 24 h of pharmacological treatment, a significant increase in ctnnb1, ctnnb2, and tcf7l2 expression was observed in the 1 μM BIO-treated group, indicating induction of Wnt signaling, whereas 10 μM IWR-1 significantly inhibited the expression of these genes  transgenic line that expresses GFP in the HC membrane23, and the SCs were marked and counted by Sox2 immunostaining. Beginning at 48 hpf, embryos were treated with BIO or IWR-1 in the presence of BrdU for a period of 24 h. Quantification showed significantly more BrdU+ cells, GFP+ HCs, and Sox2+ SCs in the BIO-treated neuromasts than in the DMSO-treated control neuromasts 25, and the second was a heat-shock–inducible dkk1 transgenic line 26. In these experiments, myosin VI was used to label mature HCs. Consistent with the pharmacological results, the numbers of proliferating cells and differentiated cells (HCs and SCs) in neuromasts were significantly higher in Apcmcr larvae relative to those in wild-type (wt Apc) at 72 hpf   Fig. . WISH re28. We, therefore, examined whether FGF signaling plays an important role in neuromast development in the zebrafish pLL. We first conducted a comprehensive expression analysis by means of WISH to examine the expression of known FGF signaling-related genes. In 72 hpf zebrafish larvae, expression of the FGF signaling receptor gene (fgfr1) and target gene (pea3) was clearly detectable within the neuromast  for 24 h, with DMSO treatment as the control. At the highest dose (15 μM), we observed evident larval death (10/12), whereas 5 μM SU5402 did not cause developmental abnormalities or death. We then measured the expression of pea3 and fgfr1, and WISH showed that the expression of both genes was significantly downregulated after 5 μM SU5402 treatment for 24 h compared to expression in DMSO controls  for 24 h, the expression of pea3 and fgfr1 was robustly upregulated compared to the expression in unstimulated neuromasts, and no deformities were detected  and FGF  signaling pathways. As expected, in BIO-treated larvae, the presumed Wnt target genes ctnnb1, ctnnb2, and tcf7l2 were markedly upregulated in the neuromast, which was consistent with sustained activation of Wnt/β-catenin signaling, whereas significant decreases were detected for these genes in the IWR-1 treatment group   larvae with 400 μM neomycin for 1 h, followed by incubation in 6-well plates with or without the presence of different agents for a period of 24 h or 48 h (24 hpa and 48 hpa). We then performed a BrdU (10 mM) incorporation assay and quantified the number of regenerated HCs. The BIO-treated larvae showed significantly more BrdU incorporation in the neuromasts than did DMSO-treated controls at both 24 and 48 hpa . Loss of HCs and SCs following inhibition of Wnt signaling was also partly restored by ectopic FGF activation (Fig. To determine if the increase in proliferation after Wnt activation during regeneration requires FGF signaling, we simultaneously activated Wnt signaling and inhibited FGF signaling using BIO and SU5402, respectively. Indeed, the BIO-induced increase in total proliferation was reduced to below normal levels after the simultaneous addition of SU5402, indicating that the majority of extra BrdUoup Fig. . This cooup Fig. g2, G2, bIn the present study, we used a zebrafish model to gain new insights into the mechanism underlying the control of cell proliferation and fate determination in developing and regenerating neuromasts in the zebrafish pLL, and we provide evidence that activation of the Wnt and FGF pathways is crucial for promoting the proliferation and regeneration of HCs in the pLL. Our current study represents the first analysis indicating that Wnt acts through the FGF signaling pathway to promote progenitor cell proliferation and regenerative cell proliferation in the zebrafish pLL neuromasts. These findings provide strong evidence that manipulation of the Wnt and FGF pathways will be useful for promoting HC regeneration in the mammalian inner ear.34 however, the exact mechanisms through which Wnt signaling stimulates cell proliferation and regeneration under these conditions remain unresolved. Here, we used BIO as a Wnt agonist to activate Wnt signaling in zebrafish neuromasts. Consistent with previous reports, our results indicate that activation of Wnt/β-catenin signaling promotes cell proliferation in developing and regenerating neuromasts. More HCs and SCs were observed in the neuromasts of the BIO-treated group, which suggests that both HCs and SCs were being overproduced and that most of the proliferated SCs differentiated into HCs after BIO treatment. Genetic activation of Wnt/β-catenin signaling phenocopies the pharmacological activation by promoting progenitor cell proliferation and regenerative cell proliferation in neuromasts.Recent studies in zebrafish showed that activating Wnt signaling promotes the proliferation of neuromast progenitors and HC regeneration in both developing and damaged neuromasts;35. Loss of FGF signaling in fgf3 and fgf8 double-deficient zebrafish not only causes failure of placode formation but also blocks HC development36, and Fgf3 and Fgf8 have been shown to act as upstream activators of the atoh1 gene, which is necessary for HC development during distinct inner ear developmental periods, including both the early development of the preotic placode and the later development of the otic vesicle37. Based on these studies, we hypothesized that early neuromast development requires the FGF signaling pathway. To test this hypothesis, we first performed in situ analyses and confirmed the expression of candidate ligands and receptors of the FGF signaling pathway in the neuromast, revealing that this pathway is potentially activated in the developing neuromast. Next, to test whether FGF signaling is involved in the proliferation of neuromast progenitors, we inhibited the FGF signaling pathway using SU5402. The results of our proliferation assay suggested that blockade of FGF signaling inhibited cell proliferation, similar to that observed when the Wnt signaling pathway was inactivated. Given that many similarities between developmental and regenerative processes in the pLL have been identified40, we hypothesized that FGF activation would enhance regenerative proliferation and subsequent new HC generation in the pLL neuromast, similar to the effects of FGF activation during initial development. As hypothesized, neomycin-damaged larval zebrafish exposed to bFGF during recovery periods had significantly more SCs in their neuromasts than neomycin-alone control animals, indicating that increased cell proliferation had occurred, and this was followed by increased HC production. Together, these results suggest that FGF signaling plays a key role in regulating normal neuromast development and HC regeneration by promoting proliferation, similar to that observed for Wnt signaling in HC development and regeneration.A number of studies have indicated the importance of FGF in inner ear development in many speciesTo identify the Wnt-FGF interactions that are required for cell proliferation in larval zebrafish neuromasts, we performed epistasis experiments using a combination of pharmacological and genetic approaches to manipulate Wnt and FGF activity. We compared cell proliferation with activated Wnt signaling alone or with activation of Wnt and inhibition of FGF signaling using the small molecule inhibitor SU5402. While activation of Wnt alone increased cell proliferation and induced greater HC numbers, it was unable to do so in the absence of FGF signaling. To further confirm that FGF functions downstream of Wnt signaling to promote cell proliferation in developing neuromasts, we used IWR-1 to downregulate the Wnt signal and then added bFGF to activate the FGF pathway. Our proliferation assay showed that bFGF treatment encouraged neuromast progenitors to proliferate and partly rescued the cell proliferation phenotype in the absence of Wnt signaling. In neomycin-damaged neuromasts, simultaneous manipulation of Wnt and FGF signaling activities showed that while Wnt signaling is sufficient to drive ectopic cell proliferation and subsequent HC regeneration, this pathway was able to do so only in the presence of functional FGF signaling.fgf3, fgf10, pea3, and fgfr1, whereas their expression in IWR-1-induced Wnt-inhibited neuromasts was significantly reduced. Furthermore, stimulating FGF signaling through the addition of bFGF had no effect on the expression of Wnt components. In addition, no significant changes in the expression of ctnnb1, ctnnb2, or tcf7l2 were observed in the neuromasts of the IWR-1+bFGF group compared to that in the neuromasts of the IWR-1–alone group, indicating that there had been no reactivation of Wnt signaling by bFGF treatment after IWR-1 treatment. Taken together, our results suggest that the pro-proliferative effect induced by Wnt activation was mainly mediated by the FGF pathway, which is consistent with previous reports. For instance, Fgf8 levels are reduced in the absence of Wnt activity in mouse embryos41, while FGF activity is enhanced in β-catenin overexpression embryos43, suggesting that FGF functions downstream of Wnt/β-catenin signaling. Intriguingly, FGF activation did not change the expression of fgf3 or fgf10 in developing neuromasts, and thus, Wnt can be reasonably assumed to activate the FGF signaling pathway through fgf3 and fgf10. We also found that inhibiting FGF signaling with SU5402 resulted in downregulation of fgfr1 and upregulation of fgf10 and fgf3 as well as several Wnt components, suggesting that a feedback mechanism involving Fgfr1 might operate during neuromast proliferation. A number of studies have shown that FGF components, such as fgf3 and fgf10a, have mitogenic activity;46 however, their signaling functions in cell proliferation during neuromast development have not yet been explored. We showed that active Wnt and FGF signaling exerted their function by negatively regulating the cell cycle inhibitor genes p21 and p27 and positively regulating the ccnd1 gene to permit cell proliferation. Sequential induction of Wnt signaling and inhibition of FGFR caused a complete loss of ccnd1 expression and enhanced p21 and p27 expression in the neuromast, similar to SU5402 treatment alone, indicating an essential role of FGF signaling in maintaining the expression of cell cycle genes.In this work, we characterized the expression patterns of the FGF and Wnt pathway components in developing neuromasts in detail. We showed that Wnt activation significantly increased the expression of FGF components such as 49. Therefore, defining which signals act downstream of the Wnt-FGF interactions during proliferation in neuromasts is of great interest.In this study, we showed that the Wnt and FGF pathways cooperate closely to regulate proliferation in the zebrafish neuromast. To fully understand the heterogeneity of the neuromast domain, it is necessary to identify all of the transcriptional targets that are activated or repressed by FGF and Wnt regulation during neuromast proliferation in zebrafish. Recently, many studies have shown that bFGF activates several intracellular pathways, including the RAS/ERK and PI3K/AKT cascadesfgf3 and fgf10 stimulation and that FGF signaling inhibits the Wnt pathway through the Wnt inhibitor fgfr1. Our findings contribute to studies on HC biology and provide a new avenue for understanding the signaling mechanisms that regulate cell proliferation during mammalian inner ear development.In summary, we have investigated the cross talk between the Wnt and FGF signaling pathways during cell proliferation and regeneration in the zebrafish neuromast and have shown that FGF activation enhances proliferation and promotes subsequent cell differentiation in the developing and regenerating neuromasts of the zebrafish pLL. Furthermore, we provide evidence that Wnt signaling activates FGF activity via Supplementary Figure LegendSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Table S1


Dietary fibre is well acknowledged to be critical in maintaining the gut homeostasis in human and other monogastric animals. As a small monogastric herbivorous animal, rabbit is much sensitive to the reduced intake of dietary fibre and more interestingly shows individual difference in clinical tolerance. In the present study, we fed rabbits with fibre-deficiency diet for two weeks and successfully distinguished the individual tolerances according to clinical signs and gastrointestinal gross lesions. A total of 40 treatments were classified into three groups of the full health (N = 10), moderate intestinal disorder (N = 11) and severe intestinal disorder (N = 19). Together with three controls, 43 individuals were subjected to gut microbiota profiling by 16S rRNA gene sequencing. It was revealed that the Firmicutes/Bacteroidetes ratio steadily decreased from 1.74 in healthy group to 1.03 in severe group. However, the healthy individuals that showed complete tolerance still remained a comparable Firmicutes/Bacteroidetes ratio with controls. Notably, the class Alphaproteobacteria was found to be higher abundance in healthy group than controls and other treatment groups. The results would improve our understanding of the relationship among dietary fibre, gut microbiota and host health. Over the past decades our dietary habits, especially in western countries, have dramatically shifted toward a lower intake of fibre as well as excessive consumption of the refined fats and sugars, which would be a key factor to drive incidences of obesity, diabetes and inflammatory bowel diseases worldwide4. In the livestock production, fibre is also an important dietary component for maintaining normal physiological function in the digestive tract for monogastric animals7.Dietary fibre, which is defined as carbohydrate polymers with ten or more monomeric units and indigestible in small intestine8. Because mammalian genomes fail to encode active enzyme for the structural carbohydrates, dietary fibre is absolutely degraded via microbial fermentation within either hindgut of monogastric herbivores and omnivores or rumen for ruminants10. The major fermented products of dietary fibre are short-chain fatty acids mainly consisting of acetate, propionate and butyrate11, which play important roles for modulating host immune responses and could also serve as energy sources of metabolism for both host and gut microorganisms15. Therefore the dietary fibre content and sources would have important roles in maintaining the gut homeostasis that is a mutually beneficial balance between gut microbiota and host, whereas both gut microbiota and host genetic background could conversely affect the degradation of dietary fibre18.The physiological effects of dietary fibre are associated with gut microbiota composition and metabolic profiles, gastrointestinal motility and immunity, and other systemic consequences21. Rabbit (Oryctolagus cuniculus) belongs to the family Leporidae and has been widely used as animal model in biomedical researches22. As a small monogastric herbivorous animal, rabbit is much sensitive to the dietary fibre content that low dietary fibre level could quickly and obviously induce intestinal disorders23. According to our field observation and other reports24, there are individual differences in clinical tolerance against the fibre-deficiency diet. A few candidate genes, such as Toll-like receptor 4 (TLR4) and myeloid differentiating factor 88 (MyD88), had been identified in our former studies to be associated with intestinal disorders of rabbit26. However, it still remains largely unknown about the link between gut microbiota and differential tolerance against the reduced intake of dietary fibre. In the present study, we fed rabbits with fibre-deficiency diet and successfully distinguished individual tolerance, for which the gut microbiota was analyzed comprehensively using high-throughput sequencing approach.In addition to health implication of the long-term dietary pattern, changes in gut microbiota can be immediately observed even after short-term consumption of the altered diets, such as conversion between animal-based and plant-based dietsThe study design was approved and all methods were performed in accordance with guidelines of Institutional Animal Care and Use Committee in College of Animal Science and Technology, Sichuan Agricultural University.vs. 15%, acid-detergent fibre of 13% vs. 21% and neutral-detergent fibre of 29% vs. 36%  and control (N = 6) groups, respectively. Rabbits in treatment group were fed with fibre-deficient diet for two weeks after a 7-day dietary transition, whereas control group was consecutively fed with standard diet Fig. . The desTwo indices in relation to intestinal health were taken into consideration for evaluating individual responses to the reduced dietary fibre level, including (i) each rabbit was carefully observed twice a day for precisely recording clinical signs of intestinal disorders throughout experimental period; (ii) all rabbits were slaughtered and subjected to an autopsy for checking gastrointestinal gross lesions at the end of experimental period. All rabbits were classified according to the two indices that were independently scored by three experienced colleagues and only those individuals that could be unambiguously classified (N = 40) were included for the gut microbiota profiling. Together with three randomly selected controls, a total of 43 rabbits were finally subjected to the high-throughput sequencing.We precisely observed the clinical sign in relation to intestinal disorder for each rabbit, including the disturbance of food intake, diarrhoea, constipation  and presence of mucus in excreta. Based on daily records during the whole experimental period for these clinical signs, every rabbit was empirically and averagely scored with four levels. By macroscopic observation for each part of gastrointestinal tract  at necropsy, symptoms and lesions were clearly described, including the watery content, dilatation, impaction, congestion and mucus. Similarly, four levels were used to subjectively score the gross lesions of gastrointestinal tract. The description of scoring standard was shown in Table 2·EDTA. Hematological analyses were immediately performed using an automatic hematological analyzer of Medonic CA620 . Finally, we measured six haematological indices, including the total red blood cell count (RBC), platelet count (PLT), white blood cell count (WBC), lymphocyte count (LYMs), neutrophil count (GRANS) and mid cell population count (MIDs).The whole blood samples were collected by vacuum-based blood collector with K27. The PCR condition involved an initial denaturation step at 95 °C for 4 min and 20 cycles of 95 °C for 1 min, 56 °C for 45 sec, and 72 °C for 1 min, and followed by an extension step at 72 °C for 7 min using a Bio-Rad CFX96 thermal cycler .To avoid the sampling bias, the entire caecal contents were first blended sufficiently and then collected for each rabbit. The total DNA was extracted from caecal content (about 200 mg) using QIAamp DNA Stool Mini Kit . After checking quality of the extracted DNA by NanoVue Plus , the V3 region of bacterial 16S rRNA gene was amplified using HOTSTAR Taq Plus Master Mix Kit  and the universal primers (338 F: 5′-ACT CCT ACG GGA GGC AGC AG-3′ and 533 R: 5′-TTA CCG CGG CTG CTG GCA C-3′)Each sample was independently amplified in triplicate, which were pooled and then purified using QIAquick PCR Purification Kit . Amplicons with both a total amount of ≥3 μg and OD260/280 ratio ≥1.8 were used to produce sequencing libraries using Illumina DNA Sample Preparation Kit  according to manufacturer’s instructions. Finally, the libraries were sequenced on Illumina HiSeq™ 2000 platform for generating the 150 bp paired-end reads.28. Paired-end reads were merged to produce tags using USEARCH tool with the minimum overlapping length of 25 bp and minimum total length of 100 bp; and all the produced tags were further filtered according to the expected error probability of 0.2529. After both forward and reverse primers were removed, we finally obtained the clean tags.For the raw reads of 16S rRNA gene sequencing, we unconditionally cut the first base and trimmed adaptor sequences using Cutadapt tool30, in which all singletons were also discarded. Based on the representative tag, every OTU was taxonomically annotated using RDP naïve Bayesian classifier with confidence value of 80%31. For each sample, the community richness (Chao1 and ACE) and diversity  at OTU rank were calculated using VEGAN R package32. Using STAMP tool33, all samples were clustered by principal component analysis (PCA) and statistical deduction of differentially abundant features among the four groups was conducted by one-way analysis of variance (ANOVA) and Tukey-Kramer post hoc test. The Benjamini-Hochberg procedure was used for controlling the false positive rate in multiple comparisons. Additionally, the unclassified sequences are only included for denoting the relative proportion of feature, which is calculated relative to the total number of sequences being assigned to its parent category at phylum or grandparent category at other levels.Subsequently, we employed UPARSE-OTU algorithm for clustering operational taxonomic units (OTUs) according to the recommended steps and similarity threshold of 0.03Raw files of gut microbiota sequencing reads are available at NCBI Sequence Read Archive (SRA) under Bioproject PRJNA393739.Among the 60 rabbits initially included in the treatment group, a total of 40 individuals could be unambiguously scored for both clinical signs and gastrointestinal gross lesions (Supplementary Table p < 0.05). All six haematological indices didn’t show significant differences among the control, healthy and moderate groups. The results would also support our classification of rabbits to indicate differential health states.Because we finally failed in preparing the qualified blood samples, the six haematological indices were successfully collected only for 33 rabbits .We first employed PCA method for clustering all samples based on the OTU profile Fig. , in whic3. Therefore, adequate intake of dietary fibre was recommended by American Dietetic Association with 25–38 g/day for adults34. However, the fundamental mechanism underlying health benefits of dietary fibre has remained largely unknown. Unlike human and other rodents, rabbit is much sensitive to the dietary fibre content and obviously shows individual difference in clinical tolerance against fibre-deficiency diet25. In this study, we fed rabbits with fibre-deficiency diet over a relatively short period of time and successfully established the animal model of intestinal disorders induced by deficiency of dietary fibre. This feeding experiment clearly distinguished individual outcomes in response to the reduced dietary fibre level and estimated that ~25% of individuals show complete tolerance, whereas almost half the individuals also develop into server intestinal disorders. These individual differences would be helpful for understanding the impact of dietary fibre on intestinal health.Although dietary fibre has little nutritional merit per se in human, its health benefits for preventing many chronic diseases have been well acknowledged35. In human and other mammals, the gut microbiota is typically dominated by two bacterial phyla of Firmicutes and Bacteroidetes and the Firmicutes/Bacteroidetes ratio is also apt to be influenced by dietary pattern36. By investigating fecal microbiota, it was revealed that children from rural Africa with long-term consumption of fibre-rich diet have higher abundance of Bacteroidetes and less Firmicutes than that of European children19. In a follow-up trial, healthy adults with a short-term supplementation of dietary fibre similarly showed the decreased Firmicutes/Bacteroidetes ratio16. In contrast, the decreased Firmicutes/Bacteroidetes ratio was also observed in rabbits fed with fibre-deficiency diet in the present study. However, the healthy individuals that showed complete tolerance against the reduced dietary fibre level still remained a comparable Firmicutes/Bacteroidetes ratio with controls. Our results would provide additional evidence for understanding relationship among dietary fibre, gut microbiota composition and host heath.The beneficial and detrimental impacts of gut microbiota on host health have been widely revealed38. However, the functional implications of class Alphaproteobacteria for regulating individual response to deficiency of dietary fibre should be investigated in the future.In addition to the altered Firmicutes/Bacteroidetes ratio, phylum Actinobacteria was also revealed to be highly abundant in severe group but almost completely absent from the healthy individuals and controls. Therefore, the increased abundance of Actinobacteria is likely a consequence of the developed severe intestinal disorders. Additionally, we observed that healthy rabbits with complete tolerance against the reduced dietary fibre level had the highest abundance of class Alphaproteobacteria in comparison with both controls and other treatment groups. This result would likely suggest a potential association of Alphaproteobacteria with individual tolerance against the reduced dietary fibre level. We also found that a higher abundance of Alphaproteobacteria was reported in patients with Crohn’s diseaseIn the present study, we fed rabbits with the fibre-deficiency diet and distinguished individual response to such diet intervention. Using the high-throughput sequencing approach, we comprehensively investigated gut microbiota composition and found that the Firmicutes/Bacteroidetes ratio would be associated with differential tolerance.Supplementary Table S1Supplementary Table S2


We present a case of a 68-year-old female who presented with left iliac fossa pain and postprandial vomiting for 7 days. Initial CT scan revealed a large mass within the upper abdomen in close proximity to the pancreatic tail, with central fat density, marginal enhancement, a distended vessel coursing through the centre and hazy increased density of the fat outside of the structure. A diagnosis of intra-abdominal focal fat infarction (IFFI) was made and she was treated conservatively. Subsequently, she re-presented with further pain and elevated inflammatory markers. A repeat CT scan again demonstrated the well-defined mass, but now with the development of fluid attenuation within the centre and several pockets of air. Radiologically guided percutaneous drains were inserted into the area of liquefaction and subsequent microbiology analysis revealed mixed coliform bacilli; targeted antibiotics were administered. The collection resolved on subsequent imaging. Identification of radiological features of IFFI is very helpful in establishing a diagnosis and may negate the need for surgical intervention. She reported the pain had been occurring for the past year but now had worsened. She had no change in bowel habit, haematemesis or haematochezia. She had a Whipple’s procedure 5 years previously, and 7 years previously had undergone a lumpectomy for breast malignancy. On examination, she was apyrexial and had left iliac fossa tenderness. Her white cell count was 12.8 × 10A CT scan of her abdomen and pelvis performed 4 weeks previously for follow-up of her pancreatic disease showed mild sigmoid diverticulosis. A repeat contrast-enhanced CT scan was performed to confirm suspicion of acute sigmoid diverticulitis. However, it revealed a large mass within the left upper quadrant, with central fat density, marginal enhancement, a distended vessel coursing through the centre and hazy increased density of the fat outside of the mass. Uncomplicated sigmoid diverticulosis was noted and unchanged  and 2.CT scan confirmed a diagnosis of partial greater omental infarct, an entity within the broader diagnostic category of “intra-abdominal focal fat infarction (IFFI)”. A conservative management pathway was followed and she was discharged with analgesics 2 days later.9 l–1, with a neutrophil count of 20.04 × 109 l–1 and a CRP of 210 mg l–1.The patient re-presented 2 weeks later with further pain in the left iliac fossa and epigastrium and persistent postprandial vomiting, anorexia and weight loss. Her white cell count had climbed to 22.7 × 10A repeat CT scan showed enlargement of the previously noted left upper quadrant mass, with new central fluid attenuation and several pockets of gas; no hollow viscus perforation could be identified and there was no free peritoneal gas .Other causes for the left upper quadrant collections include acute/acute-on-chronic pancreatitis—with associated collection/pseudocyst, inflammatory carcinoma of the large bowel, acute diverticulitis with perforation and "contained" gastric perforation; however, these would not demonstrate a lesion of fat density.The differentials for intra-abdominal fat density mass lesions with surrounding inflammatory change would include epiploic appendagitis (EA), omental infarction and mesenteric panniculitis.It was surmised the area of fat liquefaction had become infected. As such, percutaneous drains were inserted under ultrasound guidance, and a repeat CT scan showed the collection had decreased in size .–1), thus excluding leakage of pancreatic fluid. Symptoms improved with drainage of the liquefaction. She was discharged on day 11 after a marked reduction in drain contents and an improvement in her symptoms. A follow-up CT scan 10 months later showed complete resolution of the changes (Microbiological analysis of the drained material revealed mixed coliform bacilli and targeted antibiotic therapy was administered. An amylase level performed on the sample was negative (5 IU l changes .1The term “intra-abdominal focal fat infarction” encompasses a range of conditions where infarction of fatty tissue is the underlying pathological process, including EA and segmental omental infarction, as well as rarer entities such as falciform ligament infarction.2 A radiological diagnosis based upon the specific features is helpful as IFFI can be managed conservatively with oral anti-inflammatory therapy3 and correct diagnosis negates further investigation and potential surgical treatment.4 Further imaging may be required when symptoms and signs deteriorate on a conservative management pathway, as complications such as fat necrosis, liquefaction and secondary abscess formation may be identified and prompt a change in management such as in this case. Laparoscopic resection of the inflamed areas may prevent recurrence and other complications.3IFFI mimics more common intra-abdominal pathology and is frequently misdiagnosed.T1 weighted images, although slightly less intense than normal peritoneum with loss of signal on fat-suppressed T2 weighted images. Peripheral inflammatory changes are displayed as hypointense on T1 weighted images, hyperintense on T2 weighted images and enhancing on contrast enhanced fat-suppressed T1 weighted images.5IFFI pathogenesis is torsion and vascular compromise of the fatty structure with engorgement of central vessels and inflammation secondary to ensuing ischaemia. Distinguishing features can be identified on ultrasound, CT scan and MRI.The term “intra-abdominal focal fat infarction” encompasses a range of conditions where infarction of fatty tissue is the underlying pathological process.IFFI mimics more common intra-abdominal pathology and is frequently misdiagnosed. A radiological diagnosis based upon the specific features is helpful as IFFI can be managed conservatively with oral anti-inflammatory therapy and correct diagnosis negates further investigation and potential surgical treatment.Further imaging may be required when symptoms and signs deteriorate with conservative management, as complications such as fat necrosis, liquefaction and secondary abscess formation may have resulted.


Grade 3 oral mucositis (OM) is historically observed in >90% of bone marrow transplant patients who received the cyclophosphamide + total body irradiation (CY+TBI) conditioning regimen. It was previously shown that orotopically applied adrenergic vasoconstrictor prevented up to 100% of radiation-induced oral mucositis in two preclinical animal models.Adrenergic vasoconstrictor  was orotopically applied to three patients (ages 24–29) who received the CY+TBI conditioning regimen; they were compared to five matched controls who received no orotopical vasoconstrictor. All patients received the CY+TBI conditioning regimen for acute lymphoblastic leukemia within the University of Wisconsin Adult Bone Marrow Transplant Program. Over the seven-day Cy+TBI conditioning regimen, 20 min before each treatment, either radiation or chemotherapy, vasoconstrictor was applied topically to the oral cavity, and patients then received either 1.5 Gy whole-body radiation or IV cyclophosphamide.OM severity was scored over a three-week period using: i) physican assessments, ii) daily photos of the oral cavity, iii) oral pain and oral function score sheets, and iv) recorded narcotic consumption. Both “Grade 3 OM” duration and “any OM” duration in vasoconstrictor-treated patients were substantially lower than for the five control patients. Though nasogastric tube or total parenteral nutrition were used in 3 out of 5 control patients, there was no use of these supportive care measures in the three vasoconstrictor-treated patients.Orotopically applied NG11–1 vasoconstrictor formulation substantially reduced the incidence and severity of “Grade 3” and “any” oral mucositis when compared to matched control patients, all of whom received the same CY+TBI conditioning regimen. The liquid orotopical formulation was easily tolerated by patients both in its ease of use and lack of side effects. Our earlier work showed complete suppression of radiation-induced oral mucositis [·OH) that attack DNA are largely formed by “splitting water molecules,” the molecular oxygen (O2) required to “fix” the DNA damage can be excluded from these cells by the topically-applied vasoconstrictor transiently excluding delivery of oxygen-bearing blood to the mucosal epithelium during irradiation. In this case, ROS-modified DNA nucleotides then “decay” (in milliseconds) back to their basal state. The efficacy of a topically applied vasoconstrictor can be visually observed by the “blanching” of mucosa or skin that occurs when oxygenated red blood (as well as any blood-borne chemotherapy it contains) is excluded from the ~1 mm deep layer of mucosa or skin which: i) lies above the submucosal/ subcutaneous vasculature, and ii) contains the mucosal, epidermal, or hair follicle “stem” cells, whose radiation- or chemotherapy-induced apoptosis initiates the oral mucositis, radiation dermatitis or alopecia side effects observed in cancer patients. The transient hypoxia of the surface oral or epidermal stem cells following topical application of vasoconstrictor, or the transient excluded delivery of systemic chemotherapy to oral/epidermal surface stem cells [Oral mucositis (OM) occurs commonly in 40–80% of patients receiving high-dose chemotherapy conditioning regimens prior to hematopoietic stem cell transplants, in >90% of patients who receive the cyclophosphamide plus total body irradiation (CY+TBI) conditioning regimen –3, and iucositis  in two rem cells  is seen em cells  or solidem cells . The topem cells . Topicalem cells , alopeciem cells  and radiem cells  in animaBased upon the orotopical vasoconstrictor-induced prevention of OM in animal models, an initial clinical study was approved by the University of Wisconsin Health Sciences Institutional Review Board to determine if an initial dose  could suppress the incidence and severity of OM in CY+TBI patients. Three patients signed written informed consents for participation in the study and for publication of the clinical data as a case report. Institutional Review Board approval allowed study physicians to access pertinent information , etc.) from files of five patients who received the CY+TBI regimen at UW during the 12-month period preceding this study for comparison to the topical vasoconstrictor-treated patients.Baseline features of the three topical vasoconstrictor patients and the five control patients are shown in The three topical vasoconstrictor-treated patients received a formulation containing 16.3 mM phenylephrine, an α-adrenergic vasoconstrictor formulated in an aqueous-alcohol vehicle with excipients that was specifically designed  for tranThe sprayed and directed application of 3.0 ml of drug formulation was originally designed for use in pediatric patients, to avoid inadvertent swallowing of the drug. There were no enrolled children, and in future studies simple “swish and spit” administration will be used.Oral mucositis severity was scored each day by a study physician using the WHO Mucositis Scale and the RTOG common toxicity criteria. Patients were evaluated using the Oral Mucositis Daily Questionnaire (OMDQ) and the Mouth Pain Categorical Rating Scale to assess oral pain. These assessments were performed for seven days prior to bone marrow transplant and continued until any evidence of OM had resolved or the patient was discharged from the hospital. After discharge from the hospital, assessments were repeated at the 1–2 week follow-up clinic visit and the three month follow-up clinic visit to be sure OM had resolved and no unexpected side effects were noted. Use of total parenteral nutrition as well as the ability to eat and drink were recorded. Doses of opioid and non-opioid analgesics were recorded.The occurrence of oral mucositis during radiotherapy and chemotherapy is common. Clinical management of the pain and narcotic side effects, dehydration, dysphagia and weight loss, oral and septic infections, and substantial costs to HMOs are significant problems. The U.S. Cancer Pain Relief Committee concluded that “effective treatments to reduce the pain and functional impairment of oral mucositis are needed in this patient population .” There The use of oral cryotherapy to reduce OM severity  is in itAn adrenergic vasoconstrictor, orotopically applied in a one minute “swish and spit” administration, provides several advantages over the oral cryotherapy approach, including: i) there is none of the discomfort associated with holding a mouth full of ice, ii) it can be used in head and neck radiotherapy patients where ice cannot, iii) the adrenergic agonist-induced vasoconstrictor effect can be controlled in a predictable manner through standard dose and application time strategies, i.e., a single vasoconstrictor application whose transient hypoxic effect dissipates quickly after a daily radiotherapy session, or, multiple applications over several hours to sustain the exclusion of blood-borne chemotherapy from the oral mucosa through 1–2 plasma half-lives of the systemic chemotherapy drug(s), iv) protection of the oral cavity and tongue surface epithelia that contact the orotopically administered vasoconstrictor could also confer protection to taste buds.In this study, the physician photographed the patient’s oral cavity at every visit. The orotopically applied NG11–1 vasoconstrictor formulation of phenylephrine was shown to substantially suppress the incidence and severity of both the “Grade 3” and “any” oral mucositis when compared to matched control patients, all of whom received the same CY+TBI conditioning regimen within the same UW Bone Marrow Transplant Program. The liquid orotopical formulation was easily tolerated by patients both in its ease of use and lack of side effects.


Genomic selection had a higher selection response for FHB resistance than phenotypic selection, while association mapping identified major QTL on chromosome 3B unaffected by plant height and flowering date.Fhb1, though haplotype analysis highlighted the distinctiveness of both QTL. Comparison between phenotypic and genomic selection for FHB resistance revealed a superior prediction ability of the former. However, simulated selection experiments resulted in higher selection responses when using genomic breeding values for early generation selection. An earlier identification of the most promising lines and crossing parents was feasible with a genomic selection index, which suggested a much faster short-term population improvement than previously possible in durum wheat, complementing long-term strategies with exotic resistance donors.Fusarium head blight (FHB) is one of the most destructive diseases of durum wheat. Hence, minimizing losses in yield, quality and avoiding contamination with mycotoxins are of pivotal importance, as durum wheat is mostly used for human consumption. While growing resistant varieties is the most promising approach for controlling this fungal disease, FHB resistance breeding in durum wheat is hampered by the limited variation in the elite gene pool and difficulties in efficiently combining the numerous small-effect resistance quantitative trait loci (QTL) in the same line. We evaluated an international collection of 228 genotyped durum wheat cultivars for FHB resistance over 3 years to investigate the genetic architecture and potential of genomic-assisted breeding for FHB resistance in durum wheat. Plant height was strongly positively correlated with FHB resistance and led to co-localization of plant height and resistance QTL. Nevertheless, a major QTL on chromosome 3B independent of plant height was identified in the same chromosomal interval as reported for the prominent hexaploid resistance QTL The online version of this article (10.1007/s00122-018-3253-9) contains supplementary material, which is available to authorized users. Triticum durum (Desf.)) is susceptible to pathogenic fungi of the Fusarium genera such as Fusarium graminearum and Fusarium culmorum, which cause Fusarium head blight (FHB), one of the most destructive diseases of wheat worldwide. FHB leads to significant yield losses, but the major concern is the contamination of the crop with mycotoxins. Durum wheat is mostly used for human consumption, and the risk of toxin-contaminated grain entering the food chain is consequently particularly high. During the past few years, FHB on small-grain cereals has significantly increased due to changes in crop management practices, minimum or reduced tillage and intensification of maize in the crop rotation and weather patterns with more humidity and warm temperatures during anthesis  and the International Center for Agricultural Research in the Dry Areas (ICARDA) only five moderately resistant Tunisian lines were detected  were obtained for marker-assisted selection with few significant markers in an extensive study of 2325 European winter wheat lines where no large effect QTL could be detected. However, employing genomic selection increased prediction accuracies to 0.6  from Northern America, the Mediterranean, Central Europe and Australia as well as from the CIMMYT international durum wheat breeding programme was analysed in this study. The 228 lines were chosen from a larger diversity panel of 269 released varieties and advanced breeding lines, which was assembled by Maccaferri et al.  with the given population structure ; thus, for all subsequent genome-wide association mapping analyses, model  model for each trait separately:N × 1 vector of BLUEs from the phenotypic analysis, N × M matrix that contained the marker coding for the M candidate markers of each trait, respectively, and the random marker effects were assumed to follow a normal distribution N + V) × 1 vector of line effects with the genetic variance ith line, jth marker, and ith line at the jth marker. Model  simultaneously, where half of the selection candidates are discarded in the first stage either by phenotypic culling or genomic index selection, and preselected candidates are retested in disease nurseries for conducting final selection decisions.Phenotypic and genomics-assisted selection for FHB resistance was subsequently compared in several simulated selection experiments featuring three selection strategies that might be applied in a practical breeding programme Fig. :Fig. 1OnN = 160 lines from the mapping population and the latter V = 40 lines that were sampled from the validation population. It should be mentioned that these numbers were seen as appropriate given the exploratory nature of this study, although training and validation population sizes are generally much larger in applied breeding programmes.A small-scale durum breeding programme was assumed for the purpose of this study, where the budget allows assessing FHB resistance only in one artificially inoculated field trial each year. Separate training population and selection populations were 50 times sampled for all simulated selection experiments, where the former comprised, as in the assessment of the prediction abilities, Genomic-based selection relied on a G-BLUP model with a training population that was phenotyped in one trial/year, which was designated as the prediction year. Phenotypic selection was on the other hand based on phenotypic data of the selection candidates from a second trial/year designated as the selection year. Genomic-assisted selection was finally based on the combination of data obtained in the prediction and selection year, where selection was conducted with kinship enhanced phenotypic breeding values derived from model  with a f(1.1)Phenotypic selection and genomic-assisted selection among all 40 selection candidates.(1.2)Genomic-based and enhanced genomic-based selection among all 40 selection candidates, where the most promising marker for FHB severity was included as fixed effect into the latter.Given this framework, several basic approaches were initially compared for the one-stage single-trait selection with extensive disease phenotyping:(2.1)Phenotypic selection with culling in the first stage, i.e. the prediction year, where no FHB resistance information for lines in the selection population was available yet. Among the 40 selection candidates, the 10 earliest and latest lines, respectively, were culled and the leftover 20 lines were ‘advanced’ for FHB resistance phenotyping in the selection year.(2.2)Genomic-assisted selection with culling of the 20 most susceptible selection candidates according to genomic-based breeding values, followed by genomic-assisted selection among the preselected lines in the selection year.(2.3)Genomic-based and enhanced genomic-based selection among all selection candidates as described in 1.2).Moreover, the two-stage single-trait selection strategy aimed to reflect a typical scenario in many line breeding programmes where genomic selection is conducted in parallel to preliminary or observation yield trials Phenotypic selection with culling in the first stage, i.e. the prediction year, where no FHB resistance information for lines in the selection population was available yet. Among the 40 selection candidates, the 10 smallest and tallest lines, respectively, were again culled and the leftover 20 lines were ‘advanced’ for FHB resistance phenotyping in the selection year.(3.2)Genomic-assisted index selection with culling of the 20 most susceptible selection candidates according to a genomic-based selection index, followed by genomic-assisted index selection among the preselected lines in the selection year.(3.3)Genomic-based and enhanced genomic-based selection with a genomic selection index among all selection candidates as described above.The simultaneous selection for multiple agronomic traits and FHB resistance could accordingly be challenging, especially due to the often-observed negative correlation between FHB severity and plant height. Hence, the merit of genomic selection indices that took this issue into consideration was investigated in the framework of the two-stage multi-trait selection strategy by:ith trait, The culling of the smallest and tallest lines was, as a breeder would expect that lines with a very short stature will show also a strong susceptibility to FHB, while tall-statured plants will most likely be more resistance to FHB but on the other hand possess less tolerance against lodging. Genomic index selection was conducted with the previous described restriction index  aiming tr = 0.79–0.82), followed by flowering date (r = 0.51–0.59), and lowest for FHB severity (r = 0.44–0.54) (Table S1). A comparatively narrow range was observed for flowering date given a diversity panel that was constructed from a worldwide collection of durum lines, which was, however, expected due to the preselection of lines with regard to agronomically homogeneous properties under Central European conditions. The range for plant height in the retained lines was larger and had a maximum of 90 cm due to the set culling level when preselecting lines for further analyses in this study. A large genetic variance was estimated using the AUDPC as FHB severity measure, which was four times larger than the genotype-by-trial interaction variance though smaller than the residual variance  . The heritability of the latter trait was though with h2 = 0.73 slightly lower than the heritability of the AUDPC (h2 = 0.79), which further underlined the merit of repeated scorings during the field season. Despite the fact that the range of FHB severity was rather large, no resistant lines could be identified as their performance varied between medium to highly susceptible. Lines with Canadian origin displayed thereby the best performance, but also showed the highest plant height under Central European conditions . They were directly followed by lines developed in the USA whose plant height was on average lower, whereas a large range was observed for the other origins though with strong similarity in the average plant height across origins. Considering the entire panel, a negligible correlation was found between flowering date and FHB severity based on BLUEs from the across-trial analysis  , while the correlation between plant height and FHB severity was more pronounced for the entire panel  though dependant on the presence of the semi-dwarfing allele at the Rht-B1 locus .The correlation between the AUDPC and the last FHB scoring was high . Therefore, they represented the worldwide durum germplasm collection well and were regarded as suitable for validating marker–trait associations found by genome-wide association mapping.Principal component analysis did not reveal a clear population structure neither on the phenotypic level based on Euclidean distance between standardized phenotypic data for plant height, flowering date and FHB severity nor genotypically based on genome-wide distributed markers . Lines from ICARDA and CIMMYT were closely related to each other, highlighting most likely the close collaboration and germplasm exchange between these institutions. Lines with other origins were more widely spread, and especially lines from Italy could be found throughout the target genetic space. Estimation of ancestry coefficients showed accordingly an admixture between origins, while in the STUCTURE-like analysis . The 3B QTL-linked marker IWB64968 had a minimal effect on plant height (additive effect of 0.8) and no effect on flowering date, in the entire population, whereas the 6B QTL-associated marker IWB66697 had a pronounced positive influence on plant height (additive effect of 1.5) analysing all 228 lines. The contradicting results for the 6B FHB resistance QTL on plant height between the mapping and validating population were most likely due to the low resistance allele frequency of 11% and might be the result of a sampling effect (Table S3).Seven loci were found to improve FHB resistance in the international durum wheat collection at hand that were mapped to positions on chromosomes 1A, 2A, 2B, 3B, 6A and 6B. Merely, two marker–trait associations on chromosomes 3B and 6B were retained when filtering according to the permutation test based significance threshold of − logbarc133 and Xgwm493 that designates the confidence interval of the prominent resistance QTL Fhb1 in hexaploid wheat  was identified to confer resistance against fungal spread , whereas for the second PFT carrier, the susceptible French durum variety Exeldur, a missense mutation in the second exon of the gene was detected . In conclusion, the detected FHB resistance QTL on chromosome 3B in durum wheat is dissimilar to Fhb1, and only one susceptible line possessed the functional PFT gene. Nevertheless, the found marker–trait association on chromosome 3B deserves further attention due to the generally narrow genetic variation of FHB resistance in durum and might be among others a useful resource to support genomic breeding strategies.The major resistance QTL in this durum germplasm mapped to chromosome 3B between the SSR markers XRht-B1 locus among the high-confidence markers, and the 2A marker for flowering date as it was the most reliable marker–trait association for this trait according to the p value. The relative advantage of enhanced genomic-based versus marker-assisted selection in terms of prediction ability was accordingly 21% for FHB severity, 38% for plant height and 20% for flowering date. Both approaches were, however, far inferior to phenotypic selection based on BLUEs, while using the marker information to model relationship between lines in a genomic-assisted selection gave some increase in prediction ability of 4% for FHB severity and 8% for flowering date but had no effect on the prediction of plant height.The detected marker–trait associations for FHB severity and the other traits were accordingly tested for their merit in a marker-assisted selection, which was parallelly compared with phenotypic and genomic selection using the entire set of 8275 genome-wide distributed markers. Predicting the validation population that was left out from the marker discovery was feasible using the previously found marker–trait associations, although genomic selection was clearly superior to marker-assisted selection for most traits especially when enhanced by modelling one major QTL as fixed effect Fig. . For thir = 0.39 to r = 0.41 . Using a matrix based on Pearson correlations between genomic breeding values enabled on the other hand a stronger adjustment for plant height (r = − 0.05) that was though, as expected, accompanied by a reduction in prediction ability for FHB severity (r = 0.33).Aside from upweighting major QTL in the prediction of FHB severity, phenotypic imputation or trait-assisted selection with prior information about plant height and flowering trait was tested as a convenient option to increase the prediction ability as both traits are generally early available in a breeding programme. The employed multivariate model could accordingly increase the prediction ability for FHB severity from .41 Fig. a. The neons Fig. b. A neglGiven the large advantage of phenotypic over genomic-based selection in terms of prediction ability (+ 40% for FHB severity), several simulated selection experiments were conducted to further test under which circumstances genomic selection could still be useful when breeding for FHB resistance in durum. The selection among a randomly sampled set of 40 lines from the validation population showed a larger response for FHB severity by phenotypic than genomic-based selection in the selection strategy with extensive disease resistance phenotyping, reflecting the higher prediction ability of the former method Fig. a. NevertThe second simulated selection experiment aimed to reflect the need of a two-stage selection with preselection in many breeding programmes. Despite a lower prediction ability, the results showed a strong advantage of genomic over phenotypic selection due to earlier available information and even a genomic-based selection gave an average advantage of 43% over the two-stage phenotypic selection where no FHB phenotypic information was available in first stage Fig. b. MarkedEmploying a genomic selection index was able to compensate for this undesirable indirect response to plant height; it resulted though also in a lower response to selection for FHB severity Fig. c. PhenotRht-B1b. The 5A plant height QTL mapped in proximity of the gibberellin-responsive dwarfing gene Rht12 originating from the gamma ray-induced winter wheat mutant ‘Karcagi 522M7K’ . Plant height is one of the foremost morphological traits impairing FHB severity, with the general dependency—the shorter the plants the more severe are FHB epidemics, resulting in numerous co-localizations of plant height and resistance QTL Supplementary material 2 (PDF 193 kb)Supplementary material 3 (PDF 183 kb)Supplementary material 4 (PDF 171 kb)Supplementary material 5 (DOCX 18 kb)Supplementary material 6 (XLSX 53 kb)Supplementary material 7 (DOCX 27 kb)Below is the link to the electronic supplementary material.