The transition from a high forage to a highly fermentable diet can induce digestive disorders in the rumen. To date, the host mechanisms that regulate the adaption to such dietary transition are largely unknown. To understand the molecular mechanisms involved in such phenomena, RNA-sequencing was performed to identify the changes in the transcriptome of ruminal epithelia during gradual transition from a diet containing 0% to 89% grain.n = 15) fed 0%, 72% and 89% barley grain diet, respectively. The transcriptome profiles of rumen epithelia differed between low grain diet (LGD) (0% grain) and high grain diet (HGD) (72% and 89%), and HGD tended to reduce the expression of genes involved in epithelial catalytic and binding activities. When diet was changed from 72% to 89% grain, the mean ruminal pH change was significantly different among individual heifers with five of them decreased (down group (DG); from 6.30±0.09 to 5.87±0.15, P < 0.01) and five of them increased (up group (UG); from 5.84±0.42 to 6.35±0.37, P < 0.05). The functional analysis of differentially expressed (DE) genes revealed inhibited “Immune response of leukocytes”, “Attraction of phagocytes”, and “Cell movement of leukocytes” (P < 0.05) functions  in DG, and inhibited “Concentration of lipid” and “Proliferation of epithelial cells” functions in UG . In addition, the expression of genes involved in ketogenesis (HMGCL) and lipid synthesis  was increased in DG, while the expression of ketogenesis  and cholesterol synthesis related genes (HMGC and FDPS) were deceased in UG. Furthermore, the upstream regulators were found to be involved in the regulation of immune response and cell cycle progress, and SNP (g.46834311A > G) in FABP4 was identified between two groups of animals (P < 0.1).In total, the expression of 11,044, 11,322 and 11,282 genes were detected in ruminal epithelia of beef heifers  contains supplementary material, which is available to authorized users. To meet the global demand for meat consumption, it has become a common practice in the beef industry to use intensive feeding strategies such as high energy and high concentrate diets to finish cattle . HoweverNHE3) gene was different between acidosis-resistant and acidosis-susceptible steers during rapid high grain diet transition, indicating the differences in intracellular proton removal could be attributed to variation in the host response.When cattle are fed high fermentable diets, the increase and accumulation in short chain fatty acid (SCFA) production together with subsequent decrease in ruminal pH are usually observed due to increased microbial fermentation . In the To date, the underlying regulatory mechanisms for the host response to ruminal pH change has not been well defined. In this study, we aimed to identify the molecular mechanisms for variation in the response to a gradual transition to a high grain diet within the same animal by characterizing the global gene expression pattern of ruminal epithelia using RNA-seq based transcriptome profiling. The identified mechanisms may help to explain the observed animal variation in maintaining a balanced ruminal pH, which provide new insight into decreasing risk of ruminal acidosis for the beef industry.A total of 1130 million (25.11 ± 2.89 million reads per sample) high-quality 100-bp paired-end reads were obtained from 45 ruminal papillae samples collected from 15 heifers fed diets containing 0%, 72%, and 89% grain, respectively. Of these reads, ~86.1% of them were mapped to the bovine genome (UMD 3.1) and the expression of 11,044, 11,322 and 11,282 genes were detected (with reads per million (RPM) > 1 in 15 heifers fed each diet) in the ruminal epithelial tissue under each dietary condition, respectively. Among them, the expression of 10,880 genes was commonly detected from three dietary conditions Fig. . The mosThe PCA plot revealed that the rumen epithelial transcriptome profiles of heifers fed the low grain (0%) diet were different than those fed high grain diets (72% and 89%) except one outlier from 89% diet Fig. . When thP < 0.01 (down group (DG), 5 heifers); similar, from 6.17±0.25 to 6.19±0.25, P = 0.27 , 5 heifers); and higher, from 5.84±0.42 to 6.35±0.37, P < 0.05 (up group (UG), 5 heifers)  Fig. . When thrs) Fig. . The DG n = 18) showed the same change trend in both DG and UG (14 genes were down regulated and 4 gene were up regulated), while expression of 5 DE genes exhibited opposite change trend between the two groups. In particular, the expression of CYP1A1 and CYP1B1 were up regulated in UG (FC = 1.8 and 1.6) but were down regulated in DG (FC = −1.7 and −2.0), whereas the expression of CHAC1, CDSN and KLK10 were up regulated in DG  but were down regulated in UG  , the gene expression profiles displayed different changes. In total, 67 DE genes  were found in DG  Fig. .P < 0.05), and were inhibited when fed 89% grain diet   for DG after second dietary transition (Table n = 45) in the DG was related to “Innate immune response” (P < 0.0001)  Table .Table 2En = 163) in UG using DAVID revealed functions related to activity in cell proliferation, such as “Cell division”, “Mitotic metaphase plate congression”, “Chromosome segregation”, and “mitotic nuclear division” (P < 0.001) (Table n = 122) in UG were “Positive regulation of JUN kinase activity”, “Toxin metabolic process”, “Cholesterol homeostasis”, and “Drug metabolic process” (P < 0.05) , while the functions of “Cell cycle progression” and “Transmigration of phagocytes” were activated  after second dietary transition (Table Staphylococcus aureus infection” and “complement and coagulation cascades” (5 down regulated genes), and “T cell receptor signaling pathway” (3 upregulated genes)  and “TREM1 signaling” (Z-score = −2.0) Table . For theIFNγ), transmembrane protein 173 (TMEM173), toll like receptor 3 (TLR3), and tumor necrosis factor (TNF)  and prostaglandin E receptor 2 (PTGER2) being the hub nodes  Fig. . For thedes Fig. . Two and Fig. CSF and prosSMCT1 (P < 0.1) was lower while the expression of FABP4 (P < 0.05), HMGCL (P < 0.1), and SREBF2 (P < 0.1) was higher when fed the 89% grain diet than the 72% grain diet. In the UG heifers, the expression of FABP5 (P < 0.05), ABCA2 (P < 0.05), and ABCA7 (P < 0.1), ACAT2 (P < 0.05), HMGCS1 (P < 0.1), HMGCR (P < 0.1), and FDPS (P < 0.1) was lower, whereas the expression of ABCA1 (P < 0.05), and ABCA5 (P < 0.1) was higher when fed the 89% grain diet than the 72% grain diet , and MCT4 (P < 0.05) was decreased in DG, while the expression of NHE3 (P < 0.05) was increased in UG after the second dietary transition  were further investigated in the rumen epithelia , C-X-C chemokine ligand 5 (CXCL5), and NOD-like receptor C5 (NLRC5) were downregulated during the diet transition from 72% to 89% grain. The C2/C3 plays central role in the complement system and contributes to innate immunity [NLRC5 might limit the recognition of commensal bacteria, which is essential for the development and function of the immune system in the mucosal and peripheral districts [The most enriched networks of DE genes in DG and UG were related to immune response and cell cycle, respectively, suggesting that the innate immune function might be differently regulated between DG and UG heifers during second dietary transition. Although scarce information is available about the organization of the mucosal immune system in the rumen epithelia, the Langerhans cells were identified in epithelium of bovine forestomach  and boviimmunity . It’s weimmunity , 27. Theistricts . Indeed,istricts . The preistricts . The obsCDC25B), cyclin-dependent kinases regulatory subunit 2 (CKS2), cyclin B1/2 (CCNB1/2), polo like kinase 1 (PLK1), and aurora kinase A (AURKA), suggesting the enhanced cell cycle arrest in UG. Furthermore, the enriched KEGG pathway of “p53 signaling” in UG also indicates the higher cell arrest since the activated p53 could activate cell repair and apoptosis procedure to induce cell cycle arrest [ADH6), cytochrome p450 family 1 subfamily A member 1(CYP1A1), and subfamily B member 1 (CYP1B1)) in UG suggest the higher capacity to remove the ruminal toxins in these animals since these genes have been reported to be involved in the metabolism of the some ruminal toxins, such as ethanol and serials of xenobiotics [IFNG, TNF, TLR3, and TMEM173 regulate genes involved in the immune function, while the CSF2 and PTGER2 regulate genes involved in cell proliferation.In addition, the enriched pathway of “G2/M DNA damage checkpoint regulation” was activated through downregulation of genes related to cell cycle progression in UG –32, incle arrest , 34. Morobiotics , 36. TakSMCT1) tended to decrease in the DG heifers but no difference was observed in the UG heifers during the second transition period. The SMCT1 and SMCT2 play important roles in SCFA transport in addition to the SCFA−/HCO3− exchanger system [SMCT1 in the DG heifers might be decreased, which may partly account for the lower mean pH in DG heifers. In addition to SCFA absorption, SCFA metabolism could also impact on the rumen pH environment. In this study, we focused on the genes involved in butyrate catabolism in the rumen epithelia, which occurs via ketogenesis and beta-oxidation to produce ketone bodies [ACATs and HMGCS, the genes encoding the rate-limiting enzymes for ketogenesis [HMGCL increased in DG for the same dietary transition period, suggesting that UG heifers have decreased ketogenesis in the epithelia comparing to DG.Considering the fact that VFAs absorption accounts for up to 50% of the ruminal buffering capacity , the incr system , 38. Thee bodies , 40. Theogenesis , decreasFABP4 was observed in DG heifers but not in UG heifers when diet transitioned from 72% to 89% grain. Fatty acid binding proteins (FABPs) usually bind to fatty acids and/or lipids to transport and deliver them to different sites for utilization [FABP4 was responsible for the enhanced macrophage lipid accumulation in diabetic patients of human, and this effect was further paralleled with elevated intracellular total cholesterol and triacylglycerol levels [FABP4 in the ruminal epithelia of DG heifers suggest the intracellular lipid accumulation, which could be partially supported by the tended increased expression of sterol regulatory element binding protein 2 (SREBF2) in the same group of animals. The SREBF family plays important role in regulating cholesterogenic and lipogenic gene expression in the bovine liver and mammary glands [HMGCS1, HMGCR and FDPS) was decreased in UG heifers when diet transitioned from 72% to 89% grain. Steele et al. [ABCA1 was observed in the UG, but not in DG heifers. In addition, the function of “Concentration of lipid” was inhibited in the UG heifers, suggesting the UG might be more efficient in exporting cholesterol out of rumen epithelia and have less stored cholesterol. Taken together, our results suggest that the deposition of cholesterol in addition to cholesterol synthesis in the rumen epithelia is also one of the mechanisms to attribute to the variation observed between DG and UG animals  using genome wide association study (GWAS) are needed.Furthermore, this study also identified the genetic variation of genes related to lipid transport and metabolism. Association analysis indicated that 1 SNP (g.46834311A > G) in IFNG, TNF, TLR3, CSF2, and PTGER2), and FABP4-A/G SNP could be potential gene and genetic markers for selecting cattle with maintained ruminal pH during high fermentable diet transition.This is the first study that has identified varied molecular mechanisms may contribute to the individual variations in response to the high grain adaptation through analyzing the genome wide transcriptome in the ruminal epithelia of beef heifers. Understanding the whole transcriptome is essential to reveal the molecular events within cells, and to elucidate the mechanisms regulating the adaptive function and physiology of rumen in response to dietary changes. Overall, we suggest that the different genes that controlled immune function, cellular repair function, and intracellular homeostasis (cholesterol) might be the molecular mechanism accounting for individual variation in the response to gradual transition to a high grain diet. As summarized in Fig. n = 6)) treatment and 3 heifers assigned to the rapid grain adaptation (RA) treatment (n = 18). Unequal weighting of the treatment assignment was used as more variation was expected for the RA treatment than for the CON . All the RA heifers we randomly assigned to a gradual grain transition treatment (n = 18) throughout a 29-d experiment period. Briefly, heifers were initially fed a diet containing 0% grain (days 1 to 4) and transitioned to a final diet containing 89% grain using the following intermediate diets: 40% grain (days 5 to 8), 60% grain (days 9 to 12), 72% grain (days 13 to 16), 85% grain (days 17 to 20), and 89% grain (days 21 to 29). Rumen papillae biopsies were collected from the ventral sac of the rumen when the heifers were fed 0% grain on day 3 (the 3rd day of a 4-day feeding period), 72% grain on day 15 (the 3rd day of a 4-day feeding period), and 89% grain on day 26 (the 6th day of a 9-day feeding period). The biopsies were immediately washed with sterile 0.01 M phosphate-buffered saline buffer (pH 6.8) and transferred into RNA-later solution . After being kept at 4 °C overnight, all the samples were stored at −20 °C for further analysis. To identify the molecular mechanisms responsible for the varied response to a gradual high grain diet transition, the ruminal papillae samples from RA heifers were used in this study and due to three of animals did not have biopsy samples for all three treatment periods, only fifteen animals (under three dietary conditions) were subjected to transcriptome analysis.Twenty-four Angus-Hereford cross-bred yearling heifers  from the University of Alberta Kinsella Ranch  were used for this study. Detailed information on the animal study, diets, feeding of diets, and intake has been reported previously . BrieflyRuminal pH was measured using a stand-alone submersible continuous ruminal pH measurement system following the procedures as reported previously . BrieflyThe acidosis index, an indicator of the severity of ruminal acidosis, was calculated by the following formula: acidosis index (pH·min/kg) = area that ruminal pH below 5.8 (min)/dry matter intake (kg) .Ruminal papillae samples were ground while immersed in liquid nitrogen using a frozen mortar and pestle prior to RNA extraction. Total RNA was extracted from 50 mg of the resultant papillae tissue powder using Trizol reagent  following the manufacturer’s instructions. The quantity and quality of the RNA were determined using Qubit 2.0 Fluorometer  and Agilent 2100 Bioanalyzer , respectively. The samples with RNA integrity number (RIN) greater than 8.0 were used for library construction.The RNA-seq libraries were constructed using the TruSeq mRNA Sample Preparation Kit  according to the manufacturer’s instructions. Total RNA (1.0 μg each) from 45 samples (each with a unique index) were mixed and pooled for transcriptome sequencing  at Génome Québec  using the Illumina HiSeq 2000 system (Illumina) with all 45 samples loaded on a single chip with 9 samples per lane: each lane contains samples from 0%, 72%, and 89% grain diet. All reads were demultiplexed according to their index sequences with CASAVA version 1.8 (Illumina) and reads that did not pass the Illumina chastity filter were discarded.http://htseq.readthedocs.io/) based on the annotation from the ENSEMBL (http://uswest.ensembl.org/) bovine gene annotation software (v75.30). The expression levels of mRNA in each library was calculated by normalizing reads to reads per million (RPM) by the following formula: RPM =  × 1,000,000.RNA-seq reads were aligned to the bovine genome (UMD 3.1) using Tophat 2.0.10 with default parameters . The numn = 15). After that, the individual DE genes were identified by comparing the two high grain diets (89% vs. 72%) of each group (n = 10). The DE mRNA were identified by false discovery rate (FDR) < 0.05 based on Benjamini and Hochberg multiple testing correction [FABP4), lipid transport (ABCA1), Na+/H+ exchange (NHE3), and ketogenesis (ACAT2) was detected using RT-qPCR.Among the detected genes, those that were detected in at least one sampling point with more than 1 RPM in 15 heifers were considered as expressed genes. Then, differentially expressed (DE) genes were investigated by using bioinformatics tool edgeR . The DE rrection  as well -ΔΔCT  method.A total of 4 genes were selected to validate the DE genes and the primers used were listed in Additional file http://david.abcc.ncifcrf.gov) [www.ingenuity.com). In details, we used all detected genes in rumen epithelia  as background of DAVID for functional analysis  terms analysis of the commonly and highly expressed genes among 0%, 72%, and 89% die, and the ratio was calculated according to the number of ‘hits’ to the terms over the total number of ‘class hits’ . The funcrf.gov) , and IngSMCT1 and SMCT2), proton-linked monocarboxylate transporter , long chain fatty acid transporter , cholesterol efflux protein , bicarbonate transporter (PAT1), anion exchanger  were analyzed by T-test in rumen epithelia of DG and UG heifers when the diet switched from 72% to 89% grain.The expression of sodium-linked monocarboxylate transporter  that activate the volatile fatty acids, acetyl-CoA acetyl transferases (ACAT1 and ACAT2) and 3-hydroxy, 3-methylglutaryl CoA synthase (HMGCS1) that convert acetyl-CoA to 3-hydroxy, 3-methylglutaryl CoA (HMG-CoA) [HMGCL) which catalyze the synthesis of ketone bodies acetoacetate and β-hydroxybutyrate [HMGCR), farnesyl-diphosphate farnesyl-transferase 1 (FDFT1), farnesyl diphosphate synthase (FDPS), and lanosterol synthase (LSS), and sterol regulatory element binding transcription factors (SREBF1 and SREBF2) were analyzed by T-test in rumen epithelia of DG and UG heifers during diet transition (from 72% to 89%).The SCFA metabolism related genes including acyl-CoA synthetases (HMG-CoA) ,HMG-CoA butyrate , 64, choP < 0.1 was considered as significance difference. The SNP was discarded when less than 2 reads mapped to that location in at least one heifer in this study.The 5 UG and 5 DG heifers were chose for SNP analysis. RNA-Seq reads of ruminal papillae fed 0%, 72% and 89% grain diets were first combined for each heifer to increase read coverage. Then the SNP calling was performed using VarScan2 . The minAdditional file 1:Differentially expressed genes with higher expression in cattle fed low grain diets (72% vs. 0% and 89% vs. 0%); Functional annotation of HGD and LGD enriched genes using DAVID (72% vs. 0% and 89% vs. 0%) (worksheet 2); Functional annotation of HGD and LGD enriched genes using IPA (72% vs. 0% and 89% vs. 0%) (worksheet 3). (XLSX 465 kb)Additional file 2:Differentially expressed genes in Down group (89% vs. 72% grain) (worksheet 1); Differentially expressed genes in Up group (89% vs. 72% grain) (worksheet 2); Networks enriched using DE genes in Down group (89% vs. 72% grain) (worksheet 3); Networks enriched using DE genes in Up group (89% vs. 72% grain) (worksheet 4); The detected SNPs (worksheet 5). (XLSX 67 kb)Additional file 3: Figure S1.P < 0.1, ** indicated P < 0.05, and *** indicated P < 0.01. ↓ means decreased and ↑ means increased when the diet transitioned from 72% grain to 89% grain within each group. (PDF 59 kb)The patterns of ions transportation related genes expression in DG and UP. The legend represents the RPM value scaled by rows, the blue means highly expressed in the 72% grain and yellow means highly expressed in the 89% grain. The data were analyzed by T-test, * indicated Additional file 4: Figure S2.2RPM. The data were analyzed by T-test, * indicated P < 0.1, ** indicated P < 0.05. (PDF 105 kb)RT-qPCR validation of selected target genes identified by RNA-seq. The gene expressions detected by RT-qPCR are shown by line graphs on the top and values are shown on the right Y-axis as relative abundance. The gene expressions detected by RNA-seq are shown by bar graphs on the bottom and values are shown on the left Y-axis as logAdditional file 5: Figure S3.Single nucleotide polymorphisms (SNPs) associated with the varied ruminal pH response. (A) Fisher exact test of the association between SNPs and varied ruminal pH response. (B) The sequences of the SNP . (PDF 81 kb)Additional file 6: Figure S4.Illustrative comparison of the gene networks related to fatty acid transport and metabolism between Down group and Up group. ↓ means decreased and ↑ means increased when the diet transitioned from 72% grain to 89% grain within. (PDF 131 kb)Additional file 7: Table S1.Primers sequences used for RT-qPCR. (DOCX 61 kb)


Lilium lancifolium, which develops many aerial bulbils on the leaf axils of middle-upper stem, is a useful species for investigating bulbil formation. To investigate the mechanism of bulbil formation in triploid L. lancifolium, we performed histological and transcriptomic analyses using samples of leaf axils located in the upper and lower stem of triploid L. lancifolium during bulbil formation. Histological results indicated that the bulbils of triploid L. lancifolium are derived from axillary meristems that initiate de novo from cells on the adaxial side of the petiole base. Transcriptomic analysis generated ~650 million high-quality reads and 11,871 differentially expressed genes (DEGs). Functional analysis showed that the DEGs were significantly enriched in starch and sucrose metabolism and plant hormone signal transduction. Starch synthesis and accumulation likely promoted the initiation of upper bulbils in triploid L. lancifolium. Hormone-associated pathways exhibited distinct patterns of change in each sample. Auxin likely promoted the initiation of bulbils and then inhibited further bulbil formation. High biosynthesis and low degradation of cytokinin might have led to bulbil formation in the upper leaf axil. The present study achieved a global transcriptomic analysis focused on gene expression changes and pathways' enrichment during upper bulbil formation in triploid L. lancifolium, laying a solid foundation for future molecular studies on bulbil formation.Aerial bulbils are an important propagative organ, playing an important role in population expansion. However, the detailed gene regulatory patterns and molecular mechanism underlying bulbil formation remain unclear. Triploid  A bulbil, also known as a bulblet, consists of a small bud with a short stem  naturally form bulbils on the aboveground leaf axils of the stem . The only exception is L. lancifolium, which exhibits complex ploidy that includes both diploid and autotriploid (2n = 3x = 36), with triploids accounting for the majority of its distribution . The process of axillary bulbil formation includes two steps: (1) meristematic cells in the leaf axil divide continuously to form bulbil primordia; and (2) the bulbil primordia grow and differentiate to form the bulbil structure  is involved in the regulation of bulbil formation in Titanotrichum oldhamii, and its expression is down-regulated with bulbil development  occurs at bulbil initiation and increases as bulbils mature  gene confers the ability for axillary tomato meristems to undergo de novo formation of tuber-like organs  is a recently developed, effective and powerful tool for analyzing the entire transcriptome composition and the gene expression patterns of various biological processes, particularly in non-model organisms for which genomic sequences are not available  and one type of leaf axil sample from the lower stem (at the stage of swelling in the upper axil). This work provides comprehensive transcript information and gene expression data on bulbil initiation and formation, laying a solid foundation for future molecular studies on bulbil formation.To understand the process of bulbil formation in triploid Lilium lancifolium was used as the material in this study. L. lancifolium bulbs of uniform size with 1-2-cm buds were grown in soil in a greenhouse at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China, in early June of 2015. Samples of the upper and lower sectional stem nodes with ~0.4-mm petioles   according to the manufacturer's instructions, and DNA contamination was removed with RNase-free DNase I. RNA degradation and contamination were monitored using 1% agarose gels. RNA purity and concentration were measured using a NanoPhotometer® spectrophotometer  and Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer , respectively. RNA integrity (RIN) was assessed using the RNA Nano 6000 Assay Kit with the Agilent Bioanalyzer 2100 system . RNA samples with RINs ≥ 8.0, A260/280 ranging from 1.8 to 2.1, and 25S:18S ranging from 1.7 to 2.0 were used for library preparation. Three biological replicates were used for RNA extraction and further RNA-Seq.Twelve cDNA libraries (three replicates for each sample) were constructed using the NEBNext® Ultra™RNA Library Prep Kit for Illumina®  following the manufacturer's protocol. Briefly, mRNA was purified from 1.5 μg of total RNA using oligo (dT) magnetic beads, and then broken into short fragments of 300–500 bp by adding fragmentation buffer. First-strand cDNA was synthesized using random hexamer primer. Second-strand cDNA synthesis was subsequently performed using RNase H and DNA polymerase I. After adenylation of the 3′ ends of cDNA fragments, NEBNext adapter oligonucleotides were ligated to prepare for hybridization, and then the cDNA fragments were purified using AMPure XP system  to select the fragments of preferentially 150–200 bp in length. Then 3 μL of USER Enzyme  was used with size-selected and adaptor-ligated cDNA fragments. Then PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index(X) Primer. PCR products were purified with the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system. Finally, all cDNA libraries were sequenced on the Illumina HiSeq 4000 platform using the paired-end technology by Novogene Co. , and 150-bp paired-end reads were generated.de novo assembly based on the clean data was performed using the Trinity program , the Swiss-Prot protein database (http://www.ebi.ac.uk/uniprot), the Pfam database (http://pfam.sanger.ac.uk), the KEGG database (http://www.genome.jp/kegg/) . Each unigene was functionally annotated based on the protein sharing the highest sequence similarity with the given unigene. Gene Ontology  annotation was performed using Blast2GO software (version 2.5)  was then employed to produce GO functional classifications  database  < 0.05 and |log2Foldchange| ≥ 1. GO enrichment analysis (corrected p < 0.05) of the DEGs was implemented with the GOseq R package-based Wallenius noncentral hypergeometric distribution  method was used to calculate the expression of unigenes. Then, differential expression analysis of two groups was performed using the DESeq R package (1.10.1). The resulting ®; One-Step gDNA Removal and cDNA Synthesis SuperMix kit . Specific primers for qRT-PCR were designed with Primer 5.0 software, and the detailed information is listed in Table 2O. Amplification reactions were performed under the following conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 20 s, 60°C for 10 s, and 72°C for 20 s. A melting curve was generated for each PCR reaction to determine the amplification specificity and the presence of reaction contaminations. The melting curve was obtained by heating the amplification products from 60°C to 95°C in 5-s intervals. The primer efficiency was analyzed with CFX Manager™Software v3.1 (Bio-Rad). All qRT-PCR experiments were repeated with three biological and three technical replicates. The lilyActin (Gene Bank Accession Number: JX826390) gene  was used as the internal control for normalization, and the relative expression levels of the target genes were calculated using the 2−ΔΔCt method against the internal control . For cDNA synthesis, 500 ng of total RNA (without DNA contamination) was reverse-transcribed using the TransScriptSoluble sugar and starch were measured using an anthrone colorimetric method. Briefly, samples of leaf axils from the upper and lower stem at different stages were ground into powder using liquid nitrogen. Then, 0.2 g of powder was weighed in a 10-mL centrifuge tube, to which 6 mL of distilled water was added. The mixed homogenate was then incubated in boiling water for 30 min. Then, the extracted solution was centrifuged at 5,000 rpm for 10 min. The supernatant was collected, and the extraction was repeated three times . All supernatants were collected into 25-mL volumetric flasks for the determination of soluble sugar. The residual was re-dissolved in 20 mL of distilled water and incubated in boiling water for 15 min. Then, 2 mL of cold 9.2 mol/L perchloric acid was added, and the mixture was again incubated in boiling water for 15 min. The solution was finally filtered in a 50-mL volumetric flask, and the filtrate was used for the determination of starch. The soluble sugar and starch contents were measured by their absorption values at 630 nm and 485 nm, respectively, and calibration curves were constructed with standard solutions of sucrose and soluble starch, respectively. Three biological replicates for each sample and three technical replicates for each biological replicate were performed.2), and the IAA content was determined by means of enzyme-linked immunosorbent assay (ELISA)  methanol containing 1 mM butylated hydroxytoluene. The extracts were collected after centrifugation at 10,000 × g at 4°C for 20 min and were then passed through a C18 Sep-Pak cartridge (Waters). The efflux was dried with nitrogen (NISA) He, . Three bp ≤ 0.05. Non-overlapping letters (a-d) indicated significant differences between the comparisons, based on the ANOVA analysis and Duncan's multiple range test.The data for soluble sugar, starch and endogenous IAA contents and the relative expression analyses of genes were presented as the means with standard errors from three biological replicates. All statistical analyses were performed using SAS 9.1 , and statistical significance was defined at L. lancifolium, the upper and lower sectional stem nodes with ~0.4-mm petioles  and leaf axils showing no bulbil formation on the lower stem at stage S2 (D2) were obtained for an RNA-Seq analysis with three biological replicates. Sequencing of cDNA libraries from the twelve samples resulted in 46–70 million raw paired-end reads with greater than 6.5 Gb per sample , cellular component (CC), and molecular function (MF) Figure . RegardiTo further evaluate the functions of all assembled unigenes, we performed a search against the KOG/COG database. Based on sequence homology, 36,214 unigenes were assigned to a KOG functional classification, divided into 26 specific categories. Among these categories, the “general functional prediction only” (16.22%) cluster represented the largest category, followed by “posttranslational modification, protein turnover, chaperones” (13.26%), “translation, ribosomal structure and biogenesis” (9.81%), “signal transduction mechanisms” (9.54%), “energy production and conversion” (6.01%) and “intracellular trafficking, secretion, and vesicular transport” (5.98%). The “cell motility” (0.10%) and “unnamed protein” (0.01%) clusters were the smallest categories Table .KEGG pathways were also searched to allow a biological interpretation of the functions of the assembled unigenes. According to the KEGG results, 34,491 unigenes were mapped to predicted metabolic pathways Figure , and theWOX4_c109232_g2, AS1_c104426_g1, LOX1.1_c128051_g2, FTSZ1_c111017_g1, HOX21_c106431_g1, and TPS1_c100758_g1), six genes related to starch biosynthesis  and four genes involved in auxin signaling . The result showed that most of the expression patterns revealed by qRT-PCR were in good agreement with the RNA-Seq results, indicating the reliability of the RNA-Seq data .To validate the differential expression by RNA-Seq, sixteen unigenes were selected from the identified DEGs for qRT-PCR analysis, including six randomly selected genes  for “metabolic process” in “biological process (BP).” To further explore the detailed metabolic pathways, all DEGs were mapped to terms in the KEGG database, and a KEGG pathway enrichment analysis was subsequently performed. In total, 4,061 DEGs (U2 vs. U1) were assigned to 115 pathways; 773 DEGs (U3 vs. U2) to 71 pathways; 4,820 DEGs (U3 vs. U1) to 113 pathways; and 5,045 DEGs (U2 vs. D2) to 110 pathways  and in pairwise of U2 and D2. The DEGs between U2 and U1, U3 and U2, U3 and U1, and U2 and D2 were assigned to 34, 10, 104, and 108 GO terms based on “biological process (BP),” “cellular component (CC),” and “molecular function” Table . In all ys Table . Pathways Figure . The anns Figure . The anns Figure .L. lancifolium. Among starch and sucrose metabolism genes, genes encoding starch synthases, such as sucrose synthase (SUS) , HK (c115235_g3), PGM (c115630_g1), UGP (c123358_g1), AGPS (c121621_g2), SSS (c4952_g1 and c122034_g2), GBSS (c123636_g2), and SBE (c97206_g1 and c108260_g1), exhibited high expression levels during upper bulbil initiation (up-regulated in U2 vs. U1), which declined slightly in U3 , UGP2 (c123358_g1), AGPS1 (c121621_g2), SSS3 (c122034_g2), WAXY (c123636_g2), and SBE1 (c97206_g1) via qRT-PCR further confirmed our observations  from stage S0 to S2, followed by a rapid increase to 1.65% at S3  Table . Most of3 Figure . Additios Figure . In addi3 Figure . In cont3 Figure . During 3 Figure .The KEGG enrichment analysis of our transcriptome revealed that a fairly large number of DEGs were assigned to “plant hormone signal transduction” (ko04075) pathways, including auxin (IAA), cytokinin (CK), ethylene, gibberellin (GA), abscisic acid (ABA), brassinosteroid (BR), jasmonic acid (JA) and strigolactone (SL) signaling Figure . IAA andL. lancifolium, the IAA content increased from S0 to S2, peaked at S2 (47.95 ng/g FW), and then decreased significantly in S3 (21.95 ng/g FW). In contrast, the IAA content of the lower leaf axils increased continuously from S1 to S3 and was 3.5-fold greater than that in the upper part at S3  were up-regulated in U2 and then down-regulated in U3 during bulbil formation. In the IAA signaling pathway, the AUX1 (c127418_g2) gene encoding the auxin transporter protein 1 and genes encoding the auxin-responsive protein  also exhibited increased expression in U2 and reduced expression in U3. GH3 genes  encoding indole-3-acetic acid-amido synthetase, which conjugates IAA to amino acids and then reduces IAA concentrations, were down-regulated in U2 vs. U1 and up-regulated in U3 vs. U2. In addition, an auxin efflux carrier component (PIN1B_ c108190_g1) exhibited the same regulatory pattern as GH3 genes , YUC10 (c128166_g2), IAA17 (c115342_g1), and PIN1B (c108190_g1) via qRT-PCR further confirmed our observations , which catalyzes the biosynthesis of trans-zeatin, was up-regulated during upper bulbil formation and was obviously up-regulated in U2 vs. D2  related to the degradation of CK exhibited increased expression in D2 compared with U1, U2, and U3. In the CK signal transduction pathway, the expression of AHK4 (c127533_g1 and c126941_g1) and AHP1 (c101548_g1) was increased in U1, U2, and U3 compared with D2, and the expression level of AHP was the highest in U2. These results indicated that the CK signaling pathway likely played a role in promoting bulbil formation in L. lancifolium. In addition, BR signaling exhibited a promoting function. CYCD3-1 (c113663_g1), which functions downstream of BR signaling, was up-regulated during bulbil initiation and formation but showed low expression in D2  genes were slightly up-regulated during bulbil formation, but their expression levels in U1, U2, and U3 were reduced compared with D2. In the GA signaling pathway, GID1C (c119115_g1) was down-regulated in U2. GID1C levels were increased in D2 compared with U1, U2 and U3. The SLN1 gene (c127917_g2) encoding the DELLA protein, a repressor of the GA signaling pathway, exhibited a regulatory pattern opposite that of GID1C (Table D14 (c111706_g2 and c111706_g1) in the SL signaling pathway was the lowest in U2; furthermore, SL potentially inhibits bulbil initiation  and one from leaf axils located on the lower stem at the stage of swelling in the upper axil (D2) were obtained for RNA-Seq analysis. Unlike other studies on bulbil formation, our analysis was focused not only on bulbil initiation in the leaf axil but also on the reason that bulbils exclusively form on the upper part of the stem in ed Table . Starch L. lancifolium was positively related to the initiation of bulbils, according to the results of RNA-Seq analysis, qRT-PCR verification and starch and soluble sugar determination  exhibited the highest expression in U2 and the lowest expression in D2 , SSS (SSS1_c4952_g1 and SSS3_c122034_g2), GBSS (WAXY_c123636_g2) and SBE1 (c97206_g1 and c108260_g1) genes exhibited the same up-regulated expression pattern during bulbil initiation  to ADP-glucose. SSS, GBSS and SBE are key enzymes in starch synthesis  were up-regulated in U2 vs. U1 and then down-regulated in U3  and an IAA efflux carrier component (PIN1B_ c108190_g1), which reduce IAA concentrations, were down-regulated in U2 vs. U1 and then up-regulated in U3. These results are consistent with the notion that IAA accumulation induces new primordia in maize and potato and inhibits the outgrowth of axillary organs in Arabidopsis , which is involved in CK biosynthesis, was obviously up-regulated during bulbil formation in the upper leaf axil. In addition, the expression of CKX  exhibited minimal changes in the upper leaf axil during bulbil formation and was highest in D2 , which is induced by CK and plays a key role in integrating cell division and lateral organ development (Riou-Khamlichi et al., CKs play critical roles in the entire process of bulbil formation. In our study, 2 Figure . High CK2 Figure . Similar2 Figure . In addiGID1, D14, SNRK2, PYL4, ERF, JAZ, and MYC. These genes all showed the same expression pattern (Figure Based on the KEGG analysis, we also identified GA-, SL-, ABA-, ethylene-, and JA-related genes, such as n Figure : they weL. lancifolium is a complex process. Bulbil initiation is particularly important for bulbil formation and might be controlled by genes involved in hormone signal transduction and starch biosynthesis. Further studies on the functions of these candidate genes might be helpful for understanding bulbil formation in lily.Overall, bulbil formation in the upper portion of the stem of triploid JM conceived and designed the study. PY performed most of the experiments, with assistance from LX, HX, GH, YF, YC, YT, and SY. The manuscript was written by PY.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.


Vigna unguiculata (L.) Walp.] is one of the most important legumes in tropical and semi-arid regions. However, there is relatively little genomic information available for genetic research on and breeding of cowpea. The objectives of this study were to analyse the cowpea transcriptome and develop genic molecular markers for future genetic studies of this genus.Cowpea [de novo assembled to generate 47,899 unigenes with an N50 length of 1534 bp. Sequence similarity analysis revealed 36,289 unigenes (75.8%) with significant similarity to known proteins in the non-redundant (Nr) protein database, 23,471 unigenes (49.0%) with BLAST hits in the Swiss-Prot database, and 20,654 unigenes (43.1%) with high similarity in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Further analysis identified 5560 simple sequence repeats (SSRs) as potential genic molecular markers. Validating a random set of 500 SSR markers yielded 54 polymorphic markers among 32 cowpea accessions.Approximately 54 million high-quality cDNA sequence reads were obtained from cowpea based on Illumina paired-end sequencing technology and were Vigna unguiculata and a new set of genic SSR markers for further genetic studies and breeding in cowpea and related Vigna species.This transcriptomic analysis of cowpea provided a valuable set of genomic data for characterizing genes with important agronomic traits in The online version of this article (doi:10.1186/s12863-017-0531-5) contains supplementary material, which is available to authorized users. Vigna unguiculata (L.) Walp.] is a diploid Vigna crop (2n = 2× = 22), and its genome size is estimated to be 620 Mb [Cowpea [e 620 Mb . It is te 620 Mb  and is we 620 Mb . Additioe 620 Mb .Phaseolus vulgaris) and soybean (Glycine. max). However, significant progress has been made in genomic research on Medicago truncatula [Lotus japonicas [Glycine max) [Cajanus cajan) [Cicer arietinum) [Phaseolus vulgaris) [Vigna radiate) [Vigna unguiculata) [Despite its importance, cowpea has received little attention from the research perspective and is less studied than other legumes, such as common bean, chickpea, pea, and soybean. Only a relatively small number of expressed sequence tags (ESTs) and genome sequences of cowpea are available in public databases , even thuncatula , Lotus japonicas , soybeanine max) , pigeonps cajan) , chickpeietinum) , common ulgaris) , mung beradiate) , and adziculata) . Many ESan urgent need to develop numerous SSR markers that can be used by breeders in various molecular breeding strategies to increase breeding efficiency [Various molecular markers have been developed in cowpea, such as RFLP (restriction fragment length polymorphism) , RAPD . The sequences of the unigenes are listed in Additional file de novo assembly yielded 47,899 unigenes. Among these unigenes, 36,289 (75.8%) showed high similarity to known proteins in the Nr protein database, while 23,471 unigenes (49.0%) were homologous to proteins in the Swiss-Prot database  of the top BLASTx hits, followed by Medicago truncatula, Phaseolus vulgaris, Vigna unguiculata, Vigna radiata, Vigna angularis, Phaseolus aureus and Pisum sativum with 1554 (4.3%), 497 (1.4%), 258 (0.6%), 80 (0.2%), 44 (0.1%), 26 (0.1%), and 20 (0.1%) hits, respectively.According to the E-value distribution of the unigene hits in the Nr database, 24,860 (68.5%) of the unigenes had a similarity greater than 80% to known plant sequences; 26,647 (73.4%) of the mapped unigenes had high homology  with the available plant sequences; 22,778 (62.8%) of the unigenes had E-values less than 1.0E-45; and 26.6% of the homologous sequences had E-values within the range from 1.0E-5 to 1.0E-30 Fig. . In the Vitis vinifera (Vitaceae), 322 (0.9%) of the sequences in Lotus corniculatus var. japonicas (Gramineae), 287 (0.8%) of the sequences in Amygdalus persica (Rosaceae), and 71 (0.2%) of the sequences in Arabidopsis thaliana (Brassicaceae)  of the sequences matched sequences from other species, including 385 (1.1%) of the sequences in ae) Fig. .The unigenes with GO annotation were classified into three categories: biological processes, cellular components, and molecular functions Fig. . Among tAccording to Cluster of Orthologous Groups of proteins (COG) functional classification, 26,170 (30.8%) unigenes were aligned to the COG database and categorized into 25 functional categories Fig. ; among tThe pathway annotations of the unigene sequences were analysed using the KEGG mapper. A total of 20,654 unigenes were assigned to 5 major categories in 128 pathways that showed high similarity Fig. . Among tAmong the 47,899 unigenes found in this study, 4729 (9.9%) unigenes contained one or more SSR sequences. Additionally, 690 1.4%) unigenes contained at least two independent SSR sequences, and 235 (0.5%) contained compound SSRs of different motifs. These compound SSRs were not evenly distributed in various genic SSR units or groups. Tri-nucleotide (2500), di-nucleotide (1540), mono-nucleotide (1164), hexa-nucleotide (155), penta-nucleotide (137), and tetra- nucleotide (64) motifs accounted for 45.0%, 27.7%, 20.9%, 2.8%, 2.5%, and 1.2%, respectively; the tri-nucleotide motifs  were the most abundant, followed by the di-nucleotide motifs  (Table .4% unigen repeats were the most abundant (99.7% in mono-nucleotide motif) of the nucleotide repeats. The other six main motif types included the (AG/CT)n di-nucleotide repeat (71.2%), (AAG/CTT)n tri-nucleotide repeat (31.1%), (AAGAG/CTCTT)n penta-nucleotide repeat (27.7%), (AAAG/CTTT)n tetra-nucleotide repeat (23.4%), and (AAACCC/GGGTTT)n hexa-nucleotide repeat (4.5%).The number of SSR repeats per locus ranged from 4 to 24, and SSRs with five repeats were the most abundant, followed by SSRs with six, seven, and twelve random repeats. Motifs with more than 17 repeats accounted for only 6.5% of the total. The (A/T)A total of 5560 genic SSR markers were obtained from the 4729 SSR-containing sequences  varied from 0.1316 (Unigene 6390) to 0.9345 (Unigene 6224), and the average was 0.5474. The polymorphic information content (PIC) values ranged from 0.0624 (Unigene 6224) to 1.6215 (Unigene 6857), and the average was 0.8392.To confirm the 54 polymorphic genic SSRs developed in this study, we performed an extra validation in 32 diverse cowpea accessions  and adzuki bean (Vigna angularis). A few examples were genes for the auxin-responsive protein (IAA16), E3 ubiquitin protein ligase (DRIP2), ABC transporter, phosphomethylethanolamine N-methyltransferase, leucine zipper protein (ATHB-14), dof zinc finger protein (DOF1.8), ethylene-responsive transcription factor (TINY) and glycine-rich cell wall structural protein  [Vigna plants such as rice bean (986 bp) [In the present study, a total of 54.42 million clean reads with a length of 4,897,536,660 bp were obtained using Illumina paired-end sequencing technology. Additionally, 98.45% of the clean reads had Phred quality scores at the Q20 level, and the percentage of ambiguous “N” bases was 0.01%, ensuring the quality of the sequencing. Furthermore, 47,899 unigenes were assembled from the cowpea transcriptome, with an average unigene length of 871 bp, which is similar to other (874 bp) . The ave(874 bp)  but was (986 bp)  and adzu(986 bp) . This diAs a preliminary study, a total of 1071 SSRs in 15,740 cowpea unigene sequences were identified; as a result, 102 SSR markers and 1536 SNP markers were developed . Genic STo determine the polymorphism levels of the developed genic SSR markers, we evaluated 500 genic SSR loci, and 235 primer pairs (47.0%) produced successful amplicons. Additionally, 54 new polymorphic genic SSR markers were characterized and validated. A low polymorphism level was detected in 32 cowpea accessions, and the valid primer ratio was only 10.8% (54 in 500), which is lower than that from previous reports in other related legume species, including mung bean (33%) , black gIn this study, the tri-nucleotide motifs 2500, 20.9%) and di-nucleotide motifs 1540, 27.7%) were the most abundant. The types of motifs found in SSR loci were similar to the previous results on microsatellites in plants . A relat.9% and d, 27.7% wApplicable microsatellite loci were identified, and some of them may be useful in genetic research. However, there are some limitations in our study. The cDNA library was established using only one cowpea variety, which may cause an ascertainment bias. Second, only 500 of 5560 SSR markers were randomly selected for further validation, limiting their application potential. Third, more efficient RNA-Seq designs are needed to enhance marker development in transcriptomic studies. Fourth, the polymorphism rate is relatively low. Such a low rate of polymorphisms may be due to (i) a large intron between primers that would prevent amplification of the genomic sequence based on the transcribed mRNA sequence, (ii) the priming sites being disrupted by unrecognized intron splice sites, and (iii) low-quality sequences, fragmented assembly or assembly errors. The failure of 24 primer pairs to generate amplicons might be caused by long intervening introns, which would prevent PCR amplification of selected markers.Vigna unguiculata but also a new set of genic SSR markers for further genetic studies and breeding in cowpea and related Vigna species. Our analysis also showed the promise of using the RNA-Seq technique for SSR development in crop plants, such as cowpea, with few or no genomic resources.The transcriptomic analysis in our study was fruitful, providing not only a valuable set of genomic data characterizing genes for important agronomic traits in A set of 32 Chinese cowpea accessions was used in SSR marker testing, which included 14 accessions from Hunan province, 9 accessions from Hubei province, and 9 accessions from Anhui province. These cowpea germplasm accessions were obtained from the National Gene Bank . The cowpea seedlings were grown in plastic pots (20 cm × 15 cm × 15 cm) under a 14/10 h photoperiod at 25 °C (day) and 20 °C (night) in a greenhouse at the Institute of Crop Science, Chinese Academy of Agricultural Sciences . At the three-week-old seedling stage, five plants of “Zhongjiang No. 1” were transferred to a programmable incubator for dehydration for 3 h at 28 °C, termed drought stress, and the seedlings were then harvested, frozen immediately in liquid nitrogen, and stored at −80 °C for RNA-Seq.Total RNA was isolated from three different tissues, including roots, stems, and leaves, of five cowpea seedlings, and these RNA samples were mixed according to the equivalent concentration of these RNA samples were mixed, and 20 μg of RNA was quickly frozen in liquid nitrogen and then stored at −80 °C. The RNA was isolated with TRIZOL reagent  following the manufacturer’s instructions. The residual DNA was removed by incubation with RNase-free DNase I  for 30 min at 37 °C. The concentration and integrity of the total RNA was measured by a 2100 Bioanalyzer  at 260 nm and 280 nm and checked for degradation by RNase-free agarose gel electrophoresis. The RNA was further quantified with an RNA Nanochip. More than 20 μg of total RNA  ≥ 8.0) was used to create the full-length cDNA library., San Diego, CA, USA). The poly(A) RNA was then fragmented into smaller pieces using divalent cations at 94 °C for 4 min. First-strand cDNA was synthesized using SuperScript II reverse transcriptase and random primers. Second-strand cDNA was synthesized by adding GEX second strand buffer, dNTPs, RNase H, and DNA polymerase I. The cDNA fragments were further subjected to end repair and phosphorylation with T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase. The repaired cDNA fragments were then 3′-end adenylated with Klenow Exo and ligated with PE adapters. The products of this ligation reaction were purified through 2% (w/v) TAE-agarose gel, and templates of different sizes were selected for downstream enrichment. The cDNA fragments (200 ± 25 bp) were finally excised from the gel and amplified via PCR to enrich the cDNA template. Purified cDNA templates were enriched by 15 cycles of PCR amplification using PE1.0 and PE2.0 primers and Phusion DNA Polymerase . The cDNA library was constructed and sequenced in one lane on the Illumina Hiseq™ 2500 platform  with Illumina paired-end sequencing technology, and the mean fragment length was 200 bp (±25 bp) [The cDNA library was constructed using the NEBNext Ultra RNA Library Prep Kit  according to the manufacturer’s instructions . The tot(±25 bp) . Finallyde novo transcriptome assembly was performed using the program Trinity [Prior to transcriptome assembly, a stringent filtering process was carried out on the raw sequencing reads. Raw reads in fastq format were initially processed using in-house Perl scripts . All rea Trinity . The TriThe annotation of unigenes was carried out with various bioinformatics procedures. First, these unigenes were used to search various protein databases including Nr, Swiss-Prot, COG, and KEGG , retrieving proteins with the highest sequence similarity annotated to each unigene. Second, with nucleotide-based annotation, Blast2GO was used to obtain GO annotation categories according to molecular function, biological process and cellular component ontologies . Third, http://sourceforge.net/projects/primer3) under the following criteria: primer length 18–22 bp , Tm of 50–60 °C , and PCR product length in the range of 100–300 bp. Primers were synthesized by Qingke Biotech .To amplify a sequence with appropriate length (>100 bp) based on ensuring the quality of the primer, only sequences for which both ends of the SSR had lengths greater than 150 bp were used to design these primers. In this study, the minimum number of repeats for different SSR markers was chosen as follows: eleven for mono-nucleotide, six for di-nucleotide, five for tri- and tetra-nucleotide, and four for penta- and hexa-nucleotide motifs. Primers were designed based on the flanking sequences of these SSRs using Primer Premier 3.0 , 0.4 μM of each primer, and 30 ng of genomic DNA. PCR was carried out on a Bio-Rad T100™ Thermal Cycler . PCR amplification was performed as follows: 94 °C for 4 min and 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. The final extension was carried out at 72 °C for 5 min. The PCR products obtained were verified in 6.0% non-denaturing PAGE (polyacrylamide gel electrophoresis) using the silver staining method. Fragment sizes were estimated with the pBR322 DNA/MspI DNA ladder . To determine the functions of the polymorphic genic SSRs, the unigene sequences used for the development of these markers were subjected to a BLASTn analysis against a non-redundant database of legume sequences.The identified SSR markers were amplified and validated with 32 accessions of cowpea, including fourteen accessions from Hunan Province, nine accessions from Hubei Province and nine accessions from Anhui Province Additional file 2:Unigenes that were annotated in the Nr database. (XLS 7296 kb)Additional file 3:A list of 5560 cowpea genic SSR primers developed in this study. (XLS 764 kb)Additional file 4:Characteristics of 500 primer pairs derived from cowpea genic SSRs. (XLS 199 kb)Additional file 5:A list of 32 cowpea germplasm accessions used in this study. (DOC 69 kb)Additional file 6:The putative proteins identified by BLASTX of 54 unigene sequences containing polymorphic genic SSRs. (XLS 35 kb)


IGF1R), mapping on the 15q26.3 chromosome, is required for normal embryonic and postnatal growth. The aim of the present study was to evaluate the IGF1R gene expression and function in three unrelated patients with chromosome 15 structural abnormalities. We report two male patients with the smallest 15q26.3 chromosome duplication described so far, and a female patient with ring chromosome 15 syndrome. Patient one, with a 568 kb pure duplication, had overgrowth, developmental delay, mental and psychomotor retardation, obesity, cryptorchidism, borderline low testis volume, severe oligoasthenoteratozoospermia and gynecomastia. We found a 1.8-fold increase in the IGF1R mRNA and a 1.3-fold increase in the IGF1R protein expression (P < 0.05). Patient two, with a 650 kb impure duplication, showed overgrowth, developmental delay, mild mental retardation, precocious puberty, low testicular volume and severe oligoasthenoteratozoospermia. The IGF1R mRNA and protein expression was similar to that of the control. Patient three, with a 46,XX r(15) (p10q26.2) karyotype, displayed intrauterine growth retardation, developmental delay, mental and psychomotor retardation. We found a <0.5-fold decrease in the IGF1R mRNA expression and an undetectable IGF1R activity. After reviewing the previously 96 published cases of chromosome 15q duplication, we found that neurological disorders, congenital cardiac defects, typical facial traits and gonadal abnormalities are the prominent features in patients with chromosome 15q duplication. Interestingly, patients with 15q deletion syndrome display similar features. We speculate that both the increased and decreased IGF1R gene expression may play a role in the etiology of neurological and gonadal disorders.Insulin-like growth factor 1 receptor ( IGF1R) gene, made up of 315,991 base pairs, maps on the 15q26.3 chromosome. It encodes for a protein with a tyrosine kinase domain, which binds the IGF1 and is responsible for its biological activity. Chromosomal 15q structural abnormalities, such as distal duplication or ring 15, can alter the IGF1R expression and function.Insulin-like growth factor 1 receptor , he was 144.9 cm (>99th percentile) tall and had a cranial circumference (CC) that measured 56 cm (>97th percentile). At the age of 14 years, these measurements were, respectively, 97 kg (>97th percentile), 176 cm (90th percentile) and 57.5 cm (>97th percentile). The testicular volume (TV) was 4 mL bilaterally and both testes had an increased consistency. Basal serum luteotropin hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T) levels were 5.61 IU/mL : 1.5–9.3 IU/mL), 5.37 IU/mL (n.v.: 1.4–18.1 IU/mL) and 1.87 ng/mL (n.v.: 3–9 ng/mL), respectively. When he was 15 years old, the TV measured 8 mL bilaterally, and the T levels were 2.9 ng/mL. When he was 17 years old, he was at Tanner stage 3, he had a triangular distribution of pubic hair and a bilateral TV of 12 mL. Serum LH, FSH and T levels were, in turn, 4.8 IU/mL, 9.1 IU/mL and 3.96 ng/mL, and the IGF1 levels were 411.3 ng/mL (n.v.: 119–395 ng/mL). He was 180 cm  tall. At the age of 19 years, the sperm analysis detected a severe oligoasthenoteratozoospermia .Genetic analysis excluded both Prader–Willi and Kallmann syndrome . He had a 46,XY karyotype and a 568 kb pure duplication on the 15q26.3 chromosome, diagnosed by array-CGH. This duplication was not detected in the mother. The father could not be evaluated because he was not alive. The patient sister had a normal psychophysical development, and she did not show overgrowth.Patient 2 was a 16-year-old male, admitted for the first time to the Department of Pediatrics, teaching hospital ‘G. Rodolico’, University of Catania, at the age of 7 years, for clinical signs of precocious puberty. He had a marked defective speech capacity, a low-grade mental retardation (IQ 66), a benign tachycardia and a low-grade insufficiency of pulmonary valve. The abdomen ultrasound, the brain magnetic resonance and the electroencephalogram revealed no abnormality.On physical examination, performed at the age of 7 years, he weighed 39.8 kg (97th percentile), and he was 147.5 cm (>97th percentile) tall. The CC measured 54 cm (75th percentile). Seven café-au-lait spots were detected, the larger (7 × 4 cm) in the right groin, the others in the right clavicular region, left lower limb, left hip and right gluteus. He was at Tanner stage 3, and both testes had a volume of 8 mL. His serum T levels were higher for his age; the bone age was advanced by two years. The GnRH analog test showed results compatible with precocious puberty (serum hormone levels not available). On this basis, he was prescribed Gonapeptyl Depot (1 injection every 28 days), which was administered from the age of 8 to that of 11 years.At the age of 11 years, serum IGF1 levels were in the normal range . At the age of 14 years, he weighed 77 kg (95th percentile) and he was 171 cm (75th percentile) tall. He was at Tanner stage 5 and his LH and T serum levels were, respectively, 3.18 IU/mL and 3.74 ng/mL. At the age of 16 years, the TV was 11 mL, bilaterally, and the LH, FSH and T values were, in turn, 2.93 IU/mL, 1.94 IU/mL and 5.26 ng/mL. The sperm analysis detected a severe oligoastenotheratozoospermia .The genetic analysis showed a 46,XY karyotype with a 650 kb impure duplication on the 15q26.3 chromosome . The NF-1 (exons 1–58), OMG (exon 2), ASPA (exon 6), PMP22 (exon 3), TRAF4 , SSH2 , BLMH (exon 2), CPD (exon 12), SUZ12P , CRLF3 (exon 3), ATAD5 (exon 2), ADAP2 (exon 3), RNF 135 (exon 2), UTP6 (exon 14), SUZ12 (exon 10), LRRC37B (exon 1), ZNF207 (exon 9), PSMD11  and MYO1D  genes were evaluated to exclude the presence of neurofibromatosis. The genetic testing revealed no abnormality. The clinical history of his biological parents is unknown since he was adopted.Patient 3 was a 6-year-old female patient presented to the Department of Pediatrics, teaching hospital ‘G. Rodolico’, University of Catania, at the age of 6 years, complaining from developmental delay. Her birth weight was 1860 g, and her length was 44 cm. She had mental and psychomotor retardation.On physical examination, she weighed 10.3 kg (<3rd percentile), she was 92 cm (<3rd percentile) tall and the CC measured 41 cm (<3rd percentile). Café-au-lait spots in the left side of chest, in the right leg, in the groin and vitiligo in the right side of chest were detected. The diagnosis of neurofibromatosis was clinically excluded. She had triangular face, microcephaly, thin hairs, arched eyebrows, blepharophimosis, broad nasal bridge and slight superior lip. She had clinodactyly and shortness of the second finger. The X-ray showed a bilateral extra phalanx in the third finger, a bilateral hypoplasia of the phalanx of the third finger and a bilateral dysmorphic and hypoplastic phalanx in the second finger. FISH analysis detected a 46,XX r(15) (p10q26.2) karyotype. According to array-CGH results, she had a deletion of the last 5 Mb of chromosome 15 and a duplication of 2 Mb. An absence of paternal alleles in the terminal 15q chromosome (from 95.258 Mb to qter) was found at the microsatellites analysis. Thus, she did not have a paternal origin of the rearrangement. At the age of 11 years, serum IGF1 levels were 186 ng/mL (n.v.: 87.4–399.3 ng/mL). She underwent GH replacement therapy from the age of 14 years until the age of 16 years.Lymphocytes were isolated from whole blood by Ficoll, according to the manufacturer’s instructions using Ficoll-Paque Plus (Amersham). The cells obtained with this procedure were grown for 48 h in RPMI medium supplemented with 10% FBS, 1% glutamine and 2 µg/mL phytohemagglutinin, divided in two aliquots: one for RNA extraction and one for protein analysis.IGF1R  and 5′-ATT-GAA-GAA-ATT-GCA-GGC-TC-3′ (forward) and 5′-TGG-AGA-AGA-GGA-GCT-GTA-TCT-3′ (reverse) specific for the ELE1 (housekeeping gene) , on an ABI Prism 7500 (PE Applied Biosystems) using Sybr Green PCR Master Mix (PE Applied Biosystems) following manufacturer’s instructions. Amplification reactions were checked for the presence of nonspecific products by dissociation curve analysis. Relative quantitative determination of target gene levels was performed by comparing ΔCt, as described by Ginzinger. The PCR products were analyzed by 2% agarose gel electrophoresis and stained by Sybr Safe.Total RNA (5 µg) was reverse-transcribed by ThermoScript RT (Invitrogen) with Oligo dT primers. Synthesized cDNA (25 ng) was then used for a quantitative real-time PCR, using the following primers: 5′-GGG-CCA-TCA-GGA-TTG-AGA-AA-3′ (forward) and 5′-CAC-AGG-CCG-TGT-CGT-TGT-CA-3′ (reverse) specific for the g for 15 min at 4°C. Cell lysates were subjected to SDS-PAGE and the resolved proteins were transferred to nitrocellulose membranes, immunoblotted with anti-phospho-IR/IGF1R (Tyr1158/Tyr1162/Tyr1163) antibody and detected by ECL. The nitrocellulose membrane was then stripped with buffer Restore (Pierce) and, subsequently, reprobed with an anti-IGF1R rabbit polyclonal antibody. The membranes were blotted with an anti β-actin antibody to control for protein loading.Lymphocytes cultured for 48 h were serum-starved for 24 h being cultured in serum-free medium before undergoing stimulation with 10 nM IGF1 for 5 min. Cells were lysed in cold RIPA buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycolate, 10 mM sodium pyrophosphate, 1 mM NaF, 1 mM sodium orthovanadate, 2 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL pepstatin, 10 µg/mL leupeptin and the insoluble material separated by centrifugation at 10,000 s.e.m. The data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. SPSS 22.0 for Windows was used for statistical analysis (SPSS). Statistical significance was accepted when the P value was lower than 0.05.Results are expressed as mean ± Consent has been obtained from each patient or subject after full explanation of the purpose and nature of all procedures used. An approval by an ethical Committee was not required because the data presented were obtained during the routine diagnostic workout, which the three patients underwent to within the Teaching Hospital of the University of Catania.The IGF1R mRNA expression, evaluated in lymphocytes using a relative quantification by real-time PCR, is shown in To confirm the results obtained by the real-time PCR, lymphocyte lysates underwent to SDS-PAGE to evaluate the IGF1R protein relative content and the IGF1R tyrosine kinase activity after stimulation with 10 nM IGF1 .Figure After densitometric analysis and normalization by β-actin, we found that Patient 1 had a significantly higher IGF1R content (1.3 fold) than that of the control, Patient 2 had an IGF1R content similar to that of the control, whereas the IGF1R content was lower in Patient 3 compared to that of the control . After sIn the present study, we report two cases of chromosome 15q26.3 duplication and one case of ring chromosome 15 syndrome. Despite the genetic heterogeneity of these patients, we recognized some common clinical features, which became clearer after reviewing the already published cases.de novo chromosome 15q26.3 duplication has been reported to date  chromosA review of all cases of chromosome 15q duplication described up to now follows.The structural abnormalities of chromosome 15q are rare. To the best of our knowledge, up to now, 96 patients with chromosome 15q duplication have been reported in literature. Among these, 28 patients showed distal 15q tetrasomy due to a mosaicism or to a neocentromer marker chromosome (NMC) , 30 and We carefully reviewed the clinical features of all published patients with chromosome 15q duplication to estimate the prevalence of each of them. Patients with unknown measures or reports with no sufficient clinical data available were excluded from this analysis. The prevalence of each feature is summarized in Mental retardation Mental retardation is a common feature in patients with 15q distal duplication. Patients 1 and 2 share with patients previously reported in literature neurological symptoms such as developmental delay, mental and psychomotor retardation, marked defective speech capacity (capacity . As Chencapacity  58). Ho. HoMentaOvergrowth and IUGR Overgrowth is another common feature in patients with 15q distal duplication  associated with the deletion of the 15q telomere and part of the 15q26.3 band. She had a severe clinical course due to congenital heart defect, horseshoe kidney, hand contractures and clubfeet and death occurred after 20 days from birth because of cardio-respiratory failure. Curiously, she had marked IUGR. This feature was first ascribed to the 15q26.3 deletion, but FISH analysis revealed three copies of the IGF1R gene. Since this finding is not in agreement with the patient’s phenotype, the authors hypothesized that the clinical feature may depends on the global transcription dysregulation more than to the impairment of a single gene specifically correlated to the malformation  (MESP1 (OMIM: 608689 – cytogenetic locations: 15q26.1) and MESP2 (OMIM: 605195 – cytogenetic locations: 15q26.1) .patients . These i15q25.2) , 30, 60,15q26.1) . ADAMTSLGonadal and genital abnormalities Cryptorchidism  (p10q26.2) karyotype) with a deletion of the terminal 5 Mb of chromosome 15. She had IUGR, developmental delay, mental and psychomotor retardation, café-au-lait spots, vitiligo and she underwent GH administration. Her clinical feature was similar to that of patients with a chromosome 15q26-qter deletion syndrome.IGF1R gene is deleted suggesting a role of IGF1R in the observed growth retardation : these patients have short stature, low birth weight, failure to thrive, microcephaly, neurological symptoms , typical facial abnormalities , congenital cardiac anomalies , male genitalia abnormalities , hand and feet abnormalities .The clinical features of chromosome 15q26-qter deletion syndrome have been already summarized  are described both in patients with chromosome 15q duplication and in patients with ring 15 chromosome syndrome. Consequently, these clinical features may arise not from a decreased or increased expression of genes mapping on chromosome 15q, but from an imbalanced expression of these genes.Patients 1 and 2 of the present study, who share the 15q26.3 breakpoint, showed the same clinical findings commonly described in previously reported patients with 15q duplication , who genMEF2A gene (OMIM 600660) encodes for a protein that maintains the appropriate mitochondrial content and the cytoarchitectural integrity in the postnatal heart in mice  encodes for the chondroitin sulfate synthase 1 that synthetizes a glycosaminoglycan expressed on the surface of most cells and in extracellular matrices. It causes the temtamy preaxial brachydactyly syndrome  has been reported to be responsible for delayed psychomotor development with very poor or lack of speech, head nodding, mild flattening of the midface and hypotonia  development by promoting neuronal cell survival and synaptic maturation, thus facilitating functional plasticity in the brain ) is required for the development of male gonads and thus for male sexual differentiation. In fact, XY mice that were mutant for all 3 receptors developed ovaries and showed a completely female phenotype. The decreased expression of both Sry gene and the early testis-specific marker Sox9 in these mice suggests that the insulin signaling pathway is required for male sex determination  include café-au-lait spots (Finally, as far as we know, the presence of café-au-lait spots has been hardly reported in patients with chromosome 15q duplications. We describe the case of a patient with an impure chromosome 15q duplication and café-au-lait spots. This feature has also been reported in 30% of patients with ring 15 chromosome . Althougit spots . On the it spots . In the The limitations of our study include the use of a 17-year-old male as control: although it could be a good choice for patients 1 and 2, its age and sex differ from those of patient 3. However, we think that the results found in this latter patient are reliable, because they fit well with the clinical case. Furthermore, the prevalence of gonadal and genital abnormalities  may be uIn conclusion, we found that the IGF1R mRNA and protein expression were elevated in a patient with a 568 kb 15q26.3 pure duplication, normal in a patient with an impure 15q26.3 650 kb duplication and a 16p11.2 600 kb deletion and decreased in a patient with ring chromosome 15. IGF1R function was normal in both patients 1 and 2 and undetectable in patient 3. Clinically, patient 1 had overgrowth, moderate mental and psychomotor retardation, marked defective speech, right hand cryptorchidism, severe oligoasthenoteratozoospermia, obesity, borderline low TV, borderline low serum T levels, gynecomastia and triangular distribution of pubic hair. Patient 2 had overgrowth, mild mental retardation, marked defective speech, café-au-lait patches, precocious puberty and severe oligoasthenoteratozoospermia. Finally, patient 3 had IUGR, failure to thrive, mental and psychomotor retard, café-au-lait patches.We reviewed 96 patients with chromosome 15q duplication and we summarized the main clinical features in IGF1R gene may play a role in the etiology of neurological disorders and of gonadal abnormalities in these patients. Accordingly, the relevance of IGF1R in the male gonads development has been already shown in mice (IGF1R gene plays in the development of male gonads, in male sex differentiation and sperm abnormalities in humans.We speculate that the  in mice . FurtherThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Rossella Cannarella: Conception and design of the study, data acquisition, analysis and interpretation of data, drafting of the article, final approval of the version submitted. Teresa Mattina: Analysis and interpretation of data, drafting of the article, final approval of the version submitted. Rosita A Condorelli: Acquisition of data, analysis and interpretation of data, drafting of the article, final approval of the version submitted. Sandro La Vignera: Acquisition of data, analysis and interpretation of data, drafting of the article, final approval of the version submitted. Laura M Mongioì: Acquisition of data, analysis and interpretation of data, final approval of the version submitted, drafting of the article. Giuseppe Pandini: Acquisition of data, analysis and interpretation of data, drafting of the article, final approval of the version submitted. Aldo E Calogero: Conception and design of the study, analysis and interpretation of data, critical revision of the manuscript and final approval of the version submitted.


Nymphaea spp.), a perennial herbaceous aquatic plant, is divided into two ecological groups: hardy waterlily and tropical waterlily. Although the hardy waterlily has no attractive blue flower cultivar, its adaptability is stronger than tropical waterlily because it can survive a cold winter. Thus, breeding hardy waterlily with real blue flowers has become an important target for breeders. Molecular breeding may be a useful way. However, molecular studies on waterlily are limited due to the lack of sequence data.Waterlily  were detected in different contents during the color development due to the different expression levels of color-related genes, and four flavonols were detected in waterlily for the first time. Furthermore, UA3GTs were selected as the most important candidates involved in the flavonoid metabolic pathway, UA3GTs induced blue petal color formation in Nymphaea ‘King of Siam’.In this study, six cDNA libraries generated from the petals of two different coloring stages of blue tropical waterlily cultivar This study will improve our understanding of the molecular mechanism of blue flowers in waterlily and provide the basis for molecular breeding of blue hardy waterlily cultivars.The online version of this article (doi:10.1186/s12864-016-3226-9) contains supplementary material, which is available to authorized users. Flower color is one of the most important characters of ornamental plants, which determined by many factors. However, the type and content of anthocyanins are generally the primary factor , 2. AlthNymphaea spp.), a perennial herbaceous aquatic plant, occurs almost worldwide and has 45-50 species in five subgenera: Anecphya (7-10 species), Brachyceras (14-16 species), Hydrocallis (14 species), Lotos (2-3 species), and Nymphaea (8 species) [Waterlily (Nymphaea  species species) . It alsoecphya 7- species,species) , anti-inspecies) , antidiaspecies) , 14, antspecies) , antianxceras 14- species,species) . Waterlispecies) . Hardy wspecies) . Thus, bspecies) .Molecular breeding provides a new way to obtain blue hardy waterlily. Therefore, it is necessary to understand the molecular mechanism of blue characteristic formation in waterlily and identify related key genes for the purpose of modifying flower color in hardy waterlily by genetic engineering in breeding blue hardy waterlily in the future. The next-generation sequencing technology, RNA sequencing (RNA-Seq), has provided unique insights into the molecular mechanism for non-model organisms without a reference genome –23. As aNymphaea ‘King of Siam’ was performed using Illumina HiSeq™ 2500 platform. Qualitative and quantitative analysis of flavonoids were performed with I-Class ultra-high performance liquid chromatography/Xevo triple-quadrupole mass spectrometry (I-Class UPLC/Xevo TQ MS) technology and the expression patterns of differentially expressed genes (DEGs) identified in the flavonoid biosynthetic pathway were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). The above analysis results can reveal the biochemical and molecular mechanisms of blue color formation in Nymphaea ‘King of Siam’ and provide the basis for breeding blue hardy waterlily cultivars. Furthermore, this study will improve our understanding of the molecular mechanism of blue flowers and provide the evidence for molecular breeding theory concerning the basis of flower color modifications in ornamental plants.In the study, the first RNA-Seq project for the petals of two different coloring stages of tropical waterlily cultivar Nymphaea ‘King of Siam’ were selected as the experimental materials. This cultivar has petaloid stamens in deep blue-violet (RHSCC: 92A) color  flow of 800 L/h; cone gas flow of 50 L/h; collision gas flow of 0.12 mL/min; collision energy of 10 eV; the desolvation temperature of 400 °C; source temperature of 150 °C; and the scanning range of 100-1000 (m/z) units.I-Class ultra-high performance liquid chromatography/Xevo triple-quadrupole mass spectrometry (I-Class UPLC/Xevo TQ MS) was used for qualitative analysis. The liquid chromatograph was equipped with an ACQUITY UPLC HSS C18 column  . Eluent A was 1 % formic acid aqueous solution and Eluent B was acetonitrile. The following gradient profile was used: 5 % B at 0 min, 45 % B at 6 min, 90 % B at 7 min, 10 % B at 7.1 min, and 10 % B at 10 min. The flow rate was 0.2 mL/min and the injected volume was 5 μL. Column temperature was maintained at 35 °C and sample temperature was 10 °C. Chromatograms of anthocyanins and other flavonoids were acquired at 525 nm and 350 nm, respectively. The following analysis conditions of mass spectrometry were adopted: the positive-ion (PI) mode for anthocyanins and negative-ion (NI) mode for other flavonoids; capillary voltage of 3.00 kV; cone voltage of 20 V for PI mode and cone voltage of 70 V for NI mode; desolvation gas (NO-glucoside (Cy3Glc) and rutin were used as quantification standards. Mean values and SDs were calculated from three biological replicates.The contents of anthocyanins and other flavonoids were calculated in milligrams per 100 g fresh weight. Cyanidin 3-Total RNA was isolated with RN40-EASYspin plant microRNA kit , characterized on a 1 % agarose gel and examined with a NanoDrop 2000 UV-vis spectrophotometer . Agilent 2100 Bioanalyzer  was used to assess RIN (RNA integrity number) of each sample. The construction of the libraries and RNA-Seq were performed by Biomarker Biotechnology Corporation . Firstly, mRNA was enriched using Oligo (dT) attached to magnetic beads. Then mRNA was broken into short fragments through the addition of fragmentation buffer. Taking these short fragments as templates, the first-strand cDNA was synthesized with random hexamers. Buffer, dNTPs, RNase H, and DNA polymerase were mixed to synthesize second-strand cDNA. The cDNAs were purified with AMPure XP beads and then subjected to end-repair. To repair cDNAs, an ‘A’ base was inserted at the 3′ end. After that, adaptors were connected to these cDNA fragments to select different fragments. PCR amplification was performed to enrich cDNA library. Agilent 2100 Bioanaylzer and Qubit® Fluorometer  were used to assess the quality of cDNA library. At last, the six libraries were sequenced by using an Illumina HiSeq™ 2500 platform with 125 bp paired-end reads.de novo assembly using trinity platform (http://trinityrnaseq.github.io/) without digital normalization according to the parameters of min_kmer_cov = 2 and other default parameters [E-value ≤ 10−5) to various protein databases, including the NCBI non-redundant (NR) protein database (ftp://ftp.ncbi.nih.gov/blast/db/), the Swiss-Prot protein database (http://www.uniprot.org/), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/), the Clusters of Orthologous Groups of proteins (COG) database (http://www.ncbi.nlm.nih.gov/COG/), the eukaryotic Orthologous Groups of proteins (KOG) database (http://www.ncbi.nlm.nih.gov/COG/), and the Gene Ontology (GO) database (http://www.geneontology.org/). After predicting the amino acid sequences of unigenes by GetOrf (EMBOSS:v6.3.1), we aligned these sequences in Protein family  using HMMER software suite (v3.0) . The best alignment results from seven databases were chosen to determine the annotation of the unigenes.Firstly, after removing the adapter sequences, low quality sequences, unknown reads (N percentage >10 %) and ribosomes RNA from raw reads, the rest of raw reads called clean reads were obtained. Then, clean reads were used for transcriptome rameters , 27. ShoThe alignment package Bowtie (v4.4.7) was used to map reads back to unigenes. According to the comparison results, the expression levels were estimated by means of RSEM (RNA-Seq by expectation maximization) , 29. Unip-values were calculated and adjusted with the Bonferroni correction. We selected the corrected p-value of 0.05 as the threshold to determine significant enrichment of the gene sets.GO and KEGG pathway enrichment analyses for the differentially expressed unigenes were then carried out. The obtained GO annotation was enriched and refined using topGo package (v2.12.0) with the “elim” method and Kolmogorov-Smirnov test. KEGG pathways were enriched using in-house scripts according to Fisher’s exact test. Enriched For the purpose of gene validation and expression analysis, all the DEGs related to flavonoid biosynthesis were subjected to real-time quantitative PCR (RT-qPCR) on ABI StepOnePlus™ Real-Time PCR System . Primer sequences designed with Primer Premier 5.0 software are shown in Additional file Nymphaea ‘King of Siam’, we compared the metabolic profiles of petals. As expected, anthocyanins were not detected in S1, while four anthocyanins, namely delphinidin 3-O-β-galactopyranoside , delphinidin 3′-O-, delphinidin 3-O-, delphinidin 3′-O- were detected in other stages. Furthermore, we compared the intermediate products involved in flavonoid biosynthesis. A total of 12 kinds of flavonols, including myricetin derivatives, quercetin derivatives, and kaempferol derivatives were detected in petals. In addition, myricetin 3-O-α-L--rhamnopyranoside, myricetin 3-O-, quercetin 3-O-α-L--rhamnopyranoside, and kaempferol 3-O-α-L--rhamnopyranoside were first detected in waterlily. The retention time, ultraviolet-visible spectral properties and mass spectrometric data, and content of each compound are listed in Additional file To understand the color formations in −1 FW. Dp3′G (including a2 and a4) were the domain anthocyanins and the highest content of Dp3′G (74.26 mg 100 g−1 FW) was detected in S4  were constructed with blue waterlily cultivar de novo assembly generated 329,005 transcripts and 112,485 unigenes, with the N50  of 2,418 nt and 1,496 nt. In total, 63,097 unigenes (56.09 %) were between 200 and 500 nt; 38,064 unigenes (33.84 %) were between 500 and 1000 nt; 11,324 unigenes (10.07 %) were longer than 2000 nt , which accounted for about 23.3 % of 112,485 unigenes. Among them, 25,720 unigenes  could be annotated to the NCBI NR database, while 17,627 (67.26 %), 16,331 (62.32 %) and 15,308 (58.41 %) unigenes could be annotated to Swiss-Prot, Pfam and KOG databases. We annotated 13,526 (51.61 %), 7,968 (30.41 %) and 5,209 (14.39 %) unigenes to GO, COG and KEGG databases. According to the NCBI NR annotation and E-value distribution, 53.03 % of the annotated sequences had strong homology  and 33.75 % of the annotated sequences showed very strong homology   Fig. . The 25 17,627 unigenes were annotated against the SwissProt database, accounting for 67.26 % of all annotated unigenes and 121 unigenes that were not annotated against the other three databases. Pfam is the most comprehensive domain annotation of protein structure classification system. In this study, there were 16,331 unigenes noted to Pfam database.KOG analysis was used to searching orthologous classification and gene evolution rates. There were 15,308 unigenes  that aligned with 25 KOG classifications. Out of the 25 KOG categories, the cluster for general function prediction only (31.86 %) represented the largest group, followed by posttranslational modification, protein turnover, chaperones (8.16 %) and signal transduction mechanisms (7.11 %). Only a small proportion (lower than 1 %) of unigenes was assigned to the number of unigenes which involved in nuclear structure, cell motility and extracellular structure. It is worth noting that a large number of unigenes were assigned to carbohydrate transport and metabolism (4.75 %), translation, ribosomal structure and biogenesis (4.34 %), transcription (4.33 %), energy production and conversion (3.92 %), intracellular trafficking, secretion, and vesicular transport (3.80 %) and secondary metabolites biosynthesis, transport and catabolism (3.43 %)  was the threshold. There were a total of 10 enrichment GO terms among three gene ontology categories   and downstream genes   Fig. . Among t2 = 0.6634)  in six different coloring stages. These seven unigenes had similar expression patterns and their expression curves were “bell curves”. With the development of petals, their expression levels increased gradually, reached the maximum level in S4, and then decreased in S5 and S6. FLS and UA3GT-6 had the most rapid change in different stages. In Nymphaea ‘King of Siam’, changes in expression of genes encoding pathway enzymes have corresponded to changes in anthocyanin accumulation like many other cases [In order to verify the credibility of transcriptome sequencing data, the sequences of all these 33 core unigenes, which were not only associated with waterlily color formation but also had different expression levels, were subjected to fluorescent quantitative analysis of RT-qPCR with the designed primers. The results showed that transcriptome sequencing data were credible because the correlation coefficient between transcriptome sequence and RT-qPCR was high R2 = 0.634  derivatives, such as Dp3Gal, Dp3′galloylGal, Dp3acetylGlc, and Dp3′galloyl-acetylGal were detected. Therefore, we focused on the biosynthesis of Dp derivatives. Among all unigenes involved in the Dp biosynthesis process, two CHS, one CHI, five F3H, one F3′H, one F3′5′H, one DFR, three ANS, and three UA3GT homologous sequences showed up-regulated expression levels in S3 compared to those in S1. These unigenes covered almost the complete Dp biosynthetic pathway. Blue flower formation was comprehensively regulated by multiple unigenes.Due to the lack of a reference genome sequences, it is difficult to understand the molecular mechanism of blue color formation in tropical waterlily. In the study, we sequenced and comparatively analyzed the transcriptome of the different coloring stages of blue waterlily cultivar F3′H and F3′5′H are called “red gene” and “blue gene” because they are required for Cy and Dp production, respectively. F3′H and F3′5′H are the key enzymes determining the structures of anthocyanins and the color [Carnations, Tulipa, Rosa hybrida, and Chrysanthemum morifolium, do not produce Dp and thus lack violet/blue color cultivars. This is attributed to the fact that these crops do not possess the F3′5′H gene. Transgenic violet/blue carnations [F3′5′H gene. Eight F3′5′H were found in Nymphaea ‘King of Siam’, whereby one F3′5′H expression was up-regulated in S3 (log2FC = 4.17). As showed in Fig. F3′5′H was crucial in myricetin derivatives and Dp derivatives synthesis. F3′5′H expression levels increased from S1, then reached the maximum level in S4, decreased in S5 and S6 , resulting in the formation of corresponding leucoanthocyanidins. The three substrates of DFR are similar in the structure and only different in the number of hydroxyl groups on the B phenyl ring, which is not the site of enzymatic action. It is not surprising that DFRs from many plant species can utilize all three dihydroflavonols. However, in some species, DFR has strict substrate specificity . In thisNymphaea ‘King of Siam’, and the sequences of methoxylation genes involved in anthocyanin modification were not obtained in assembled results. Metabolites of anthocyanins had been compared in previous studies [Gentiana triflora and anthocyanin 3′, 5′-glucosyltransferase (CtUA3′5′GlcT) of Clitoria ternatea [Anthocyanins are modified at one or several positions by methylation, glycosylation, or acylation. These modifications endow anthocyanins with unique properties, including color shade, stability, and solubility . Methyla studies , 44–47. ternatea , 49.UA3′GT homologous unigenes were found in waterlily (Additional file Clitoria ternatea which accumulated polyacylated anthocyanins named ternatins, UA3′5′GT could be glycoslated at 3′-, 5′-position of delphindin. Ogata et al. [UA3GTs might be the most important enzymes for the accumulation of delphindin 3′-glycosides which are the main anthocyanins in blue petals.Six a et al.  had repoUA3GTs were selected as candidates involved in flavonoid metabolic pathway, UA3GTs induced blue color petals in Nymphaea ‘King of Siam’. All these results revealed the biochemical and molecular mechanisms of blue color formation and provided the basis for breeding blue hardy waterlily cultivars. This study will improve our understanding of the molecular mechanism of blue flowers and provide the evidence for molecular breeding theory concerning the basis of flower color modifications in ornamental plants.Nymphaea. Transcriptome analyses from the petals of two different coloring stages of blue tropical waterlily cultivar Nymphaea ‘King of Siam’ resulted in 112,485 unigenes. 127 unigenes could be identified as putative homologues of color-related genes in other species, including 28 up-regulated and 5 down-regulated unigenes. The composition of flavonoids and the metabolic pathways related to blue formation were identified, and UA3GTs were selected as the most important candidates involved in the flavonoid metabolic pathway, which induced blue petal color formation. This study provides a comprehensive molecular biology background for researches on Nymphaea, particularly with respect to the process of blue color formation.The transcript dataset derived by using high-throughput sequencing technology provided an efficient approach for identifying vital unigenes in


To analyze alterations in left ventricular (LV) myocardial T1 times in patients with pulmonary hypertension (PH) and to investigate their associations with ventricular function, mass, geometry and hemodynamics.Fifty-eight patients with suspected PH underwent right heart catheterization (RHC) and 3T cardiac magnetic resonance imaging. Ventricular function, geometry and mass were derived from cine real-time short-axis images. Myocardial T1 maps were acquired by a prototype modified Look-Locker inversion-recovery sequence in short-axis orientations. LV global, segmental and ventricular insertion point (VIP) T1 times were evaluated manually and corrected for blood T1.p < 0.0001; lateral, 1190 ± 45 ms vs. 1150 ± 33 ms, p = 0.0003; global, 1220 ± 52 ms vs. 1171 ± 29 ms, p < 0.0001; VIP, 1298 ± 78 ms vs. 1193 ± 31 ms, p < 0.0001). In PH, LV eccentricity index was the strongest linear predictor of VIP T1 (r = 0.72). Septal, lateral and global T1 showed strong correlations with VIP T1 .Septal, lateral, global and VIP T1 times were significantly higher in PH than in non-PH subjects (septal, 1249 ± 58 ms vs. 1186 ± 33 ms, In patients with PH, T1 times in VIPs and in the entire LV myocardium are elevated. LV eccentricity strongly correlates with VIP T1 time, which in turn is strongly associated with T1 time changes in the entire LV myocardium.• Native T1 mapping detects left ventricular myocardial alterations in pulmonary hypertension• In pulmonary hypertension, native T1 times at ventricular insertion points are increased• These T1 times correlate strongly with left ventricular eccentricity• In pulmonary hypertension, global and segmental myocardial T1 times are increased• Global, segmental and ventricular insertion point T1 times are strongly correlated Patients with pulmonary hypertension (PH) are at risk for progressive heart failure , 2. The Myocardial T1 relaxation time mapping is an emerging technique for tissue characterization that provides information about tissue composition on a standardized scale , 10. IncThe aim of the present study was, therefore, to assess LV global, segmental and VIP T1 times in patients with normal and elevated mean pulmonary arterial pressure, and to investigate their correlations with ventricular function, mass, geometry and hemodynamic parameters.This prospective, explorative study was approved by the local ethical review board, and all subjects gave written informed consent. Patients with contraindications to magnetic resonance imaging were not enrolled.From November 2012 to January 2015, a total of sixty-four patients with suspected PH underwent right heart catheterization (RHC) and non-contrast CMR imaging. Patients with known cardiomyopathies or PH owing to left heart diseases . LVEI remained the strongest correlate of non-normalized VIP T1 time for patients with PH .Correlation coefficients of non-normalized VIP T1 time with septal, lateral and global non-normalized myocardial T1 times remained similar to the blood normalized values [all patients, The major findings of the present study were that 1) septal, lateral, global and VIP T1 times were significantly higher in patients with PH than in those without PH; 2) VIP T1 time was the strongest linear predictor of septal, lateral and global T1 times; and 3) in patients with PH, LVEI was the strongest linear predictor of VIP T1 time, and RVCI was the only additional significant parameter in bilinear regression.As a consequence of ventricular interdependence, PH impairs both RV and LV function –24: ProgAccordingly, we presume that the significantly higher VIP T1 times found in patients with PH can be explained by focal myocardial remodelling in all (anterior and posterior) VIPs at the basal, mid-ventricular and apical levels.To lessen the impact of interobserver variability, analysis was restricted to mean VIP T1 times. In agreement with relationships previously reported between LGE volume and hemodynamic, function, mass and geometry parameters –5, 29, uIn our study, alterations of LV myocardial T1 times in patients with PH were not limited to the VIPs and IVS. T1 times were higher in PH patients in all LV segments, whereas significance was reached for all but the apical lateral segment. Segmental averaging reduced variances of determined T1 times, such that septal, global and lateral T1 values were all significantly higher in patients with PH than in patients without PH. No differences were found between PAH and non-PAH PH patients. We conjecture that the presence of higher T1 times throughout the LV myocardium in patients with PH could be related to interstitial fibrosis; in turn, such fibrosis could explain the increased LV wall stiffness, the reduced LV longitudinal contractility and the reduced circumferential strain even at LV non-septal segments reported in patients with chronic PH , 20, 21.The current study cannot unmask the underlying mechanisms leading to LV tissue alterations. However, given that VIP T1 time was strongly correlated with septal, global and lateral T1 times and no simple multilinear model of hemodynamic, ventricular function, mass and geometrical parameters could similarly predict these T1 values, it appears that PH involves the total LV myocardium to a large extent in parallel. This is compatible with the fact that VIP, septal, global and lateral T1 times showed similar significances in univariate correlations with hemodynamic, ventricular function, mass and geometrical parameters. Especially, significant correlations of lateral T1 times with RVMMI, RVEDVI, RVESVI and RVEF can be understood in this context, as these RV parameters are related to mPAP and PH. Moreover, consistently, neither PAWP as a surrogate for left atrial pressure, nor LV functional parameters exhibited dominant association with septal, global or lateral T1 times similar to the association of these parameters with VIP T1 times.k = 0.30 derived from healthy volunteers at 3T was expectedly different from the one at 1.5T [Segmental, myocardial and VIP T1 times were normalized to the mean blood T1 time of the study population. The blood normalization coefficient  at 1.5T . Blood nThe present study had a number of limitations. A non-contrast CMR protocol was applied, and, therefore, LGE and post-contrast T1 times could not be assessed. Analysis of LGE volume would have allowed proving the association of VIP T1 with the extent of LGE at VIPs. Post-contrast T1 and calculation of ECV might have provided additional information on the underlying nature of tissue alterations observed in native T1, as a recent pre-clinical study  suggestsIn patients with PH, T1 times in VIPs and also in the entire LV myocardium are elevated. LV eccentricity strongly correlates with VIP T1 time, which in turn is strongly associated with T1 time changes in the entire LV myocardium. Further research is needed to determine if elevated LV T1 times have prognostic importance in patients with PH.